@article {pmid41104514,
year = {2026},
author = {Hu, Y and Zeng, Z and Ming, X and Yin, S and Guan, Y and Gao, L and Li, D},
title = {Construction of pathogenic Sec16a mutation mouse model using CRISPR/Cas9.},
journal = {Animal models and experimental medicine},
volume = {9},
number = {3},
pages = {537-545},
doi = {10.1002/ame2.70082},
pmid = {41104514},
issn = {2576-2095},
support = {HZKY20220076//The Chunhui Program of the ministry of Education/ ; 21JC1402200//the Shanghai Municipal Commission of Science and Technology/ ; 22YF1437700//the Shanghai Municipal Commission of Science and Technology/ ; 23HC1400700//the Shanghai Municipal Commission of Science and Technology/ ; 32200732//National Natural Science Foundation of China/ ; 2022YFC3400200//National Key Research and Development Program of China/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Disease Models, Animal ; *Mutation ; Mice ; *Vesicular Transport Proteins/genetics ; Humans ; Mice, Inbred C57BL ; Male ; },
abstract = {BACKGROUND: SEC16A is a pivotal protein that facilitates the transport of proteins from the endoplasmic reticulum to the Golgi apparatus. Utilizing the protein structure function database, a potentially pathogenic mutation site (NM_014866.1: c.4606C>G(p.L1536V)) was pinpointed within the conserved central core region of the human SEC16A protein, a component integral to the COPII complex assembly.<br><br>METHODS: Leveraging information on human gene mutations and aligning human and mouse protein amino acid sequences, the Sec16aL1551V/L1551V mouse model was successfully developed using CRISPR/Cas9 technology.<br><br>RESULTS: Two behavioral experiments, namely novel object recognition and cued fear conditioning, revealed that Sec16aL1551V/L1551V mice demonstrated a phenotype of neurological impairment, evidenced by diminished abilities in learning and memory. Furthermore, while undergoing tail suspension, the Sec16aL1551V/L1551V mice displayed a distinctive limb clasping behavior, a characteristic typically associated with mouse models of chronic neurodegenerative diseases.<br><br>CONCLUSION: The Sec16aL1551V/L1551V mouse model developed in this study providing a powerful tool for better understanding of the pathogenic mechanisms of Sec16a gene mutations in brain dysfunction diseases.},
}
@article {pmid41700779,
year = {2026},
author = {Alberio, V and Savy, V and Felipe, MY and Fernandez-Martín, R and Salamone, DF},
title = {Simultaneous CRISPR-on activation of TFAP2C and SMARCA4 promotes development of trophoblast-fate cells in bovine embryos†.},
journal = {Biology of reproduction},
volume = {114},
number = {5},
pages = {1598-1609},
doi = {10.1093/biolre/ioag041},
pmid = {41700779},
issn = {1529-7268},
support = {PICT 2018-04632//ANPCyT from the Ministry of Science and Technology, Argentina/ ; },
mesh = {Animals ; *Trophoblasts/physiology/metabolism/cytology ; Cattle/embryology ; *Transcription Factor AP-2/genetics/metabolism ; Gene Expression Regulation, Developmental ; Female ; *Embryonic Development/genetics ; *Transcription Factors/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; Cell Differentiation/genetics ; *Nuclear Proteins/genetics/metabolism ; Embryo, Mammalian ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Pregnancy ; },
abstract = {Assisted reproductive techniques are widely used to produce domestic animal embryos for commercial or research purposes. In cattle, abnormal trophoblast cell differentiation during embryo development causes pregnancy and placentation failures. The CRISPR-on system has been successfully used in bovine embryos to individually activate early trophoblast lineage genes TFAP2C and SMARCA4. This study evaluates the effect of CRISPR-on activation of early and advanced trophoblast-related genes in bovine zygotes to promote trophectoderm differentiation. In one experiment, RNA was microinjected to simultaneously activate TFAP2C and SMARCA4; in another, circular DNA was microinjected to induce CDX2 or GATA3 expression separately. Controls included groups without small guide RNAs (sgRNAs, SHAM) and non-injected embryos (IVF). RNA microinjection transiently and simultaneously increased TFAP2C, SMARCA4, and downstream gene expression, producing a prolonged effect beyond individual gene activation. This led to a significant increase in trophectoderm cells at the blastocyst stage. Individual activation of CDX2 and GATA3 was effective, also inducing upstream genes without altering trophectoderm cell percentages. Neither RNA nor DNA microinjection affected blastocyst production compared to IVF. This work demonstrates the successful increase of trophoblast cells in bovine embryos using CRISPR-on, offering a useful strategy for IVF or SCNT embryos. The CRISPR-dCas9VP160 system may also aid understanding of trophoblast lineage signaling during development.},
}
@article {pmid41712528,
year = {2026},
author = {Magdy, M and Tinker-Kulberg, R and Josephs, EA},
title = {Polyvalent Guide RNAs Enhance the CRISPR-Mediated Suppression of a Human Coronavirus.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1738-1750},
doi = {10.1021/acssynbio.5c00574},
pmid = {41712528},
issn = {2161-5063},
support = {R35GM133483/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Coronavirus 229E, Human/genetics ; Gene Editing/methods ; RNA, Viral/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; },
abstract = {While CRISPR enzymes have become important tools for targeted gene editing in mammalian cells, they can also be used to specifically target and deplete viral nucleic acids to treat infections; this can be accomplished by delivering an RNA-targeting CRISPR effector like Cas13 along with a guide RNA (gRNA) that recognizes sequences from the genomes of single-stranded RNA (ssRNA) viruses. Previously, we hypothesized that by designing individual gRNAs able to target multiple, similar-but-not-identical viral sequences simultaneously ("polyvalent" guide RNAs or pgRNAs), gRNA's polyvalency would overcome any deficits caused by mispairing between the gRNA and the viral targets and, hence, still increase Cas13's antiviral potency and prevent mutagenic escape. We subsequently demonstrated this was the case using a model of viral infection in plants; however, it was not determined whether this strategy would also work against a human virus. Here, pgRNAs were designed to target multiple RNA sequences within human coronavirus 229E (hCoV-229E) and delivered along with Cas13 into a human lung epithelial cell line infected by hCoV-229E. CRISPR antiviral treatments using pgRNAs exhibited significant viral suppression in a CRISPR-dependent manner─more so than their single-target gRNA counterparts, even when multiple single-target gRNAs were used simultaneously. This improvement was also observed even as Cas13 with those same pgRNAs exhibited less "collateral" or nonspecific RNase activity relative to their single-target counterparts, which could imply that they may have greater specificity and safety profiles as therapeutic agents. Our findings demonstrate a computational and experimental pipeline by which pgRNAs, created using an unconventional gRNA design strategy, can be generated and validated to target human viruses using CRISPR antiviral biotechnologies more effectively.},
}
@article {pmid41874563,
year = {2026},
author = {Fang, J and Simon, JM and Wang, T and Gao, Y and Bi, X and Hu, L and Liao, C and Zhang, C and Adachi, Y and Zhou, J and Liu, H and Liang, Q and Nathan, JA and Mani, R and Brugarolas, J and Zhang, Q},
title = {Genome-wide CRISPR screen identifies a cytokine-enhancer circuit driving HIF-2α activation in renal cancer.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI201639},
pmid = {41874563},
issn = {1558-8238},
support = {P50 CA196516/CA/NCI NIH HHS/United States ; },
mesh = {*Basic Helix-Loop-Helix Proteins/genetics/metabolism/biosynthesis ; Humans ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; STAT3 Transcription Factor/metabolism/genetics ; Mice ; Animals ; Suppressor of Cytokine Signaling 3 Protein/genetics/metabolism ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Janus Kinase 1/metabolism/genetics ; *CRISPR-Cas Systems ; *Enhancer Elements, Genetic ; *Cytokines/metabolism/genetics ; Signal Transduction ; *Neoplasm Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Von Hippel-Lindau Tumor Suppressor Protein/genetics ; },
abstract = {Resistance to HIF-2α inhibitors such as belzutifan underscores the need to better understand how HIF-2α is transcriptionally regulated in clear cell renal cell carcinoma (ccRCC). Here, we uncover a cytokine-driven enhancer mechanism that sustains HIF-2α expression through the JAK1/STAT3 signaling pathway. Using a genome-wide CRISPR screen in von Hippel-Lindau-deficient (VHL-deficient) ccRCC cells, we identified SOCS3 as a key negative regulator of HIF-2α. Mechanistically, loss of SOCS3 activates JAK1/STAT3 signaling, leading to the recruitment of STAT3 to distal enhancers upstream of endothelial PAS domain-containing protein (EPAS1) that physically loop to its promoter to drive HIF-2α transcription. This cytokine-enhancer circuit was recapitulated in samples from patients with ccRCC and functionally validated using CRISPR interference (CRISPRi), which disrupted enhancer-promoter looping and reduced tumor growth in HIF-2α-dependent models. SOCS3 overexpression or pharmacologic inhibition of JAK1/STAT3 markedly suppressed HIF-2α expression and tumor progression both in vitro and in vivo. Unlike prior studies focusing on VHL/HIF occupancy-driven enhancer activation, this work defines a trans-acting cytokine-JAK1/STAT3 pathway that transcriptionally controls EPAS1. Together, these findings reveal a targetable enhancer mechanism that sustains HIF-2α expression and suggest that combined inhibition of JAK1/STAT3 and HIF-2α may overcome therapeutic resistance in kidney cancer.},
}
@article {pmid41915438,
year = {2026},
author = {Ma, Z and Jia, W and Zhou, X and Liu, J and Li, Q and Chang, R and Shiqi, G and Yuan, N and Chen, Z and Lan, P},
title = {In vivo CRISPR screens identify CBX4 as an epigenetic regulator for cancer immunotherapy.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI200564},
pmid = {41915438},
issn = {1558-8238},
mesh = {Animals ; Mice ; Humans ; *Epigenesis, Genetic/immunology ; *Immunotherapy ; *Tumor Microenvironment/immunology/genetics ; *CRISPR-Cas Systems ; *Polycomb-Group Proteins/genetics/immunology ; *Ligases/genetics/immunology ; Cell Line, Tumor ; *Neoplasm Proteins/genetics/immunology ; *Neoplasms/genetics/immunology/therapy ; *Liver Neoplasms/immunology/genetics/therapy ; },
abstract = {Epigenetic dysregulation is associated with immune evasion and immune checkpoint blockade (ICB) resistance. Here, using in vivo CRISPR/Cas9 screens targeting epigenetics-related factors in mouse tumor models treated with ICB, we identified chromobox 4 (CBX4) as a key negative regulator of the immune tumor microenvironment (TME). Single-cell RNA-seq and spatial transcriptomics analyses of patients receiving neoadjuvant anti-programmed cell death protein 1 (anti-PD-1) therapy revealed high CBX4 expression in both tumor cells and immunosuppressive tumor-associated macrophage subpopulations, with preferential accumulation in nonresponders. Deficiency of CBX4 in macrophages or tumor cells induced robust antitumor immunity and increased infiltration and the cytotoxic activity of CD8+ T cells and NK cells, thereby heightening the sensitivity of ICB treatment. Mechanistically, CBX4 targeted H3K9me3- and H3K27me3-marked endogenous retroelements such as RLTR4-Mm-int. Loss of CBX4 derepressed retrotransposons, activating cytosolic RNA-sensing pathways and triggering the type I IFN response, ultimately leading to a robustly inflamed TME. Moreover, we uncovered a negative correlation between CBX4 expression, immune responses, and retrotransposon levels, and were able to determine the prognosis of patients with hepatocellular carcinoma (HCC) undergoing ICB therapy. Our study establishes CBX4 as an epigenetic immune checkpoint through the epigenetic silencing of retrotransposons, remodeling the immune TME and thus providing a promising therapeutic target to enhance tumor immunogenicity and overcome immunotherapy resistance.},
}
@article {pmid41926741,
year = {2026},
author = {Driscoll, CS and Kim, J and Knott, JG},
title = {CRISPR-mediated editing of cis-regulatory elements in early mouse embryos: a tool for studying pluripotency gene regulation.},
journal = {Reproduction (Cambridge, England)},
volume = {171},
number = {5},
pages = {},
doi = {10.1093/reprod/xaag042},
pmid = {41926741},
issn = {1741-7899},
support = {HD095371//National Institutes of Child Health and Development/ ; //Michigan State University AgBioResearch/ ; HD087166//T32 doctoral fellowship from the NICHD/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; Female ; *Transcription Factor AP-2/genetics/metabolism ; *Blastocyst/metabolism ; *Embryonic Development/genetics ; Octamer Transcription Factor-3/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Pluripotent Stem Cells/metabolism ; },
abstract = {In brief Cis-regulatory elements and transcription factor binding motifs play crucial roles in regulating the spatial and temporal patterns of gene expression during development. This study tested the utility of CRISPR/Cas9 as a tool to interrogate the function of transcription factor AP2 gamma motifs in pluripotency gene expression during mouse preimplantation embryo development. Abstract Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) is a highly efficient tool that enables the generation of gene knockouts, knock-ins, and single base substitutions in a variety of organisms. Recently, we used CRISPR to examine the activity of cis-regulatory elements (CREs) in mouse preimplantation embryos. However, there is limited information on the feasibility of using CRISPR in preimplantation embryos to interrogate the function of select transcription factor (TF) binding motifs located within critical CREs in pluripotency genes. In the current study we employed CRISPR to disrupt TF AP2 gamma (TFAP2C) binding motifs located within key CREs involved in the regulation of Pou5f1 and Sox2 expression in early embryos. Microinjection of ribonucleoprotein complexes containing Cas9 and single guide RNAs (sgRNAs) targeting TFAP2C motifs located within a distal enhancer and proximal promoter substantially impaired Pou5f1 and Sox2 expression, respectively. Quantification of the editing efficiencies at each targeted CRE revealed that the targeting sgRNA sequences and the number of sgRNAs injected influenced the overall editing rates. Lastly, we investigated whether TFAP2C-induced activation of Sox2 expression in 2-cell embryos required TFAP2C motifs located within the Sox2 proximal promoter. CRISPR-mediated editing of these motifs diminished the activation of Sox2 expression. In summary, these findings indicate that CRISPR/Cas9 is a feasible approach for editing TF motifs in preimplantation embryos and provide evidence that TFAP2C directly contributes to Pou5f1 and Sox2 expression in preimplantation embryos.},
}
@article {pmid41957993,
year = {2026},
author = {Duhalde, MA and Martino, RA and Smania, A and Alvarez, HM and Hernández, MA},
title = {CRISPR/Cas9 and Cytidine Base-Editing Systems for Efficient Genome Engineering in Oleaginous Rhodococcus.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1926-1942},
doi = {10.1021/acssynbio.6c00019},
pmid = {41957993},
issn = {2161-5063},
mesh = {*Rhodococcus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Cytidine/genetics/metabolism ; Plasmids/genetics ; Genome, Bacterial/genetics ; DNA End-Joining Repair/genetics ; },
abstract = {Oleaginous Rhodococcus strains can degrade diverse compounds and synthesize and accumulate large amounts of intracellular lipids, making them attractive platforms for biotechnological applications. However, efficient genome editing in Rhodococcus remains challenging, and new molecular tools are needed to advance the understanding of its metabolism, stress responses, and cellular physiology. Here, we describe the nonhomologous end joining (NHEJ) system in rhodococci and implement an efficient genome-editing system based on a CRISPR/Cas9 nuclease approach that utilizes this repair mechanism, eliminating the need for donor DNA templates. In addition, we report the first implementation of a cytidine base-editing (CBE) system, enabling precise single-nucleotide substitutions (C•G → T•A) in oleaginous Rhodococcus strains. Both strategies rely on a dual-plasmid CRISPR platform, resulting in two plasmid sets: pTipCas9/pCA71sgRNA and pTipBE/pCA71sgRNA. These systems enabled high rates of INDEL formation and C•G → T•A base conversions, with efficiencies of 70-80% and 75-85% at native genomic targets, respectively. Finally, for the CBE system, we implemented a Csy4-mediated sgRNA-processing module to support multiplex genome editing, enabling the simultaneous modification of multiple loci. Together, these tools outperform recombination-based approaches and will facilitate the study of complex metabolic pathways and the development of genetic strategies for biotechnological applications in Rhodococcus, while also being transferable to other actinobacteria.},
}
@article {pmid41974167,
year = {2026},
author = {Peng, Z and Liu, Z and Wei, S and Zhu, Y and Jia, X and Xiao, W and Dong, Z and Hu, H and Lu, C and Pan, M and Chen, P},
title = {Genome-Wide CRISPR/Cas9 Screening Reveals BmM-ALP Orchestrates the Antioxidant Response and Metabolic Adaptations for Heat Resistance in Bombyx mori.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1968-1979},
doi = {10.1021/acssynbio.6c00053},
pmid = {41974167},
issn = {2161-5063},
mesh = {Animals ; *Bombyx/genetics/metabolism/physiology ; *CRISPR-Cas Systems/genetics ; Reactive Oxygen Species/metabolism ; *Antioxidants/metabolism ; *Thermotolerance/genetics ; Adaptation, Physiological/genetics ; *Insect Proteins/genetics/metabolism ; Heat-Shock Response/genetics ; Animals, Genetically Modified ; },
abstract = {Climate change-induced heat stress represents an increasing threat to biodiversity, engendering the need to understand evolutionary adaptations to thermal extremes. Using Bombyx mori as an economically important insect and research model, we employed genome-wide CRISPR/Cas9 screening integrated with high-throughput sequencing to systematically identify adaptations for heat resistance. The analysis identified the Bombyx mori membrane-bound alkaline phosphatase-like gene (BmM-ALP) as a vital thermoregulator. BmM-ALP triggered a pleiotropic protective cascade by significantly decreasing reactive oxygen species (ROS), suppressing apoptosis, and reprogramming mitochondrial metabolism via the phosphorylation of Vitamin B1 (VB1), suggesting a potential role in sustaining oxidative phosphorylation and enhancing energy metabolism under stress. Transgenic validation experiments confirmed the consistency of this strategy, and the BmM-ALP-OE strain displayed significantly enhanced thermal tolerance and prolonged survival under high-temperature stress. Beyond establishing BmM-ALP as a key gene of heat resistance in silkworms, this study uncovered a regulatory axis linking BmM-ALP to Vitamin B1 metabolism, illustrating a novel connection between energy homeostasis and thermal adaptation. The findings provide new insights that can be applied to breeding resistant strains in agriculture and biodiversity conservation in the context of global warming.},
}
@article {pmid41979903,
year = {2026},
author = {Stuckless, EE and Gai, LS and Slattery, SS and Dempsey, KH and Browne, TS and Gloor, GB and Edgell, DR},
title = {PHYCUT: Scalable Multiplex CRISPR/Cas9 Editing for Genome Engineering in the Diatom Phaeodactylum tricornutum.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1850-1865},
doi = {10.1021/acssynbio.5c00843},
pmid = {41979903},
issn = {2161-5063},
mesh = {*Diatoms/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Glycosylation ; Plasmids/genetics ; Fucose/metabolism ; },
abstract = {Diatoms are globally significant microalgae that contribute ∼20% of oxygen production and exhibit remarkable metabolic diversity. The marine diatom Phaeodactylum tricornutum has emerged as a promising synthetic biology platform for the bioproduction of recombinant proteins, supported by a human-like N-linked glycosylation pathway. However, its α(1,3)-linked core fucose is potentially immunogenic in humans and thus limits its biopharmaceutical applications. One hurdle to efficient genome engineering in P. tricornutum is the lack of a robust system for simultaneous CRISPR/Cas9 editing at multiple sites. To overcome this limitation, we develop PHYCUT (Phaeodactylum tricornutum Csy4-Cas9 multiplex tool), a versatile plasmid-based CRISPR/Cas9 system that uses the Csy4 endoribonuclease to process multiguide RNA arrays. To highlight PHYCUT applications, we demonstrate multiplex editing of all three FucT genes responsible for α(1,3) fucosylation in P. tricornutum, yielding strains with reduced fucosylation of secreted proteins. PHYCUT enables facile, multiplexed genome engineering in diatoms and provides a foundation for humanizing the P. tricornutum glycosylation pathway to support next-generation algal biotechnology.},
}
@article {pmid42014015,
year = {2026},
author = {Li, R and Xie, L and Hu, J and Liu, B and Zhang, H and Qian, H},
title = {Genome-wide CRISPR knockout screening identifies novel disease-associated genes in retinal pigment epithelium cells.},
journal = {Experimental eye research},
volume = {268},
number = {},
pages = {111032},
doi = {10.1016/j.exer.2026.111032},
pmid = {42014015},
issn = {1096-0007},
mesh = {*Retinal Pigment Epithelium/metabolism/pathology ; Humans ; Gene Knockout Techniques ; *Retinal Degeneration/genetics/metabolism/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome-Wide Association Study ; Cell Survival ; *CRISPR-Cas Systems ; *Gene Expression Regulation/physiology ; Cells, Cultured ; *Eye Proteins/genetics ; },
abstract = {Dysfunction and degeneration of retinal pigment epithelium (RPE) cells are common pathological features observed in various retinal degenerative diseases. It has been proposed to treat these diseases by either protecting RPE cells or replacing them with new RPE cells derived from stem cells. However, the development of effective therapeutic strategies is still limited due to the insufficient understanding of the pathogenic factors involved in retinal degeneration and their impact on the function and survival of RPE cells. In this study, we employed genome-scale CRISPR knockout (KO) screening in human RPE cells to identify genes critical for RPE cell survival. Over 300 genes were identified, including well-established housekeeping genes as well as several candidate genes previously linked to retinal degeneration, many of which still lack comprehensive investigation. Among these, we further validated PRPF38B, which was both enriched in our screening and highlighted in a prior family-based linkage study, as essential for RPE cell survival, thus confirming the effectiveness of our approach. As a component of the spliceosome, we found that PRPF38B is crucial for functions specific to RPE cells, offering new insights into its role in retinal degeneration. Our study presents a novel approach for investigating risk genes associated with retinal diseases and may inspire future research on RPE cells and vision disorders.},
}
@article {pmid42044361,
year = {2026},
author = {Huang, J and Zhang, C and Li, J and Ren, H and Yang, K and Zhang, Y},
title = {Comparative Evaluation of Engineered Bacteria and Yeast for Oral Delivery of CRISPR/Cas9 Systems in Colon Cancer Therapy.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1915-1925},
doi = {10.1021/acssynbio.6c00006},
pmid = {42044361},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Colonic Neoplasms/therapy/genetics ; Mice ; *Escherichia coli/genetics/metabolism ; Humans ; Administration, Oral ; Gene Editing/methods ; Cell Line, Tumor ; Female ; Genetic Therapy/methods ; Mice, Inbred BALB C ; },
abstract = {Colorectal cancer (CRC) poses a serious threat to human health. CRISPR/Cas9 technology offers new therapeutic strategies for the management of this disease, but its oral application is severely hindered by the limitations of suitable delivery systems. Herein, we develop and compare two separate orally delivered, genetically and chemically modified CRISPR/Cas9 delivery platforms based on E. coli BL21 and P. pastoris X33, which upon colonization in the intestine, secreted extracellular vesicles carrying the Cas9 protein and ART1-targeting sgRNA for tumor-specific gene disruption. Arginine ADP-ribosyltransferase 1 (ART1) plays a crucial role in the biological regulation of colon cancer, which was for the first time to the best of our knowledge, employed in vivo as a target gene in this study. Furthermore, we employed polydopamine (PDA) coating and gastrointestinal synthetic epithelial lining systems to facilitate microbial viability and intestinal retention, establishing on site cell factories for sustained CRISPR secretion. In subcutaneous tumor-bearing murine models, both delivery systems demonstrated comparable antitumor efficacy with significant tumor suppression. Taken together, the genetically modified microbial platform using bacterial and yeast strategies shows great potential and broad therapeutic versatility, offering a promising CRISPR-based solution for CRC treatment.},
}
@article {pmid42052944,
year = {2026},
author = {Chen, Z and Hong, W and Wei, X and Li, Y and Feng, T and Ke, X and Li, X and Wang, Y and Hang, H and Tian, X and Chu, J},
title = {A Versatile tRNA-gRNA Array-Based CRISPR/Cas9 Platform Enabling Multiplex Genome Editing and Large-Fragment Engineering in Acremonium chrysogenum.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1955-1967},
doi = {10.1021/acssynbio.6c00052},
pmid = {42052944},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Acremonium/genetics/metabolism ; *RNA, Transfer/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Cephalosporins/biosynthesis ; Metabolic Engineering/methods ; Gene Knockout Techniques ; },
abstract = {Cephalosporin C (CPC)-derived antibiotics have played a vital role in improving human health and extending life expectancy. Acremonium chrysogenum remains the only microorganism capable of industrial-scale CPC production to date. However, the lack of efficient multiplex genome-editing tools has limited studies on its gene function, high-yield mechanisms as well as metabolic engineering. To overcome this limitation, a rapid and efficient CRISPR/Cas9-based multiplex genome-editing system was developed, driven by endogenous tRNA promoters, enabling one-step multilocus knockout, large-fragment DNA deletion, and gene overexpression in A. chrysogenum. Given that many strains lack visible phenotypes associated with specific genes, we introduced a visually distinguishable red phenotype by expressing the heterologous protein mCherry under a strong promoter. In the wild-type strain, 20 endogenous tRNA promoters were evaluated and compared to the heterologous Aspergillus nidulans PgpdA and Aspergillus fumigatus U6 promoters. The endogenous tRNA[Val] promoter showed the highest knockout efficiency (95.5%). The tRNA-gRNA array-based CRISPR/Cas9 system enabled double- and triple-site knockouts without donor DNA in industrial strain, with efficiencies of 50.0-83.3% and 14.3%, respectively. This is the first demonstration of simultaneous triple-site knockout in A. chrysogenum, especially in industrial strain. Using this system, we successfully deleted a 50.7-kb DNA fragment containing the sorbicillinoids biosynthetic gene cluster with nearly 100% efficiency and achieved overexpression of the key gene pcbAB involved in CPC biosynthesis in high-yield strain, increasing CPC titer from 5.59 g/L to 6.92 g/L with an improvement of 23.8%. Overall, this tRNA-gRNA array-based CRISPR/Cas9 multiplex gene-editing system provides an efficient and versatile platform for functional genomics and industrial strain engineering in A. chrysogenum.},
}
@article {pmid42068556,
year = {2026},
author = {Ban, H and Rondthaler, SN and Lebovich, M and Lora, MA and Ugbesia, B and Andrews, LB},
title = {Cross-Strain Transferability of CRISPRi Systems and Design Rules from Laboratory to Clinical Escherichia coli Strains.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1993-2010},
doi = {10.1021/acssynbio.6c00075},
pmid = {42068556},
issn = {2161-5063},
support = {NSF DMR-1904901//NSF National Science Foundation/ ; NSF EES-1824090//NSF UMass ADVANCE program National Science Foundation/ ; NSF CBET-1943695//NSF National Science Foundaion/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {CRISPR interference (CRISPRi) has emerged as a versatile approach for targeted gene repression in many organisms, including microbes and bacteria, due to the simple design of sequence-specific transcriptional silencing of gene expression. However, the strain-specific effects on repression efficiency and the host when translating a CRISPRi system from a laboratory strain to nonmodel strains are not well understood, yet they can present important limitations to its use. Here, we investigated the repression efficiency and toxicity of three CRISPRi systems (one dCas9 and two dCas12a variants) across four different Escherichia coli strains, including a laboratory K-12 strain (MG1655) and three nonmodel strains that are clinical isolates (probiotic Nissle 1917, uropathogenic CFT073, and uropathogenic UMN026). We evaluated the repression in each strain using sets of guide RNAs (gRNAs) targeting along the gene sequence and assayed cytotoxicity of expressing each dCas protein. Growth toxicity from expression of the different dCas proteins notably differed and showed high variation between some host strains. We also observed variable repression among the strains and notably poorer repression in multiple clinical strains. Therefore, we developed a dual gRNA CRISPRi system for enhanced gene silencing among the strains, which achieved up to 824-fold repression in CFT073. The results demonstrate that strain-specific design considerations can arise when a CRISPRi genetic system is transferred to a closely related bacterial strain. These findings provide insight into the relationships between criteria used for CRISPRi genetic design and in vivo activity across nonmodel E. coli strains, providing guidelines for diverse applications of these tools.},
}
@article {pmid42084552,
year = {2026},
author = {Huang, X and Liu, M and Chen, Y and Fang, L and Cao, Y},
title = {Genome-Scale CRISPRi Screening Identifies Gene Targets for Enhanced Octanoic Acid Tolerance and Production in Escherichia coli.},
journal = {ACS synthetic biology},
volume = {15},
number = {5},
pages = {1771-1778},
doi = {10.1021/acssynbio.6c00105},
pmid = {42084552},
issn = {2161-5063},
mesh = {*Escherichia coli/genetics/metabolism/drug effects ; *Caprylates/metabolism/pharmacology ; Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Escherichia coli Proteins/genetics/metabolism ; Reactive Oxygen Species/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; NADP/metabolism ; Genome, Bacterial ; },
abstract = {Medium-chain fatty acids (MCFAs) are valuable precursors for biofuels and other commodity chemicals; however, the microbial biosynthesis of these compounds is severely constrained by cytotoxic effects. Here, we employed a genome-scale CRISPR interference (CRISPRi) library to systematically identify gene targets whose repression enhanced the tolerance to octanoic acid (C8) in Escherichia coli. Among the identified targets, repression of ygaM, gluQ, gatY, and talA enabled a 1.1- to 1.7-fold increase in C8 production relative to the parental strain MS-1. Mechanistic analyses revealed that the enhanced tolerance was associated with improved membrane properties, reduced reactive oxygen species (ROS) levels, and a shorter cell morphology. Further metabolic engineering to optimize NADPH availability increased C8 production to 1083 mg/L, representing a 2.3-fold increase over that of the MS-1 strain. This study provides new insights into engineering robust E. coli strains for MCFAs production and highlights the utility of genome-scale CRISPRi screening for identifying genetic determinants of microbial stress tolerance.},
}
@article {pmid42136289,
year = {2026},
author = {Sorourian, S and Behbahani, AB},
title = {CRISPR-Cas Systems and CHO in Biopharmaceuticals: Unlocking New Possibilities in Gene Editing.},
journal = {Current pharmaceutical biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.2174/0113892010408149251016112221},
pmid = {42136289},
issn = {1873-4316},
abstract = {The CRISPR-Cas system has significantly advanced genome editing, offering superior efficiency, precision, and ease of use compared to traditional technologies such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs). CHO cells are a widely used mammalian cell line for large-scale therapeutic protein manufacturing due to their ability to produce human-like glycosylation patterns and grow in serum-free media. Recent CRISPR-based CHO cell engineering enables precise genetic modifications, improving productivity, stability, scalability, and reducing costs. This article highlights the transformative role of CRISPR technologies in addressing genetic disorders and expanding the frontiers of multiple scientific fields. It offers a comprehensive analysis of several CRISPR-Cas systems, including Cas9, Cas12, Cas13, and Cas14, emphasizing their unique structural features and functional capabilities. While Cas9 has dominated many genomeediting applications, the use of Cas13 in Chinese Hamster Ovary (CHO) cells has opened up promising RNA-targeting strategies. Moreover, the compact Cas14 system presents notable potential for applications requiring ultra-precise genome manipulation. With their critical role in therapeutic protein production, CHO cells have greatly benefited from CRISPR-enabled engineering, leading to measurable improvements in productivity, stability, and cost-efficiency. Key advancements in CRISPR delivery platforms, including both viral and nonviral vectors, are discussed alongside ongoing challenges such as off-target effects and regulatory considerations. Emerging trends such as base editing, prime editing, and the integration of artificial intelligence for system optimization are also explored. Altogether, the discussion underscores the pivotal contribution of CRISPR technologies to CHO cell engineering and their broader impact on the future of biopharmaceutical manufacturing.},
}
@article {pmid42137038,
year = {2026},
author = {Koshi, N and Kobayashi, M and Ezura, H and Miura, K},
title = {Enhancement of parthenocarpy and fruit set through genome editing in tomato variety for processing use.},
journal = {Plant biotechnology (Tokyo, Japan)},
volume = {43},
number = {1},
pages = {127-131},
pmid = {42137038},
issn = {1342-4580},
abstract = {Tomatoes are extremely important plants that are cultivated worldwide, with various varieties grown in different regions. The traits required can vary depending on the region and intended use. Parthenocarpy, a trait that confers numerous advantages, reduces the labor required for pollination and minimizes the incidence of poor fruit set owing to temperature fluctuations. Mutations in SlIAA9 induce parthenocarpy in tomatoes, and the introduction of this trait into processed varieties via genome editing suggests its potential to markedly shorten the breeding timeline. Genome editing has gained considerable attention as a breeding technique because it enables precise mutations in specific genes. However, only a few recent studies have reported examples of genome editing in Japanese tomato varieties for processing. In this study, we employed a genome-editing technique targeting SlIAA9 to induce parthenocarpy in the Japanese tomato variety Natsunokoma for processing purposes, thereby reducing the labor required for pollination. The null-segregant Sliaa9 mutant exhibited enhanced parthenocarpy and fruit set. These results suggest that improvements in fruit-bearing and parthenocarpic traits enhance the quality of tomato varieties that are mainly used for processing.},
}
@article {pmid42137600,
year = {2026},
author = {Schönberg, PY and Muñoz-Ovalle, &#xc1; and Saleh, HA and Crespo, E and Kuhnert, R and Michen, S and Loureiro, L and Temme, A and Feldmann, A and Buchholz, F},
title = {Epigenetic editing balances TCR suppression and persistence in CAR T cells.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201712},
pmid = {42137600},
issn = {3117-387X},
abstract = {Allogeneic chimeric antigen receptor (CAR) T cell therapies offer a scalable, off-the-shelf option for cancer treatment, but their clinical use is limited by the risk of graft-versus-host disease (GvHD), mediated by the endogenous T cell receptor (TCR). Conventional strategies to eliminate TCR expression rely on genome editing tools such as CRISPR-Cas9 or base editing, which introduce permanent DNA changes and pose safety concerns. Here, we present an epigenetic editing approach that enables efficient, specific, and reversible silencing of the CD3ε gene, a critical component of the TCR complex, without altering the genome. We systematically optimized the epigenetic editor and guide RNA in a cell line and achieved robust TCR silencing in primary T and CAR T cells while preserving CAR expression, activation, and effector function. Transcriptome analysis confirmed minimal off-target effects. In vivo observations suggest the epigenetically silenced T cells to prevent GvHD while persisting longer than TCR-knockout cells, supporting the notion that transient TCR suppression may help balance safety and long-term efficacy. Our findings establish epigenetic editing as a non-genotoxic alternative to genome editing, offering a flexible and safer route to generate next-generation allogeneic CAR T cells.},
}
@article {pmid42137606,
year = {2026},
author = {Campbell, JM and Korpela, DM and Han, H and Zhao, S and Webster, DA and Nguyen, YAH and Koes, N and Aune, RO and Dagan, H and Milliken, R and Watts, JK and Murthy, N and Carlson, DF},
title = {Swine reporter model for preclinical evaluation and characterization of gene delivery vectors.},
journal = {Molecular therapy. Advances},
volume = {34},
number = {2},
pages = {201729},
pmid = {42137606},
issn = {3117-387X},
abstract = {Delivery of gene therapy vectors efficiently targeted to any somatic cell remains a key barrier for the development of genetic medicines. While rodent models provide insights into vector biodistribution and cellular tropism, their anatomical and physiological differences from humans limit their translational potential and studies in large animal models are often required. In this study, we developed a swine reporter model (SRM-1) to evaluate both viral and non-viral vector delivery in a large animal system. The SRM-1 model harbors a tdTomato reporter at the ROSA26 locus that can be activated by Cre recombinase or CRISPR-Cas reagents and allows for tracking of gene delivery vectors in vivo. To evaluate this model, we administered adeno-associated virus serotype 9 (AAV9) and lipid nanoparticles (LNPs) carrying messenger RNA (mRNA) systemically and found successful in vivo reporter activation across a variety of tissues. Intracerebroventricular (i.c.v.) administration of LNP-mRNA was also performed and demonstrated localized activation in cortical brain cells. In addition to systemic biodistribution studies, this model has utility for testing clinically relevant local administration methods, surgical and non-surgical, of delivery vectors. Our findings support the SRM-1 model as a valuable tool for advancing gene therapies from preclinical testing to clinical application.},
}
@article {pmid42137980,
year = {2026},
author = {Xiong, W and Zhou, E and Qi, Q and Liu, X and Zhang, K and Huang, S and Li, M and Wang, C and Zhou, X and Tian, T},
title = {Harnessing a single molecule for dual bioorthogonal regulation of RNA function and m6A methylation.},
journal = {Nucleic acids research},
volume = {54},
number = {9},
pages = {},
doi = {10.1093/nar/gkag438},
pmid = {42137980},
issn = {1362-4962},
support = {22377094//National Natural Science Foundation of China/ ; 22177089//National Natural Science Foundation of China/ ; 22037004//National Natural Science Foundation of China/ ; 91853119//National Natural Science Foundation of China/ ; 91753201//National Natural Science Foundation of China/ ; 22177088//National Natural Science Foundation of China/ ; 22377095//National Natural Science Foundation of China/ ; 22407104//National Natural Science Foundation of China/ ; 22407106//National Natural Science Foundation of China/ ; 22407107//National Natural Science Foundation of China/ ; 22377094//National Natural Science Foundation of China/ ; 2022YFA1502902//National Key Research and Development Program of China/ ; },
mesh = {Methylation ; Click Chemistry/methods ; *Adenosine/analogs &amp; derivatives/metabolism/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Humans ; *RNA/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Azides/chemistry ; RNA Processing, Post-Transcriptional ; },
abstract = {This study presents a multifunctional RNA regulation strategy that enables RNA molecules to undergo both bioorthogonal ligation and cleavage reactions within the same system. Using guide RNA (gRNA) as an example, we demonstrate on-demand inactivation via click chemistry and subsequent reactivation by light exposure. Applied to CRISPR-mediated, site-specific RNA methylation, this technology enables continuous, multistep programmable control, overcoming the one-way limitation of traditional methods. Results show that gRNA can stably tolerate both azide and photodegradable groups, facilitating efficient targeting of M3M14-dCas9 and dCas13b-M3M14 methylation systems to introduce m6A at specific RNA sites. Small molecules can turn off methylation through in situ click chemistry, while 365-nm light exposure rapidly restores gRNA function, allowing precise control over RNA methylation. This strategy highlights the efficiency and flexibility of progressive bioorthogonal RNA modulation and paves the way for multisite, dynamic regulation in complex biological systems.},
}
@article {pmid42138081,
year = {2026},
author = {Chang, LC and Eyler, CE and Lee, CL},
title = {Chromosomal instability induced by CRISPR/Cas9: implications for pancreatic cancer therapy.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI206028},
pmid = {42138081},
issn = {1558-8238},
mesh = {Humans ; *Pancreatic Neoplasms/genetics/therapy/pathology/metabolism ; *Chromosomal Instability ; *CRISPR-Cas Systems ; *DNA Breaks, Double-Stranded ; },
abstract = {Clinical management of pancreatic cancer (PC) remains severely limited, primarily due to the complex tumor microenvironment. Emerging DNA damage-targeted strategies have demonstrated considerable therapeutic potential in PC. In this issue of the JCI, Teh et al. employed cancer-specific multitarget sgRNAs to induce DNA double-strand breaks (DSBs), resulting in lethal effects in PC cells. Integrative bioinformatic and cytogenetic analyses revealed that CRISPR/Cas9-mediated DSBs provoked persistent chromosomal instability, ultimately leading to chromosome catastrophe and cell death. Compared with equivalent radiation-induced DSBs, these sgRNAs exhibited superior cytotoxicity and were able to eliminate cells resistant to a specific sgRNA via subsequent targeting at distinct genomic sites, highlighting a promising and innovative precision therapeutic approach for clinical treatment of PC.},
}
@article {pmid42138085,
year = {2026},
author = {Teh, SSK and Kotwal, A and Bennett, A and Halper-Stromberg, E and Morsberger, L and Zamani, S and Shi, Y and Skaist, A and Zhu, Q and Bowland, K and Liang, H and Hruban, RH and Hung, CF and Anders, RA and Roberts, NJ and Scharpf, RB and Goldstein, M and Zou, YS and Eshleman, JR},
title = {Simultaneous CRISPR/Cas9-induced double-strand breaks are lethal in models of pancreatic cancer.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {10},
pages = {},
doi = {10.1172/JCI190121},
pmid = {42138085},
issn = {1558-8238},
mesh = {*Pancreatic Neoplasms/genetics/pathology/therapy/metabolism ; *CRISPR-Cas Systems ; Humans ; *DNA Breaks, Double-Stranded ; Cell Line, Tumor ; Animals ; Mice ; Gene Editing ; },
abstract = {While radiation is an effective oncologic therapy, killing cancer by inducing DNA double-strand breaks (DSBs), it lacks specificity for neoplastic cells. We have previously adapted the CRISPR/Cas9 gene-editing technology as a cancer-specific treatment modality targeting somatic mutations in pancreatic cancer (PC). However, its tumoricidal potential remains unclear, especially in comparison with therapeutic doses of radiation. Here, we demonstrate that CRISPR/Cas9-induced DSBs are more cytotoxic in PCs than a comparable number of radiation-induced DSBs. We observed more than 90% tumor growth inhibition by targeting 9 sites with cancer-specific sgRNAs. Through both bioinformatics and cytogenetics analyses, we found that CRISPR/Cas9-induced DSBs triggered ongoing chromosomal rearrangements, with 87% of structural variants not directly produced from the initial CRISPR/Cas9-induced DSBs, and chromosomal instability peaking before cell death. By comparing the cytotoxicity of CRISPR/Cas9- and radiation-induced DSBs, we demonstrated that the number of DSBs required to achieve equitoxic effects was approximately 3 times higher for radiation than CRISPR/Cas9. Finally, we showed that PC cells that had survived CRISPR/Cas9 targeting retained susceptibility to subsequent CRISPR/Cas9-induced DSBs at different genomic sites with more than 87% growth inhibition. Together, our data support the therapeutic potential of CRISPR/Cas9 as an anticancer strategy.},
}
@article {pmid42138264,
year = {2026},
author = {Gao, M and Jiang, T and Si, W and Rong, Y and Hu, Y and He, X and Liu, BF and Chen, P and Yang, Y and Deng, Y and Wang, FB},
title = {Harnessing CRISPR-Cas12 and Microfluidics Chips for Multiplex Respiratory Pathogens Diagnosis.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.5c03226},
pmid = {42138264},
issn = {2379-3694},
abstract = {Respiratory pathogens jeopardize population health, particularly high-risk groups. CRISPR-Cas systems, as novel nucleic acid detection platforms, offer timely identification and have become a major research focus. This study presents a novel diagnostic workflow that combines recombinase polymerase amplification (RPA) for pre-amplification of pathogen nucleic acids with CRISPR-based detection. By combining microfluidic technology and portable imaging devices, this study developed a multiplex assay capable of simultaneously detecting seven clinically relevant pathogens in a single sample, including influenza A virus (FluA), influenza B virus (FluB), respiratory syncytial virus (HRSV) A and B, mycoplasma pneumoniae (MP), adenovirus (HAdv), and parainfluenza virus (HPIVs). Utilizing the POCT-CRISPR platform, simultaneous detection of seven respiratory pathogens can be achieved within approximately 30 min, achieving detection limits of 0.1-1 fM. This method streamlines the detection process, significantly reducing both the complexity of operations and the overall detection time. Clinical cohort validation demonstrated a detection efficiency of 99.63% sensitivity and 100% specificity. These results confirm the effectiveness and reliability of the detection method. Additionally, the 7-virus panel is estimated at approximately $32 per sample, a cost competitive with commercial multiplex qPCR detection kits ($15-$110 per sample) and substantially more economical than integrated cartridge-based syndromic platforms. The platform features simple operation, cost-effectiveness, short turnaround time, and reliable detection performance, making it highly suitable for point-of-care testing (POCT) at the grassroots level.},
}
@article {pmid42138716,
year = {2026},
author = {Lefrançois, G and Lavallée, E and Rowell, MC and Bourdeau, V and Mohebali, F and Bertomeu, T and Duman, AM and Nikolova, M and Tyers, M and Gravel, SP and Schmitzer, AR and Ferbeyre, G},
title = {The role of ATP synthase subunit e (ATP5I) in mediating the metabolic and antiproliferative effects of metformin in cancer cells.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
doi = {10.7554/eLife.102680},
pmid = {42138716},
issn = {2050-084X},
support = {TFRI Project #1123//Terry Fox Research Institute/ ; Operating Grant 2016//Cancer Research Society/ ; 935858//Cancer Research Society/ ; 840633//Cancer Research Society/ ; RGPIN-2021-03128//Natural Sciences and Engineering Research Council of Canada/ ; Ganotec/Marc-Andr&#xe9; Pigeon Fund//Cancer Research Society/ ; 1054571//Cancer Research Society/ ; },
mesh = {*Metformin/pharmacology/metabolism ; Humans ; Cell Line, Tumor ; *Mitochondrial Proton-Translocating ATPases/metabolism/genetics ; *Cell Proliferation/drug effects ; Oxidative Phosphorylation/drug effects ; *Antineoplastic Agents/pharmacology ; Mitochondria/drug effects/metabolism ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Hypoglycemic Agents/pharmacology ; },
abstract = {Here, we identify the subunit e of F1F0-ATP synthase (ATP5I) as a target of metformin, a first-in-class antidiabetic biguanide. ATP5I maintains the stability of F1F0-ATP synthase dimers, which is crucial for shaping cristae morphology. We demonstrate that ATP5I interacts with a biguanide analogue in vitro, and disabling its expression by CRISPR-Cas9 in pancreatic cancer cells leads to the same phenotype as biguanide-treated cells, including mitochondrial morphology alterations, reduction of the NAD[+]/NADH ratio, inhibition of oxidative phosphorylation (OXPHOS), rescue of respiration by uncouplers, and a compensatory increase in glycolysis. Notably, metformin disrupts F1F0-ATP synthase oligomerization, leading to the accumulation of vestigial assembly intermediates in pancreatic and osteosarcoma cancer cells, a phenotype also observed upon ATP5I inactivation in pancreatic cancer cells. Moreover, ATP5I knockout (KO) cells exhibit resistance to the antiproliferative effects of biguanides, but reintroduction of ATP5I rescues the metabolic and antiproliferative effects of metformin and phenformin. Finally, a genome-wide CRISPR screening in NALM-6 lymphoma cells revealed that metformin-treated cells exhibit genetic interaction profiles similar to those observed with the F1F0-ATP synthase inhibitor oligomycin, but not with the complex I inhibitor rotenone. This provides unbiased support for the relevance of the newly proposed target.},
}
@article {pmid42139236,
year = {2026},
author = {Quiroz-Huanca, A and Vargas-Reyes, M and López, JD and Flores-Jimenez, K and Saldarriaga-Morán, S and Cifuentes, K and Alcántara, R},
title = {Thermal optimized PCR coupled to CRISPR-Cas12a for rapid detection of blaOXA-1 resistance gene.},
journal = {PloS one},
volume = {21},
number = {5},
pages = {e0337675},
doi = {10.1371/journal.pone.0337675},
pmid = {42139236},
issn = {1932-6203},
mesh = {*beta-Lactamases/genetics ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/drug effects/isolation &amp; purification/enzymology ; *Polymerase Chain Reaction/methods ; Animals ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The β-lactams are critically important broad-spectrum antibiotics, widely used as first-line treatments; however, their effectiveness is increasingly compromised by β-lactamase enzymes. Among these, OXA-type enzymes have expanded to over 400 variants and are highly prevalent in Enterobacteriaceae. Current phenotypic and molecular detection tests have long turnaround times or require specialized equipment, respectively. In this study, we optimize a rapid molecular assay combining a PCR with modified thermal ramp rate (TRR) along with CRISPR-Cas12a fluorescence detection for blaOXA-1-harboring E. coli isolates. Using a commercial DNA Taq polymerase (TRR: 2.2 °C/s, annealing and extension hold time: 1 s), amplification time was reduced from 80 to 30 min, enabling detection within 50 min (PCR: 30 min; CRISPR: 20 min). With a locally produced enzyme (hold: 10 s), amplification time was 44 min. To demonstrate the practical application of the assay, we evaluated spiked poultry fecal samples achieving an analytical sensitivity of 8 CFU/reaction using commercial DNA Taq polymerase. The accelerated PCR:CRISPR workflow delivers results in less than one hour without compromising technical sensitivity (attomoles range), not requiring high technical expertise, and can be implemented in laboratories with basic molecular biology equipment.},
}
@article {pmid42141309,
year = {2026},
author = {Qin, L and Liu, D and Wang, Z and Sun, X and Zhao, X},
title = {Molecular biology of Pleurotus mushrooms: genomic resources, genetic manipulation, and regulation mechanisms.},
journal = {Antonie van Leeuwenhoek},
volume = {119},
number = {6},
pages = {},
pmid = {42141309},
issn = {1572-9699},
support = {32402651//National Natural Science Foundation of China/ ; 2024AFB274//Hubei&#x3000;Provincial&#x3000;Natural&#x3000;Science&#x3000;Foundation&#x3000;of&#x3000;China/ ; Q20231511//Youth Talent Project of Scientific Research Plan of Hubei Provincial Education Department of China/ ; K2023058//Scientific Research Fund of Wuhan Institute of Technology/ ; },
mesh = {*Pleurotus/genetics ; *Genomics/methods ; *Genome, Fungal ; Gene Editing ; CRISPR-Cas Systems ; Gene Expression Regulation, Fungal ; RNA Interference ; },
abstract = {As one of the most widely cultivated edible mushrooms in the world, Pleurotus mushrooms are popular among people for their delicious taste and rich nutritional value. Because of their great economic value, the research on the molecular biology of Pleurotus spp. has been deepening in recent years. The study first summarized the current situation of genomic resources available for this genus. The whole genome sequencing of 14 species, including Pleurotus tuoliensis and Pleurotus ostreatus, provides reference data for mining functional genes. Although the genomic data for Pleurotus mushrooms are continuously increasing, actual instances of successful genetic transformation remain restricted. Research on the regulatory mechanisms of key genes at different developmental stages and under various environmental stresses is insufficient. Then, the application of gene editing methods (CRISPR/Cas9, RNAi, and gene overexpression) in Pleurotus mushrooms was systematically described. RNAi and gene overexpression technologies have become well-established and are routinely used in most Pleurotus mushrooms. However, the application of CRISPR/Cas9 technology is still limited to P. eryngii and P. ostreatus. This limitation is attributed to the difficulties in establishing genetic transformation systems and the low efficiency of homologous recombination. Furthermore, this review explored the value of multi-omics technologies in elucidating the molecular mechanisms of morphogenesis and stress responses. To address the lack of specific antibodies for non-model organisms, we evaluated the application potential of DAP-seq technology in Pleurotus mushrooms and discussed its limitations, including the risk of false positives arising from the absence of a genuine environment in vivo. The purpose of this review is to evaluate the current molecular biology research on Pleurotus spp., and to provide systematic technical support and insights for functional genomics research and the analysis of molecular mechanisms of complex traits in Pleurotus mushrooms.},
}
@article {pmid41397899,
year = {2026},
author = {Chowdhury, A and Garcia, BG and Zahoor, MA and ElMawla, NF and Davidson, AR and Wyatt, HDM and Maxwell, KL and Mahassine, A and Gehring, A and Feld, JJ},
title = {A Rapid Assay for Hepatitis C Virus RNA Detection Using Reverse-Transcription Loop-Mediated Isothermal Amplification-Coupled CRISPR-Cas12b-Based Strategy.},
journal = {The Journal of infectious diseases},
volume = {233},
number = {5},
pages = {840-847},
doi = {10.1093/infdis/jiaf609},
pmid = {41397899},
issn = {1537-6613},
mesh = {Humans ; *Hepacivirus/genetics/isolation &amp; purification ; *Hepatitis C/diagnosis/virology ; *RNA, Viral/genetics/blood ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Genotype ; Limit of Detection ; },
abstract = {BACKGROUND: Hepatitis C virus (HCV) diagnosis usually requires detection of antibody followed by HCV RNA. The requirement for 2 tests leads to major drop-offs in the cascade of care. Existing near-care HCV RNA tests have slow turnaround time and are expensive with limited availability. We aim to develop a cost-effective, rapid, and sensitive test for detection of HCV RNA to enhance screening, particularly in marginalized and remote populations.<br><br>METHODS: After RNA extraction from plasma, HCV RNA is reverse-transcribed and amplified using loop-mediated isothermal amplification with HCV-specific primers. The amplified HCV DNA is then detected via CRISPR-Cas12b with a fluorescence readout.<br><br>RESULTS: HCV RNA from patient samples with genotypes 1a, 1b, 2, 3a, and 4 was detected with high sensitivity and specificity. The lower limit of detection (LLOD) with HCV JFH1 plasmid (genotype 2) is 250 plasmid copies/mL (approximately 100&#x2005;IU/mL). For clinical samples, we determined the LLOD for genotypes 1 and 3, the most common in North America. Using 500&#x2005;&#x3bc;L of plasma, genotype 1 RNA &#x2265;100&#x2005;IU/mL was detected within 40-45&#x2005;minutes, while genotype 3 had an LLOD of 5000&#x2005;IU/mL. The clinical sensitivity was 100% in 72 HCV patient samples, including acute HCV and HCV/hepatitis B virus (HBV) coinfection. The specificity was 100%, with no false-positives in 33 HCV-negative samples, including those with HBV or human immunodeficiency virus/HBV coinfection.<br><br>CONCLUSIONS: Our assay shows high specificity and sensitivity to detect HCV RNA directly from plasma within 45&#x2005;minutes and hence could be used for efficient screening and diagnosis of HCV infection globally.},
}
@article {pmid41604419,
year = {2026},
author = {Martínez Rivas, FJ and Smith, MA and Zangishei, Z and Alseekh, S and Usadel, B and Plaxton, WC and Fernie, AR},
title = {Malate matters: disrupting bacterial-type phosphoenolpyruvate carboxylase (BTPC) rewires tomato fruit development.},
journal = {Plant physiology},
volume = {201},
number = {1},
pages = {},
pmid = {41604419},
issn = {1532-2548},
support = {452682775//Deutsche Forschungsgemeinschaft/ ; 390686111//Deutsche Forschungsgemeinschaft/ ; //PlantaSyst/ ; 664621//European Union's Horizon 2020 Research and Innovation Programme/ ; 739582//European Union's Horizon 2020 Research and Innovation Programme/ ; 664620//European Union's Horizon 2020 Research and Innovation Programme/ ; RGPIN-2024-06467//Natural Sciences and Engineering Research Council of Canada/ ; //Queen's Research Chair/ ; },
mesh = {*Solanum lycopersicum/growth &amp; development/genetics/enzymology/metabolism ; *Malates/metabolism ; *Fruit/growth &amp; development/genetics/enzymology/metabolism ; *Phosphoenolpyruvate Carboxylase/metabolism/genetics ; *Plant Proteins/metabolism/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Plant phosphoenolpyruvate carboxylases (PEPCs) are ubiquitously expressed as cytosolic Class-1 PEPC homotetramers composed of 107 kDa plant-type PEPC (PTPC) subunits that are highly sensitive to allosteric inhibition by malate. Class-2 PEPC heterooctameric complexes that are desensitized to malate inhibition also exist in certain sink tissues due to the interaction of a Class-1 PEPC with unrelated 118 kDa bacterial-type PEPC (BTPC) polypeptides. Class-2 PEPCs dynamically associate with the mitochondrial outer envelope and have been hypothesized to support sustained anaplerotic flux and respiratory CO2 refixation in malate-rich sink tissues, including immature tomato fruit. The current study generated CRISPR-Cas9-edited tomato lines with targeted disruption of the BTPC gene and investigated the impact on fruit development, metabolism, and transcriptional regulation. Immunoblotting and co-immunoprecipitation confirmed the absence of BTPC polypeptides and Class-2 PEPC complexes in the edited lines. Fruits from the edited plants were 25% smaller and 40% lighter and required up to 10 additional days to complete ripening compared to the WT. Metabolomic analysis across ripening stages revealed substantial reductions in malate and citrate, with elevated sugars and amino acids, indicating reprogrammed carbon flux. RNA-seq data showed downregulation of genes for cell wall remodeling, sugar transport, and ethylene-responsive transcription factors. These results provide direct evidence that BTPC is essential for organic acid balance, sugar metabolism, and ripening regulation in tomato. Its absence perturbs metabolic homeostasis and developmental progression, positioning BTPC as a strategic target for enhancing fruit quality traits through genetic engineering.},
}
@article {pmid41734599,
year = {2026},
author = {Kim, GH and Kim, MM},
title = {Effect of p53 gene mutation with loss of function on the expression of genes and proteins involved in cell proliferation.},
journal = {Mutation research},
volume = {832},
number = {},
pages = {111931},
doi = {10.1016/j.mrfmmm.2026.111931},
pmid = {41734599},
issn = {1873-135X},
mesh = {Humans ; *Cell Proliferation/genetics ; *Tumor Suppressor Protein p53/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Loss of Function Mutation ; Gene Expression Regulation, Neoplastic ; *Mutation ; Cell Cycle/genetics ; },
abstract = {The tumor suppressor gene TP53 plays a vital role in preserving genomic integrity by regulating cell cycle progression, DNA repair mechanisms, and apoptosis. This study aims to examine how CRISPR/Cas9-induced loss-of-function mutations in the p53 gene influence cellular processes on cell cycle regulation and tumorigenic signaling in HT1080 human fibrosarcoma cells. Successful TP53 gene disruption was confirmed by Sanger sequencing, and its structural modelling using AlphaFold2 and ChimeraX confirmed alterations in the predicted TP53 protein structure compared to that of wild type. Gene expression analyses, conducted via RT-PCR and qPCR, demonstrated a marked decrease in TP53 mRNA expression within the modified cells. Despite the mutation, the edited cells elevated activity of the senescence marker β-galactosidase (SA-β-gal). They decreased the production of collagen, suggesting that the structural disruption caused by CRISPR/Cas9 leads to the loss of functional p53 activity. Western blotting and immunofluorescence assays further revealed a remarkable downregulation of key cell cycle and tumorigenesis-related proteins, including TP53, phosphorylated TP53 (p-TP53), acetylated TP53 (ac-TP53), MMP-2, cyclin D, cyclin E, AKT, BAX, MDM2, and phosphorylated Rb (p-Rb) in the edited cells relative to the wild-type counterpart. Our results suggest that the TP53 mutation may disrupt essential pathways related to cell proliferation and stress responses. This provides new insights into TP53 functionality and underscores its potential as a therapeutic target in cancer biology.},
}
@article {pmid41882360,
year = {2026},
author = {Smith, QM and Whittle, S and Aramayo, RJ and Rollins, DE and Jalal, ASB and Egharevba, DI and Morris, KL and Pyne, ALB and Rueda, DS},
title = {Structural basis of supercoiling-induced CRISPR-Cas9 off-target activity.},
journal = {Nature},
volume = {653},
number = {8114},
pages = {627-635},
pmid = {41882360},
issn = {1476-4687},
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/ultrastructure ; *CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; DNA, Circular/chemistry/ultrastructure/genetics/metabolism ; *DNA, Superhelical/chemistry/metabolism/ultrastructure/genetics ; *Gene Editing/methods ; Models, Molecular ; Protein Domains ; RNA/chemistry/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 is a powerful genome-editing tool[1], but genome-wide off-target activity can hinder therapeutic applications. Negative supercoiling ((-)SC) has been implicated in off-target activity, but a molecular-level understanding is lacking. Here, using (-)SC DNA minicircles, we observe supercoiling-driven structural defects in the DNA that are resolved by Cas9 binding. Cryo-electron microscopy structures of Cas9 bound in both the on-target and off-target configurations highlight that the Cas9 HNH domain is poised in a more catalytically competent conformation. New DNA-RNA mismatch geometries are accommodated across the protospacer and structural plasticity in the protospacer adjacent motif distal region of the protospacer is topology dependent. Together, our study reveals the molecular basis for (-)SC-induced Cas9 targeting and provides a framework for the design of next-generation high-fidelity CRISPR effectors with topological context.},
}
@article {pmid41913646,
year = {2026},
author = {Streiber, M and Liu, N and Simon, L and Adermann, F and Bachmann, V and Gath, L and Hoeppener, S and Schubert, S and Werz, O and Lapinte, V and Morille, M and Bauer, M and Press, AT and Schubert, US and Traeger, A},
title = {Extrahepatic Gene Editing In Vivo Using Organic Solvent-Free Lipid Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {27},
pages = {e11489},
pmid = {41913646},
issn = {1613-6829},
support = {13XP5034A//Bundesministerium f&#xfc;r Bildung und Forschung/ ; 03RU2U071H//Bundesministerium f&#xfc;r Bildung und Forschung/ ; 2021 FGI 0005//Th&#xfc;ringer Aufbaubank/ ; 2023FGR0077//Th&#xfc;ringer Aufbaubank/ ; //Joachim Herz Stiftung/ ; P2024-02-016//Carl-Zeiss-Stiftung/ ; 57604510//German Academic Exchange Service/ ; 2018FGI0025//European Regional Development Fund/ ; ANR- 20-CE09-0011-01//Agence Nationale de la Recherche/ ; 514006196//Deutsche Forschungsgemeinschaft/ ; 316213987//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Nanoparticles/chemistry/ultrastructure ; Humans ; Animals ; *Lipids/chemistry ; *Gene Editing/methods ; Mice ; Solvents/chemistry ; CRISPR-Cas Systems/genetics ; Polyethylene Glycols/chemistry ; Transfection ; Liposomes ; },
abstract = {Targeted therapy, which modifies genes and their expression, holds great promise for treating a variety of diseases, including cancer, inborn errors of metabolism, and acute and chronic inflammatory and infectious conditions. However, it also presents challenges related to RNA delivery, immune responses, side effects of delivery vectors, and the need for individualized formulations. To overcome these limitations, the choice of lipids and formulation processes might be re-evaluated, with a focus on eliminating critical components, such as poly(ethylene glycol) (PEG) and ethanol. Thus, a purely water-based formulation for lipid nanoparticles was developed, offering a material-efficient, time-saving process with high reproducibility. Initially, a stealth lipid containing poly(2-methyl-2-oxazoline) (PMeOx) was used, and the formulation was later expanded to include approved lipids. These nanoparticles not only efficiently transfect primary human immune cells but also effectively deliver multiple nucleotides in CRISPR-Cas9 applications. Moreover, an in vivo comparison revealed that the nanoparticles exhibited preferential transfection in extrahepatic tissues. This distinguishes them from conventional cholesterol-rich lipid nanoparticles, which primarily target the liver regardless of the application route.},
}
@article {pmid41931906,
year = {2026},
author = {Pedersen, A and Blay-Cadanet, J and Storgaard, J and Hernaez, B and Thyrsted, J and Bach-Nielsen, CS and Twayana, K and Jørgensen, SE and Rio-Bergé, C and Poulsen, C and Thielke, AL and Thomsen, EA and Kalucka, J and Olagnier, D and Luo, Y and Reggiori, F and Mogensen, TH and Alcamí, A and Hansen, AL and Holm, CK},
title = {NRF2 controls a diverse network of antiviral effectors with p62 acting as a central restriction factor effective across virus families.},
journal = {Redox biology},
volume = {93},
number = {},
pages = {104135},
pmid = {41931906},
issn = {2213-2317},
mesh = {*NF-E2-Related Factor 2/metabolism/genetics ; Humans ; *SARS-CoV-2/physiology ; Virus Replication/drug effects ; *Sequestosome-1 Protein/metabolism/genetics ; Influenza A virus/physiology ; Herpesvirus 1, Human/physiology ; *COVID-19/virology/genetics/metabolism ; Animals ; Autophagy ; Vaccinia virus/physiology ; Antiviral Agents/pharmacology ; CRISPR-Cas Systems ; Chlorocebus aethiops ; },
abstract = {The transcription factor erythroid 2 (NFE2)-related factor 2 (NRF2) is a key regulator of cellular homeostasis. Recent discoveries have identified agonists of NRF2 as inducers of broad cellular resistance to viral infection including SARS-CoV-2. Nevertheless, it is still unclear to what extent NRF2 itself is an inducer of anti-viral immunity and its downstream antiviral effectors have not been mapped. Here, we first demonstrate through specific genetic activation and silencing that NRF2 restricts SARS-CoV-2 replication. We then used a focused CRISPR-activation screen to map antiviral NRF2-inducible effector genes that restrict replication of SARS-CoV-2, Influenza A virus (IAV), Herpes Simplex virus 1 (HSV1) and Vaccinia virus (VACV). This approach allowed us to identify a range of antiviral effectors each of which restrict members of one or more virus families. Importantly, we identified the NRF2-inducible selective autophagy receptor p62/SQSTM1 as a broadly effective restriction factor across all the tested viruses. Importantly, p62 inhibited SARS-CoV-2 replication in cells treated with the lysosomal inhibitor bafilomycin A1, as well as in cells deficient in the autophagy protein ATG5. Similarly, p62 inhibited replication of HSV1 and IAV independently of ATG5 and ATG16L1 respectively. Thus, NRF2 restricts viral replication through a hitherto underappreciated network of antiviral restriction factors effective across multiple virus families. Importantly, we identify p62 as a broadly acting antiviral effector that restricts viral replication independently of canonical autophagy.},
}
@article {pmid41942711,
year = {2026},
author = {Wang, W and Li, Y and Dong, S and Liu, Y and Guo, C and Su, Y and Tian, W and Hu, X and Wang, Z},
title = {The exonic SNP rs11676272-C risk allele mediates diet-induced obesity and reduces enhancer activation.},
journal = {EMBO reports},
volume = {27},
number = {9},
pages = {2462-2490},
pmid = {41942711},
issn = {1469-3178},
support = {32470645//MOST | National Natural Science Foundation of China (NSFC)/ ; 32070567//MOST | National Natural Science Foundation of China (NSFC)/ ; 32202840//MOST | National Natural Science Foundation of China (NSFC)/ ; C2023201032//| Natural Science Foundation of Hebei Province ()/ ; 050001-5000019//Hebei University ()/ ; ZJ2025118//Zhejiang Province Human Resources and Social Security Department ()/ ; },
mesh = {Animals ; Humans ; *Obesity/genetics/etiology/metabolism ; HEK293 Cells ; Mice ; *Polymorphism, Single Nucleotide ; *Alleles ; *Adenylyl Cyclases/genetics/metabolism ; *Enhancer Elements, Genetic ; Diet, High-Fat/adverse effects ; Genetic Predisposition to Disease ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Gene Expression Regulation ; Male ; },
abstract = {Genome-wide association studies (GWASs) have identified hundreds of obesity-associated SNPs, but establishing their causality remains challenging. Here, we demonstrate that rs11676272, located in the ADCY3 gene, is a functional causal variant for obesity susceptibility. Bioinformatic analyses and dual-luciferase reporter assays indicate that the rs11676272 region may act as a human-gained enhancer regulating ADCY3 expression. In HEK293T cells, CRISPR-Cas9-mediated single-nucleotide editing of rs11676272 (T > C) reduces ADCY3 expression. Moreover, the rs11676272-T allele is preferentially bound by the transcription factor E2F3 to upregulate ADCY3 expression, whereas the rs11676272-C risk allele loses this binding. In vivo, the rs11676272 T > C variant in human ADCY3 (hADCY3) knock-in mice accelerates weight gain under high-fat diet conditions and shortens primary cilia in the ventromedial hypothalamus (VMH). CRISPRa-mediated activation of the hADCY3 promoter region rescues ciliary length in both the VMH and hypothalamic arcuate nucleus of Mut-hADCY3 mice. Our data reveal a causal role for rs11676272 in obesity, offering insight into potential therapeutic strategies.},
}
@article {pmid42020604,
year = {2026},
author = {Calvo Fernández, E and Tomassoni, L and Zhang, X and Wang, J and Obradovic, A and Laise, P and Griffin, AT and Vlahos, L and Minns, HE and Morales, DV and Simmons, C and Gallitto, M and Wei, HJ and Martins, TJ and Becker, PS and Crawford, JR and Tzaridis, T and Wechsler-Reya, RJ and Garvin, J and Gartrell, RD and Szalontay, L and Zacharoulis, S and Wu, CC and Zhang, Z and Califano, A and Pavisic, J},
title = {Systematic design of combination therapy by targeting master regulators of coexisting diffuse midline glioma cell states.},
journal = {Nature genetics},
volume = {58},
number = {5},
pages = {1112-1125},
pmid = {42020604},
issn = {1546-1718},
mesh = {Humans ; *Glioma/genetics/drug therapy/pathology ; Animals ; Mice ; Cell Line, Tumor ; Pyrazoles ; *Brain Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use/pharmacology ; Pyrimidines ; Gene Expression Regulation, Neoplastic/drug effects ; Nitriles ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {Intratumor heterogeneity fundamentally challenges cancer treatment, as coexisting, molecularly distinct cell states with non-overlapping drug sensitivities can drive therapeutic resistance. We establish and validate a generalizable, network-based framework to systematically identify combination therapies targeting complementary tumor cell states. Applied to diffuse midline glioma (DMG)-a universally fatal pediatric malignancy-this approach identified master regulator protein dependencies in seven coexisting cell states, confirmed by pooled CRISPR-Cas9 assays. Perturbational transcriptional profiles for 372 clinically relevant drugs prioritized candidates predicted to invert state-specific master regulator activity. State-selective drug sensitivity was validated for eight out of nine (89%) drugs in vivo, including avapritinib, ruxolitinib and larotrectinib. Compared with monotherapy, co-administering drugs targeting complementary states significantly prolonged survival across virtually all combinations, with avapritinib plus ruxolitinib extending median survival nearly threefold versus vehicle and 1.5-fold versus avapritinib alone. These findings establish clinically actionable DMG combinations and a tumor-agnostic and mutation-agnostic framework for rational combination therapy design.},
}
@article {pmid42062574,
year = {2026},
author = {Xu, Y and Stubbendieck, RM and Viswanatha, R and Krč, A and Baik, LS and Suh, WS and Hu, Y and Wang, H and Yin, L and Mameli, E and van der Meij, A and Carlson, JR and Doxey, AC and Stenmark, P and Perrimon, N and Currie, CR and Dong, M},
title = {Streptomyces produce a diphtheria toxin-like exotoxin that targets insects.},
journal = {Nature microbiology},
volume = {11},
number = {5},
pages = {1271-1285},
pmid = {42062574},
issn = {2058-5276},
support = {R01AI170835//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01AI189789//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01NS080833//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; },
mesh = {Animals ; *Streptomyces/metabolism/genetics ; Drosophila melanogaster/drug effects ; Virulence Factors/metabolism/genetics ; *Diphtheria Toxin/genetics/metabolism/chemistry ; *Insecta/drug effects/microbiology ; Bacterial Proteins/genetics/metabolism/chemistry ; *Exotoxins/metabolism/genetics/chemistry/toxicity ; Phylogeny ; *Insecticides/metabolism ; Peptide Elongation Factor 2/metabolism ; CRISPR-Cas Systems ; Insect Proteins/metabolism ; },
abstract = {Streptomyces and insects engage in complex interactions shaped by millions of years of evolution. While many beneficial relationships are well recognized, it remains unknown whether Streptomyces produce virulence factors targeting insects specifically. Here, through bioinformatic analysis, we identified diphtheria toxin (DT) homologues, which we named Streptomyces antiquus insecticidal proteins (SAIP), within a monophyletic lineage of Streptomyces that emerged more than 100 million years ago. SAIP is cytotoxic to insect cells and lethal to Drosophila melanogaster, suppressing neuronal activity and immune responses in vivo. Structural and functional studies validated that SAIP is homologous to DT and acts by ADP ribosylation of eukaryotic elongation factor 2. CRISPR-Cas9 screening identified the insect protein Flower as the SAIP receptor across a range of insects. Toxigenic Streptomyces can consume dead insects and produce bioactive secondary metabolites while growing on insect carcasses. These findings establish an insecticidal toxin in Streptomyces and demonstrate that Streptomyces have evolved highly specific virulence factors against insects.},
}
@article {pmid42065511,
year = {2026},
author = {Chen, Y and Tang, D and Zhan, J and Wang, Y and Song, Z},
title = {Study on the detection of prostate cancer using MIRA-CRISPR/Cas12a technology.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {18},
pages = {3848-3857},
doi = {10.1039/d6ay00025h},
pmid = {42065511},
issn = {1759-9679},
mesh = {Humans ; Male ; *Prostatic Neoplasms/diagnosis/urine/genetics ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Antigens, Neoplasm/genetics/urine ; *Molecular Diagnostic Techniques/methods ; Biomarkers, Tumor/urine/genetics ; Sensitivity and Specificity ; },
abstract = {Background: Prostate cancer (PCa) is the most diagnosed cancer among men globally and a leading cause of cancer-related mortality. However, current conventional prostate diagnostic methods fail to meet the growing demands of clinical practice in terms of speed, simplicity, sensitivity, and specificity. To address these limitations, we established a molecular detection system based on MIRA-CRISPR/Cas12a technology. Using reverse transcription-multienzyme isothermal rapid amplification (RT-MIRA) to amplify minute PCA3-specific fragments in samples, we employ CRISPR/Cas12a to detect the fluorescence signal released by these fragments, enabling the detection of trace PCA3 molecules in urine. Methods: PCA3 standard strain cultivation and total RNA extraction; establishment and optimization of the MIRA amplification system using primers designed for the PCA3 molecular marker; design of crRNA targeting optimal sites within the detection sequence, combined with CRISPR/Cas12a technology to establish the detection system; preliminary validation of the technology's sensitivity and specificity. Results: the MIRA-CRISPR/Cas12a technology was successfully established for visual detection of PCA3 molecules in prostate cancer. Primer and crRNA sequences within the reaction system were determined. The detection sensitivity for PCA3 molecules in urine reached 1 × 10[0] copies per µL with excellent specificity. Conclusion: the MIRA-CRISPR/Cas12a technology enables specific detection of PCA3 molecules in urine. This technique features high sensitivity, high specificity, visual results, and simple operation. It does not require specialized laboratory UV imaging equipment; results are visible to the naked eye under LED blue light. Following further optimization, it offers a feasible technical solution for rapid molecular screening of prostate cancer.},
}
@article {pmid42084031,
year = {2026},
author = {Ma, R and Xiao, Y and Yu, W and Huo, C and Meng, S and Zhao, Z and Guo, Z and Ren, X and Zhang, H and Li, B and Wang, Y and Liu, S and Huang, J},
title = {A novel high-sensitivity fluorescence detection technology for zearalenone based on the PER-triggered crRNA conformational change and CHA-coordinated energy supply.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {18},
pages = {3865-3873},
doi = {10.1039/d6ay00474a},
pmid = {42084031},
issn = {1759-9679},
mesh = {*Zearalenone/analysis ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Nucleic Acid Conformation ; Spectrometry, Fluorescence/methods ; Fluorescence ; },
abstract = {Zearalenone (ZEN), frequently encountered in corn, is a hazardous mycotoxin capable of impairing liver and kidney function, compromising immune responses, and potentially inducing carcinogenesis. Current detection methodologies are hampered by elevated costs, complex workflows, limited sensitivity, and poor specificity. There is a pressing need to develop simple, rapid, and ultrasensitive assays that combine high specificity with operational convenience, thereby facilitating precise biotoxin surveillance and control. This study developed a novel biosensing strategy for ultrasensitive detection of zearalenone (ZEN) by engineering a blocked Primer Exchange Reaction (PER) dumbbell-hairpin structure integrated with a Catalytic Hairpin Assembly (CHA)-based DNA machine. We constructed a highly specific and sensitive fluorescence biosensor for zearalenone (ZEN) by integrating a Primer Exchange Reaction (PER) with the trans-cleavage activity of CRISPR/Cas12a. This strategy significantly simplifies the operational procedure compared to conventional techniques. Furthermore, its modular design establishes a versatile and efficient platform adaptable for the detection of various trace analytes, offering a promising proof-of-concept for mycotoxin screening in agricultural products, although further extensive validation across diverse realistic matrices is warranted.},
}
@article {pmid42132069,
year = {2026},
author = {Liu, Z and Qi, J and Sun, L and Su, J and Li, W and Wu, L and Liang, Y and Wei, C and He, F and Han, Y and Sun, Y and Yan, H and Li, H and Xiao, R},
title = {Oxygen-Vacancy-Engineered WOX Nanowire-Based Surface-Enhanced Raman Scattering Biosensor with Lyophilized CRISPR/Cas13a Platform for CHIKV Detection.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00934},
pmid = {42132069},
issn = {2379-3694},
abstract = {The outbreak of Chikungunya virus (CHIKV) has caused widespread acute morbidity with severe polyarthralgia or chronic arthritis, placing a great challenge to public health and socioeconomic development. Establishing a rapid and highly sensitive detection technology is crucial for achieving precise control and prevention. Here, we established a CRISPR/Cas13a-mediated SERS lateral flow immunoassay platform for rapid and highly sensitive detection of CHIKV. One-dimensional nanowires loading Au nanoparticles were used to prepare SERS tags, presenting excellent SERS-enhanced performances and superior applicability to directional flow on the test strip. For the best performance, the structure of WOX nanowires was regulated by adjusting the addition concentration of ascorbic acid during the synthesis process, resulting in a stronger LSPR effect derived from more Au NPs in situ grown on highly reducing WOX. Furthermore, the lyophilized CRISPR/Cas system greatly simplified the workflow. In the optimal conditions, the limit of detection reached 0.56 and 1.03 copies/μL for the CHIKV plasmid and inactivated viruses by this method, respectively. Furthermore, 34 clinical serum samples were accurately diagnosed by our proposed method, 100% consistent with qPCR. This platform with the advantages of simple operation and rapid response provides a reliable technical tool for the early precise identification and efficient monitoring of CHIKV.},
}
@article {pmid42133116,
year = {2026},
author = {Rajan, A and Raveendran, M and Shanmugam, V and Arul, L and Kumar, KK and Subramanian, A and Mannu, J and Eswaran, K},
title = {Engineering crop determinacy: CRISPR/Cas based advances in self-pruning gene function and application.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42133116},
issn = {1573-4978},
support = {BT/INF/22/SP45584/2022//Department of Biotechnology, Government of India/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics/growth &amp; development ; Gene Editing/methods ; Flowers/genetics/growth &amp; development ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; Genetic Engineering/methods ; Plant Proteins/genetics ; },
abstract = {The transition from indeterminate to determinate growth represents a key achievement in crop improvement, as it enhances agricultural productivity by synchronizing flowering, facilitating uniform harvest, and improving overall efficiency. In tomato and other crops, this shift is largely governed by mutations in the SELF-PRUNING (SP) gene, a key member of the CENTRORADIALIS (CEN), TERMINAL FLOWER 1 (TFL1), and SELF-PRUNING (SP) (CETS) gene family that regulates the vegetative to reproductive phase transition and influences overall shoot architecture. With increasing labour constraints, climate variability and rising global food security challenges, the ability to engineer optimized plant architectures has become increasingly important. CRISPR-based genome editing provides a precise and efficient strategy to modify SP/TFL1 homologs, enabling targeted transition from indeterminate to compact, determinate growth forms that exhibit synchronized flowering and enhanced mechanical harvestability. These genome editing approaches have been successfully applied across diverse crop species, including tomato, legumes, cotton, cereals and horticultural crops. This review consolidates current understanding of the molecular mechanisms governing determinacy, with emphasis on the central role of SP/TFL1 genes and their interactions with hormonal pathways such as auxin and cytokinin. By integrating these insights with recent advances in CRISPR-based editing platforms, this review provides a practical framework for researchers and breeders aiming to leverage CRISPR technology for next-generation crop improvement. Such strategies hold significant potential for enhancing productivity, resilience and sustainability within modern agricultural systems.},
}
@article {pmid42133523,
year = {2026},
author = {Nammi, B and Madugula, SS and Jayasinghe-Arachchige, VM and Pham, T and Liu, J and Wang, S},
title = {Robust CRISPR-Cas Protein Identification using Max-Margin Regularized Transformer Models.},
journal = {IEEE transactions on computational biology and bioinformatics},
volume = {PP},
number = {},
pages = {},
doi = {10.1109/TCBBIO.2026.3693528},
pmid = {42133523},
issn = {2998-4165},
abstract = {The discovery of CRISPR-Cas system has significantly advanced genome editing, offering vast applications in medical treatments and life sciences research. Despite their immense potential, the existing CRISPR-Cas systems still face challenges concerning size, delivery efficiency, and cleavage specificity. Addressing these challenges requires a deeper understanding of CRISPR-Cas proteins to advance the design and discovery of novel Cas proteins. Here, we study CRISPR-Cas proteins extensively using deep-learning techniques to build classification models that can differentiate between Cas and non-Cas proteins, as well as identify subfamilies Cas9 and Cas12. We developed two types of deep learning models: 1) a transformer encoder-based classification model, trained from scratch; and 2) a large protein language model fine-tuned on ProtBert, pre-trained on more than 200 million proteins. To boost learning efficiency for the model trained from scratch, we introduced a novel margin-based loss function to maximize inter-class separability and intra-class compactness in protein sequence embedding latent space of a transformer encoder. Our results show that the Fine-Tuned ProtBert-based (FTPB) classification model achieved accuracies of 99.06%, 94.42%, 96.80%, 97.57% for Cas9 vs. non-Cas, Cas12 vs.non-Cas, Cas9 vs. Cas12, and multi-class classification of Cas9 vs. Cas12 vs. non-Cas proteins, respectively. The Latent Space Regularized Max-Margin Transformer (LSRMT) model achieved classification accuracies of 99.81%, 99.81%, 99.06%, and 99.27% for the same tasks, respectively. These results demonstrate the effectiveness of the proposed Max-Margin-based latent space regularization in enhancing model robustness and generalization capabilities. Remarkably, the LSRMT model, even when trained on a significantly smaller dataset, outperformed the fine-tuned state-of-the-art large protein model. The high classification accuracies achieved by the LSRMT model demonstrate its proficiency in identifying discriminative features of CAS proteins, marking a significant step towards advancing our understanding of CAS protein structures in future research endeavors.},
}
@article {pmid42134322,
year = {2026},
author = {Lei, L and Kaufmann, MM and Lao, J and Thoulass, G and Ammann, S and Xiao, H and Rhiel, M and Dettmer-Monaco, V and Grünewald, J and Andrieux, G and Alzubi, J and Miller, BR and Weißert, K and Gräßel, L and Schell, C and Illert, AL and Joung, JK and Boerries, M and Cornu, TI and Ehl, S and Erlacher, M and Aichele, P and Cathomen, T},
title = {Genotoxicity profiling reveals distinct platform-and cell type-specific effects in therapeutic gene editing for genetic hyperinflammation.},
journal = {Cell stem cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.stem.2026.04.014},
pmid = {42134322},
issn = {1875-9777},
abstract = {Base editors enable precise correction of point mutations without requiring DNA double-strand breaks, yet platform- and cell type-specific genotoxicities remain incompletely characterized. Here, we applied cytosine base editing (CBE) to disrupt a cryptic splice-site mutation in the Unc13d locus of Jinx mice, a model of familial hemophagocytic lymphohistiocytosis type 3 (FHL3). Efficient editing (62%-89%) in fibroblasts, T cells, and hematopoietic stem cells (HSCs) restored Unc13d splicing, reconstituted cytotoxic T cell function, and protected mice from virus-triggered hyperinflammation after transplantation of edited HSCs. Comparative genotoxicity profiling revealed distinct platform- and cell type-specific patterns: hyperactive CBE induced broader off-target activity and more structural variants than CRISPR-Cas9. Although off-target sequence edits persisted, the stability of CBE-induced chromosomal translocations differed between cell types. These findings establish base editing as a therapeutic strategy for a genetically predisposed hyperinflammatory syndrome and underscore the importance of context-specific safety profiling to guide the clinical translation of genome editors.},
}
@article {pmid41237872,
year = {2026},
author = {Abdul-Rahman, T and Roy, P and Garg, N and Nazir, A and Mehta, KS and Doshi, NA and Hassnain, S and Reddi, R and Kanagala, SG and Ashinze, P and Lavie, CJ and Gupta, R},
title = {Gene editing for inherited cardiac conditions: A new frontier in cardiology.},
journal = {Trends in cardiovascular medicine},
volume = {36},
number = {4},
pages = {232-244},
doi = {10.1016/j.tcm.2025.11.002},
pmid = {41237872},
issn = {1873-2615},
mesh = {Humans ; *Gene Editing/ethics/methods ; *Genetic Therapy/adverse effects/methods ; Genetic Predisposition to Disease ; *Cardiology/methods/trends ; Animals ; *Heart Diseases/genetics/therapy/diagnosis ; Phenotype ; CRISPR-Cas Systems ; Treatment Outcome ; Epigenesis, Genetic ; Mutation ; Heredity ; },
abstract = {Inherited cardiac conditions (ICCs) such as hypertrophic cardiomyopathy and Marfan syndrome pose significant global health challenges, often rooted in complex genetic mutations. Recent advances in gene editing, particularly the CRISPR/Cas9 system, have opened new avenues for precise, personalized interventions. This review examines the current landscape of gene editing in cardiology, with emphasis on its scientific promise, integration with epigenetics, gene therapy, and artificial intelligence, and its potential to transform clinical outcomes. Key gene editing strategies are analyzed for their efficacy and translational potential. The review also explores ongoing clinical trials and emerging research, offering practical insights for future studies. Ethical implications are critically evaluated, with proposed frameworks to address concerns around safety, equity, and long-term consequences. By synthesizing these developments, this review underscores the urgent need for continued interdisciplinary research in the quest to mitigate inherited cardiac diseases.},
}
@article {pmid41722572,
year = {2026},
author = {Abadie, FMC and Suiter, CC and Smith, NT and Daza, RM and Rominger, MC and Parrish, P and McDiarmid, TA and Lalanne, JB and Martin, B and Calderon, D and Ellison, A and Berger, AH and Shendure, J and Starita, LM},
title = {A multiplex, prime editing framework for identifying drug resistance variants at scale.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101167},
doi = {10.1016/j.xgen.2026.101167},
pmid = {41722572},
issn = {2666-979X},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Point Mutation/genetics ; *Drug Resistance, Neoplasm/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Kinase Inhibitors/pharmacology ; Cell Line, Tumor ; },
abstract = {CRISPR-based genome editing has revolutionized functional genomics, enabling thousands of perturbations to be concurrently assayed in single experiments. However, for methods such as saturation genome editing (SGE), which aims to generate and assay libraries of point mutations, a challenge is that only one region (e.g., one exon) is studied per experiment. Here, we describe prime-SGE, a prime editing-based framework in which libraries of specific point mutations are installed into genes throughout the genome and then functionally assessed by sequencing of prime editing guide RNAs (pegRNAs) rather than the mutations themselves. We apply prime-SGE in two cell lines to assay thousands of point mutations in eight oncogenes for their ability to confer drug resistance to four tyrosine kinase inhibitors. Our prime-SGE strategy, combined with ongoing improvements in prime editing efficiency, opens the door to efficient positive selection screens of large numbers of point mutations at locations throughout the genome.},
}
@article {pmid41872214,
year = {2026},
author = {Khadake, RM and Shinde, K and Rode, AB},
title = {Engineering ligands for theophylline riboswitches expands its regulatory dynamic range in prokaryotic and eukaryotic systems.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41872214},
issn = {2041-1723},
support = {BT/ PR45158/DRUG/134/119/2022//Department of Biotechnology, Ministry of Science and Technology (DBT)/ ; CRG/ 2022/004570//DST | Science and Engineering Research Board (SERB)/ ; },
mesh = {*Theophylline/metabolism/pharmacology/chemistry ; *Riboswitch/genetics ; Ligands ; Aptamers, Nucleotide/metabolism/genetics/chemistry ; Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation ; Genetic Engineering/methods ; },
abstract = {The theophylline riboswitch has been a foundational tool in synthetic biology for three decades, yet its regulatory performance remains constrained by the modest affinity of its native ligand. Enhancing the dynamic range of riboswitches is critical for precise gene regulation in biotechnological applications. Here, we show that synthetic 4-quinazolinone derivatives, designed through a structure-based approach, are significantly better than theophylline in both binding and functional activation across multiple biological systems. We demonstrate that these derivatives bind the theophylline aptamer with up to 30-fold higher affinity, thereby expanding regulatory performance. In the bacterial system, these ligands enhance "ON" gene expression by up to 380-fold, compared to 75-fold with theophylline. This superior control extends to diverse organisms; in mycobacteria, the activation ratio reached 20-fold, and in eukaryotes, expression increased 11-fold. Furthermore, in riboswitch-mediated conditional CRISPR-Cas9 applications, these ligands achieve 70% genome editing efficiency at 10-fold lower concentrations than theophylline. These results demonstrate that ligand optimization is a crucial driver for enhancing riboswitch performance for advanced biomedical engineering.},
}
@article {pmid41887225,
year = {2026},
author = {Drepanos, LM and Srikanth, S and Kaplan, EG and Shah, ST and Velasco, BE and Merzouk, S and Doench, JG},
title = {Balancing off-target and on-target considerations for optimized CRISPR-Cas9 knockout library design.},
journal = {Cell genomics},
volume = {6},
number = {5},
pages = {101190},
doi = {10.1016/j.xgen.2026.101190},
pmid = {41887225},
issn = {2666-979X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Gene Library ; Mice ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Human/genetics ; },
abstract = {The continued development of high-dimensional CRISPR screen readouts, such as single-cell RNA sequencing and high-content imaging, necessitates compact libraries to enable functional interrogation at genome scale. Improved genome annotations cause library deprecation over time, further motivating an updated genome-wide design effort. Additionally, while on-target efficacy and off-target avoidance are often optimized in isolation, we lack a robust framework for simultaneously weighing and balancing these competing priorities. Here, we present a selection strategy that identifies guides with sufficient off-target activity to justify omission from the library, thus avoiding the unnecessary exclusion of active guides, allowing the inclusion of those with maximal on-target activity. We create, validate, and make available to the community the Jacquere library for knockout screens of the human genome, as well as its mouse counterpart, Julianna, to facilitate gene function discovery at scale.},
}
@article {pmid41985719,
year = {2026},
author = {Liang, G and Li, G and Liang, T and Zhen, Z and Teng, C and Xiong, J and Guangqiang, J and Zheng, M and Pan, Y and Zhong, S and Wu, C and Li, J and Huang, W and Wei, Z},
title = {Construction of an RAA-CRISPR detection platform for differentiation of Brucella abortus A19-∆VirB12 vaccine strain from wild-type strains.},
journal = {Journal of microbiological methods},
volume = {245},
number = {},
pages = {107512},
doi = {10.1016/j.mimet.2026.107512},
pmid = {41985719},
issn = {1872-8359},
mesh = {*Brucella abortus/genetics/isolation &amp; purification/classification ; Animals ; Cattle ; Recombinases/metabolism/genetics ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Brucellosis, Bovine/diagnosis/microbiology ; *Brucella Vaccine/genetics ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Brucella abortus is a primary etiological agent of bovine brucellosis, a zoonosis posing significant threats to livestock industries and public health. The recently developed A19-∆VirB12 vaccine strain, which carries a deletion of the VirB12 gene, complicates serological differentiation from wild-type infections. This study aimed to establish a rapid, accurate, and economical detection strategy targeting the VirB12 gene to distinguish the A19-∆VirB12 vaccine strain from wild-type B. abortus. We developed a recombinase-aided amplification (RAA) coupled with CRISPR/Cas12a-based detection method. Following optimization, primer pair C and crRNA1 were selected as optimal components, with 150 nM each of Cas12a protein and crRNA identified as the ideal concentrations in a 50 μL reaction. The assay demonstrated high analytical specificity, showing no cross-reactivity with six non-target bacterial pathogens. Sensitivity analysis established a limit of detection of 10[2] copies per reaction. When evaluated on 52 clinical samples, the RAA-CRISPR assay detected two positive samples, outperforming conventional PCR which detected only one. Crucially, the method yielded no positive signal when challenged with the A19-∆VirB12 gene-deficient vaccine strain, while successfully detecting wild-type strains A19 and S2, confirming its discriminatory capability. The entire workflow, comprising RAA amplification (30 min) and CRISPR-mediated cleavage (20 min), can be completed within one hour, with results visualized via fluorescence or lateral flow strips. This study successfully establishes a rapid, sensitive, and specific diagnostic method for distinguishing the A19-∆VirB12 vaccine strain from wild-type B. abortus, offering a practical tool for field surveillance and eradication programs.},
}
@article {pmid42003616,
year = {2026},
author = {Theriault, ME and Wong, AI and DeJesus, MA and Pisu, D and Nae Rin Lee, B and Kirukubar, G and Li, S and Wallach, JB and Schnappinger, D and Lê-Bury, G and Russell, DG and Rock, JM},
title = {Utilization of a CRISPRi-based ex vivo challenge model to reveal temporally dependent gene essentiality in intracellular Mycobacterium tuberculosis.},
journal = {mBio},
volume = {17},
number = {5},
pages = {e0061026},
doi = {10.1128/mbio.00610-26},
pmid = {42003616},
issn = {2150-7511},
support = {R01 AI155319/AI/NIAID NIH HHS/United States ; AI155319//National Institute of Allergy and Infectious Diseases/ ; AI162598//National Institute of Allergy and Infectious Diseases/ ; INV-055894//Bill and Melinda Gates Foundation/ ; //Mueller Health Foundation/ ; //Rita Allen Foundation/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/growth &amp; development ; Animals ; Mice ; Macrophages/microbiology ; *Genes, Essential ; *CRISPR-Cas Systems ; *Tuberculosis/microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Inbred C57BL ; Lung/microbiology ; Disease Models, Animal ; },
abstract = {UNLABELLED: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. Using a library comprising ~20,000 sgRNAs covering >96% of Mtb open reading frames, we identified genes required for growth within the changing immune microenvironment. Mutant depletion patterns varied across immune environments sampled at 2, 4, and 6 weeks post-infection, which revealed a weighted dependency on cell wall biosynthesis genes early and the reliance on cholesterol catabolism and iron acquisition across all time points. Functional validation of three genes-embB, fadE29, and mbtI-confirmed their temporal significance in vivo. This screen provides increased resolution of the differential metabolic vulnerabilities in Mtb in the evolving immune environments during infection, stressing the temporal nature of conditional essentiality in vivo.<br><br>IMPORTANCE: Mycobacterium tuberculosis (Mtb) remains a leading cause of infectious disease mortality worldwide, largely due to its ability to survive within host macrophages. Despite advances in understanding the environmental pressures Mtb encounters in vivo, the genetic requirements for adaptation and survival within the intracellular niche remain incompletely defined. Here, we employed a genome-wide CRISPR interference (CRISPRi) screen in an ex vivo model exploiting single-cell suspensions from Mtb-infected mouse lung homogenates to identify genes critical for intracellular survival at different time points in the infection continuum. This novel approach enabled us to identify how different bacterial metabolic pathways were of greater importance to the bacterium at different time points post-infection. The results provide insights into how the evolving immune response to infection shapes the metabolic and replicative status of the bacterium. This information has significance in the design of therapeutic strategies toward cure.},
}
@article {pmid42031246,
year = {2026},
author = {Han, T and Long, K and Hu, W and Liu, M and Guo, M and Huo, D and Hou, C},
title = {Dual-locked probes inhibit off-target circularization: Enhancing specificity in rolling circle transcription for nucleic acid detection.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152180},
doi = {10.1016/j.ijbiomac.2026.152180},
pmid = {42031246},
issn = {1879-0003},
mesh = {CRISPR-Cas Systems/genetics ; *Transcription, Genetic ; *Nucleic Acids/genetics/analysis ; Human papillomavirus 16/genetics ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Associated Proteins/genetics ; },
abstract = {Rolling Circle Replication (RCR) holds significant potential for detecting low-abundance nucleic acids. However, its practical application is often hindered by nonspecific ligation, which leads to high background signals and false-positive results. Herein, we designed a novel dual-locked circular template, termed no-bilateral-overhang dual-locked probe (ndRC). This probe employed steric hindrance from its dual hairpins lacking overhangs to create a "dual-locked" mechanism that specifically prevents off-target circularization. By integrating this template with exonuclease purification and a CRISPR/Cas12a module, we constructed the dual-locked RCT-CRISPR/Cas12a (DL-RCT-Cas12a) system, in which RCT generates long RNA transcripts containing crRNA repeats. These transcripts then activate Cas12a-mediated trans-cleavage, thereby amplifying the detection signal. The established assay achieved an ultralow detection limit of 40.31 aM, with a detection range from 100 aM to 1 μM. It demonstrated robust performance in complex matrices for the detection of partial HPV16 L1 gene sequences and exhibited a superior ability to discriminate high-concentration targets compared to qPCR. This work presents a versatile strategy to enhance the specificity of highly sensitive detection, improving performance in low-abundance nucleic acid analysis.},
}
@article {pmid42054542,
year = {2026},
author = {Gu, T and Xue, J and Zhang, Z and Cao, J and Song, J and Li, G and Ming, L and Zhu, Z and Wang, H},
title = {Mechanisms of Resistance to ALS Inhibitors and Bentazone in Fimbristylis littoralis and Rapid Identification of the ALS Trp-574-Leu Mutation Using LAMP-CRISPR/Cas12a.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {18},
pages = {14311-14321},
doi = {10.1021/acs.jafc.5c17149},
pmid = {42054542},
issn = {1520-5118},
mesh = {*Acetolactate Synthase/genetics/antagonists &amp; inhibitors/metabolism ; *Herbicides/pharmacology ; *Plant Proteins/genetics/metabolism/antagonists &amp; inhibitors/chemistry ; Herbicide Resistance ; Mutation ; CRISPR-Cas Systems ; *Enzyme Inhibitors/pharmacology ; },
abstract = {Fimbristylis littoralis Gaudich., a harmful sedge weed in Chinese rice paddy, impairs rice productivity and quality. In this study, we identified a resistant population (FL2) displaying multiple resistance to pyrazosulfuron-ethyl and bentazone, alongside cross-resistance to other acetolactate synthase (ALS)-inhibiting herbicides. The other population (FL6) showed exclusive resistance to bensulfuron-methyl. Sequencing demonstrated that FL2 carried a Trp-to-Leu mutation at codon 574 of ALS, whereas no mutations were detected in the psbA gene of bentazone-resistant FL2 or the ALS gene of bensulfuron-methyl-resistant FL6. Studies on nontarget-site resistance (NTSR) mechanisms indicated that FL2's resistance to pyrazosulfuron-ethyl was associated with neither PBO-inhibited P450s nor NBD-Cl-inhibited GSTs. In contrast, FL6's resistance to bensulfuron-methyl and FL2's resistance to bentazone were linked to P450 activity. A loop-mediated isothermal amplification (LAMP) coupled with CRISPR/FnCas12a assay was established for rapid detection of the Trp-574-Leu mutation, facilitating resistance management. These findings provide insights for managing resistant F. littoralis populations.},
}
@article {pmid42061542,
year = {2026},
author = {Yuan, Y and Ni, B and Tian, X and Cui, J and Zhang, Y and Hu, Y and Zheng, D and Zou, Y and Yu, X and Liu, C and Liu, S and Ren, W and Chang, X and Wang, Y and Ge, S and Wei, R and Chen, Y and Wu, X and Li, J and Wang, Z and Huang, B},
title = {Development of a point-of-care diagnostic method for FMDV SAT2 using RT-RAA-CRISPR technology.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152271},
doi = {10.1016/j.ijbiomac.2026.152271},
pmid = {42061542},
issn = {1879-0003},
mesh = {*Foot-and-Mouth Disease Virus/genetics/isolation &amp; purification ; Animals ; *Foot-and-Mouth Disease/diagnosis/virology ; *CRISPR-Cas Systems ; Cattle ; Swine ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; Sensitivity and Specificity ; Recombinases/metabolism ; },
abstract = {In recent years, the foot-and-mouth disease virus (FMDV) serotype SAT2 has expanded beyond its traditionally recognized endemic areas on the African continent, leading to continuous dissemination in Middle Eastern countries and presenting a risk of further spread to Asia. In this study, the highly conserved sequence of the FMDV SAT2 genome was initially selected as the target, and five pairs of reverse transcription-recombinase-aided amplification (RT-RAA) primers were designed. By comparing fluorescence signal intensities, the CRISPR RNA (crRNA) with the highest sensitivity and specificity was identified and subsequently integrated with the CRISPR/Cas13a gene-editing system to establish a novel nucleic acid detection method. This method possesses a robust capacity for differential diagnosis and shows no cross-reaction with other serotypes of FMDV. Moreover, this method demonstrated high specificity and no cross-reactivity with the nucleic acid sequences of various common pathogens in porcine and bovine populations. The test results are readily interpretable and can be directly visualized using a fluorescence reader or lateral flow test strips (LFSs). In simulated clinical samples, this method achieved a concordance rate of 100% with the detection results of fluorescence quantitative RT-PCR. This study successfully developed a highly sensitive and specific FMDV SAT2 nucleic acid detection method based on RT-RAA-CRISPR/Cas13a technology. This method is straightforward to perform, does not require costly experimental equipment, and is suitable for rapid onsite detection, offering a convenient and efficient diagnostic tool for the early diagnosis, prevention, and control of FMDV SAT2 epidemics.},
}
@article {pmid42061934,
year = {2026},
author = {Gunasekaran, H and Najwa, KV and Nidarshan, NC and Porkodi, M and Singh, LS and Rasal, KD and Brahmane, MP and Goswami, M and Sonwane, AA},
title = {Characterization of Trachinotus blochii mstnb gene and construction of cognate gRNA vector.},
journal = {International journal of biological macromolecules},
volume = {364},
number = {},
pages = {152225},
doi = {10.1016/j.ijbiomac.2026.152225},
pmid = {42061934},
issn = {1879-0003},
mesh = {Animals ; *Myostatin/genetics/chemistry ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Fishes/genetics ; Amino Acid Sequence ; Base Sequence ; *Fish Proteins/genetics ; },
abstract = {Skeletal muscle development is negatively regulated by the myostatin b (mstnb) gene in finfishes. CRISPR-Cas9-mediated knockout of mstnb has been used to develop fish strains with increased muscle mass. Trachinotus blochii (Silver Pompano), a high-value, moderately sized (~500 g), and cultivable marine finfish, is a promising candidate for mstnb knockout using CRISPR/Cas9. However, limited studies exist on the characterization of T. blochii mstnb and no CRISPR-based knockout studies have been reported in this species. This study aimed to partially characterize the mstnb gene of T. blochii and develop a CRISPR/Cas9-based guide RNA (gRNA) expression vector for its knockout. The T. blochii mstnb sequence available in NCBI was used as a reference to design exon- and intron-specific primers for PCR amplification and sequencing. The sequence revealed three exons and two introns. A predicted 1131 bp open reading frame encodes a 376 amino acid protein containing conserved domains typical of the TGF-β family which includes an N-terminal signal peptide, a propeptide region, a conserved RARR cleavage motif, and a C-terminal GF domain containing nine conserved cysteine residues. Secondary and 3D structure predictions confirmed the protein's functional integrity. Sequence analysis revealed novel putative polymorphisms, including SNPs and a (CA)n microsatellite. Phylogenetic analysis clustered T. blochii with related Trachinotus and other Carangiformes species. A gRNA targeting exon 1 was designed using CRISPOR and successfully cloned into expression vectors. This novel sequence information can aid population-level studies and genetic marker discovery. The constructed gRNA vectors can facilitate CRISPR/Cas9-mediated mstnb knockout in T. blochii to study gene function and develop a fleshy strain.},
}
@article {pmid42067160,
year = {2026},
author = {Dai, S and Niu, L and Lv, Y and Zhang, H and Xu, L and Liao, Y and Hu, X and Xie, X and Yan, J and Yan, Y},
title = {Semi-artificial photobiocatalysis via genetically modified sulfur metabolism to in situ assembly of a solar-biohybrid for antibiotic degradation.},
journal = {Bioresource technology},
volume = {454},
number = {},
pages = {134750},
doi = {10.1016/j.biortech.2026.134750},
pmid = {42067160},
issn = {1873-2976},
mesh = {*Sulfur/metabolism ; *Anti-Bacterial Agents/metabolism ; Sulfides/metabolism ; Biodegradation, Environmental ; Nanoparticles/chemistry ; Gene Editing ; CRISPR-Cas Systems/genetics ; Solar Energy ; Tetracycline/metabolism ; },
abstract = {The solar-driven semi-artificial biohybrid system incorporates semiconductor materials with microbial metabolism, affording an innovative strategy for antibiotic degradation via photocatalysis. In this study, the sulfur metabolic pathway of biological cells was rationally engineered using CRISPR-Cas9 and Cre-loxP site-specific gene editing systems, successfully achieving intracellular accumulation of sulfide up to 552.84 ppm. Based on this capability, In(Ⅲ) was adsorbed in situ onto the cell surface, leading to the self-assembly of photosensitive In2S3 nanoparticles (NPs). The resulting inorganic-biological hybrid system of In2S3-cell exhibited a broad-spectrum light-harvesting capability with an ideal optical bandgap of 1.96 eV. Photoelectrochemical analysis confirmed the charge transfer process and the semiconductor biointerface based regeneration mechanism of redox cofactors in the cytosol. Photogenerated electrons directly form ROS for tetracycline oxidation, and are transferred to cells for enhance the regeneration of intracellular reducing cofactors. This light-driven photocatalytic biohybrid system enabled efficient tetracycline degradation of over 98% within 4 h and demonstrated excellent stability over consecutive cycles. Transcriptomic analysis identified key genes involved in solar energy capture, electron transport, and metabolic regulation, elucidating their functional roles in biomanufacturing processes and photocatalytic degradation. This study presents a bottom-up paradigm for the biotic-abiotic system from electronic and molecular perspectives to develop efficient and sustainable technologies for antibiotic remediation and solar energy conversion.},
}
@article {pmid42121843,
year = {2026},
author = {Siddika, A and Husseiny, FE and Rousseau, J and Tremblay, JP},
title = {Successful In Vitro Modification of the Dmd Gene Using Prime Editing.},
journal = {Cells},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/cells15090740},
pmid = {42121843},
issn = {2073-4409},
support = {53320215//Defeat Duchenne Foundation/ ; CIHR; Application No. 492510/CAPMC/CIHR/Canada ; },
mesh = {*Gene Editing/methods ; *Dystrophin/genetics ; Animals ; Mice ; *Muscular Dystrophy, Duchenne/genetics ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Myoblasts/metabolism ; Base Sequence ; Mutation/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Duchenne muscular dystrophy (DMD) is a fatal X-linked neuromuscular disorder caused by mutations in the dystrophin gene. Prime editing is a versatile genome editing technology capable of introducing precise nucleotide changes without generating double-strand DNA breaks, making it a promising approach for correcting pathogenic point mutations. In this study, we applied prime editing to modify mdx-4cv and mdx-5cv mutation-equivalent sites in mouse C2C12 myoblasts in vitro. Initial editing efficiencies were unexpectedly low and were associated with the presence of a 5'-TTCT-3' motif within engineered prime editing guide RNAs (epegRNAs). epegRNA designs containing this motif exhibited reduced prime editing efficiency, whereas silent substitution eliminating the motif significantly improved editing outcomes, indicating that specific sequence features within epegRNAs can influence editing performance. Rational redesign of epegRNAs to remove this motif substantially enhanced editing efficiency, achieving up to 20% modification at the 4cv target site using an NGG PAM and 21% editing at the 5cv locus using an NGAG PAM. These findings highlight an important sequence-dependent constraint in epegRNA design and provide practical guidance for optimizing prime editing strategies targeting Dmd mutations in vitro.},
}
@article {pmid42121869,
year = {2026},
author = {Siwak, JF and Connelly, JP and Pruett-Miller, SM},
title = {Essential HDRescue: A Co-Targeting Strategy to Enhance Precision Genome Editing by Co-Editing Essential Genes.},
journal = {Cells},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/cells15090768},
pmid = {42121869},
issn = {2073-4409},
support = {N/A//American Lebanese Syrian Associated Charities/ ; NA//St. Jude Graduate School of Biomedical Sciences/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Recombinational DNA Repair/genetics ; *Genes, Essential/genetics ; DNA End-Joining Repair/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded ; Induced Pluripotent Stem Cells/metabolism ; Animals ; },
abstract = {Genome editing is widely used and conceptually simple, yet in practice, it is hindered by laborious workflows and high costs. These challenges stem from the difficulty of identifying and isolating cells that contain the desired user-defined modifications, a problem compounded by the wide variability in editing efficiencies across cell types. While homology-directed repair (HDR) provides a mechanism for precise genome modification following nuclease-induced double-strand breaks (DSBs), it is frequently outcompeted by the dominant mutagenic non-homologous end-joining (NHEJ) pathway in mammalian cells. Therefore, we developed a novel enrichment method, Essential HDRescue, to increase the frequency of HDR events at a target site by co-targeting an essential genomic locus. Using both intrinsic positive and negative selection at a common essential gene, we enabled enrichment of precise editing events at a second, unlinked target site. We demonstrated that co-targeting essential genes in cancer cell lines and iPSCs increased HDR rates without the need for an exogenous reporter or selective drug. Analysis of resulting clones revealed that Essential HDRescue produced up to a 6-fold increase in single-allele edits and an ~4-fold increase in homozygous edits relative to single-targeted controls. By harnessing the intrinsic cellular dependencies that arise from DSB repair at essential loci, Essential HDRescue offers a widely applicable method to improve precise genome editing outcomes in mammalian cells, leaving only a minimal, protein-silent scar at the essential gene.},
}
@article {pmid42122867,
year = {2026},
author = {Wen, Y and Li, Y and Bao, S and Cao, G and Li, M and Wang, J and Ding, B and Xie, X and Qiu, L},
title = {The Auxin Response Factor TaARF18-A Negatively Regulates Salt Tolerance in Common Wheat (Triticum aestivum L.).},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {9},
pages = {},
doi = {10.3390/plants15091375},
pmid = {42122867},
issn = {2223-7747},
support = {32301817//National Natural Science Foundation of China/ ; 22JCQNJC01470//Natural Science Foundation of Tianjin/ ; 26CXNE010//Gansu Provincial Science and Technology Program Project/ ; KLIBMC2507//Open Fund from the Tianjin Key Laboratory of Intelligent Breeding of Major Crops/ ; },
abstract = {Soil salinization is one of the major abiotic stresses that influences agricultural production and the environment. Auxin response factors (ARFs) are key components of the auxin signal transduction pathway, while their role in wheat salt stress responses remains unclear. In this study, we identified TaARF18 as a negative regulator of salt tolerance in wheat. The coding sequences of TaARF18-A, TaARF18-B, and TaARF18-D were 2106, 2088, and 2088 bp, respectively. TaARF18 is a hydrophilic protein featuring typical Auxin-resp and B3 DNA-binding domains and exhibits relatively high evolutionary conservation among Poaceae species. The expression of TaARF18 was upregulated under salt stress. TaARF18 predominantly accumulated in the nucleus. Silencing of TaARF18 via the BSMV-VIGS approach enhanced salt tolerance in wheat seedlings. In addition, haplotype analysis based on resequencing data from 355 wheat accessions identified 25, 31, and 16 haplotypes for TaARF18-A, TaARF18-B, and TaARF18-D, respectively. Fourteen wheat accessions carrying different haplotypes were evaluated under salt stress, and HapIII of TaARF18-A exhibited the highest level of salt tolerance, which can act as a strong selection locus in global wheat breeding. Our findings provide insight into the function of ARFs in salt stress responses and offer a potential target for CRISPR/Cas-mediated salt-tolerant wheat breeding programs.},
}
@article {pmid42123509,
year = {2026},
author = {Li, Y and Yao, Y and Xu, Z and Xiong, Y and Zhang, C and Yu, L and Gao, H and Fei, T},
title = {Genome-Wide CRISPR Screening Identifies Genetic Modulators of Amyloid Precursor Protein Processing.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27093926},
pmid = {42123509},
issn = {1422-0067},
mesh = {Humans ; *Amyloid beta-Protein Precursor/metabolism/genetics ; *CRISPR-Cas Systems ; *Alzheimer Disease/genetics/metabolism ; Amyloid beta-Peptides/metabolism/genetics ; HEK293 Cells ; },
abstract = {The proteolytic processing of the amyloid precursor protein (APP) is a core pathological event in Alzheimer's disease (AD) pathogenesis, yet the global genetic regulatory networks modulating this process have not been fully characterized. To systematically identify novel regulators of APP cleavage, we performed a genome-wide CRISPR/Cas9 knockout screen utilizing an optimized UAS-GAL4-based cellular reporter, and identified genetic modulators governing amyloidogenic and non-amyloidogenic processing. The screen uncovered distinct functional gene clusters regulating the APP, prominently involving cellular metabolism, protein modification, and vesicular trafficking. Specifically, LDHB, PIAS2, CCDC53, and TRIM61 emerged as novel functional modulators. Biochemical validation confirmed that ablating these genes significantly alters the metabolic balance between sAPPα and amyloid-β (Aβ) production. Finally, integration with human AD transcriptomic datasets demonstrated that these identified modulators undergo significant dysregulation in clinics. Together, these findings establish a reporter-based functional screening framework for APP processing and identify candidate regulatory nodes linked to metabolism, protein modification, and vesicular trafficking. These candidates provide a resource for future mechanistic investigation and validation in more disease-relevant AD models.},
}
@article {pmid42123512,
year = {2026},
author = {Yuan, Y and Yuan, J and Deng, D and Wu, J and Zhou, X and Jiang, A and Wang, J and Wang, X and Li, M and Long, K and Zhao, L},
title = {CRISPR/Cas9-Mediated Knockout of CGNL1 Confers Resistance to Aflatoxin B1 in Porcine Intestinal Epithelial Cells via Suppressing ROS Generation.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27093928},
pmid = {42123512},
issn = {1422-0067},
support = {XZ202501ZY0147//Tibet Autonomous Region Science and Technology Agency/ ; sccxtd-2025-08-13//the Program for Pig Industry Technology System Innovation Team of Sichuan Province/ ; 32472884//National Natural Science Foundation of China/ ; 32573165//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Aflatoxin B1/toxicity ; *CRISPR-Cas Systems ; Swine ; *Epithelial Cells/metabolism/drug effects ; *Reactive Oxygen Species/metabolism ; Oxidative Stress/drug effects ; *Intestinal Mucosa/metabolism/drug effects ; Gene Knockout Techniques ; Cell Line ; Cell Survival/drug effects ; Signal Transduction ; },
abstract = {Aflatoxin B1 (AFB1) is a prevalent and highly toxic mycotoxin in the food and feed chain and can directly injure the intestinal epithelium. Yet, its upstream determinants linking epithelial stress to cytotoxicity remain insufficiently defined. Here, we used porcine intestinal epithelial IPEC-J2 cells to characterize AFB1-induced cytotoxic and transcriptomic responses and to determine the role of the tight-junction scaffold, Cingulin-like 1 (CGNL1), a candidate gene identified through genome-scale CRISPR knockout library screening. The results showed that AFB1 exposure reduced cell viability in a dose-dependent manner and induced oxidative stress. RNA-seq profiling analysis revealed broad transcriptional remodeling, with activation of inflammatory pathways (including NF-κB and JAK-STAT signaling). Based on our constructed CGNL1-knockout IPEC-J2 cell line (CGNL1-KO IPEC-J2) using CRISPR/Cas9, it was found that CGNL1 deficiency markedly alleviated AFB1-induced cytotoxicity and oxidative stress. Comparative transcriptomics analysis showed that CGNL1 knockout attenuated AFB1-triggered aberrant expression of some CGNL1-dependent AFB1-responsive genes related to immune response under AFB1 challenge. Together, these findings identify CGNL1 as a potential modulator of epithelial susceptibility to AFB1 and support its involvement in the regulation of toxin-induced oxidative response.},
}
@article {pmid42123673,
year = {2026},
author = {Jing, L and Roy, D and Kalischuk, M},
title = {Advances in CRISPR Plant Applications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {9},
pages = {},
doi = {10.3390/ijms27094095},
pmid = {42123673},
issn = {1422-0067},
support = {Alliance #44842//Natural Sciences and Engineering Research Council of Canada/ ; 45402//Solanum International Inc./ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Crops, Agricultural/genetics ; Plants, Genetically Modified/genetics ; *Plants/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The ability to precisely edit genetic characteristics with a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immunity complex is a revolutionary advance in science. Originally discovered in bacteria as part of a natural defense mechanism against viruses, CRISPR/Cas provides a precise, efficient, and relatively simple method for editing genes in microbes, plants, animals, and humans. The process relies on the Cas protein, an enzyme that cleaves and unwinds DNA at targeted locations. This process is guided by RNA sequences complementary to the DNA or RNA sequence of interest, allowing for changes to the genome through innate non-homologous end joining (NHEJ) and homology-directed repair (HDR). The potential applications of CRISPR/Cas are immense and, in agriculture, is facilitating crop development with resistance to abiotic, biotic, and agronomic characteristics that improve yield, quality, and food security. Gene editing also facilitates the relatively rapid modification of regulatory and complex pathways that enable studies to advance our understanding of gene function. This review provides an update of the fast-evolving CRISPR/Cas modification of important crops to address emerging global population, as well as environmental and climate challenges.},
}
@article {pmid42124669,
year = {2026},
author = {Sanderson, EM and Peralta, J and Nouwens, S and Oriolt, L and Hayes, VM and Kaiser, BK and Meeske, AJ},
title = {Phage-encoded CasPRs transcriptionally silence diverse CRISPR-Cas systems.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.02.23.707548},
pmid = {42124669},
issn = {2692-8205},
abstract = {Anti-CRISPRs (Acrs) are diverse proteins or RNAs that protect invading phages and plasmids from host CRISPR-Cas immunity. Most Acrs neutralize their cognate Cas proteins via direct physical interaction. Here we describe CasPRs, a particularly widespread family of DNA-binding Acrs that recognize specific sequence motifs within cas gene coding regions, thereby blocking RNA polymerase and silencing transcription. We demonstrate that eight diverse CasPRs bind to the cas8b gene to repress the type I-B CRISPR-Cas system in its native host, Listeria seeligeri . Meanwhile, a CasPR from Streptococcus dysgalactiae silences type II-A CRISPR-Cas immunity by binding to the cas9 coding sequence. We found that one CasPR is required to inhibit CRISPR immunity during lysogeny by its host prophage. Taken together, our results indicate that members of the CasPR family have diverged to silence completely unrelated CRISPR types, and suggest transcriptional repression is a common mode of phage-mediated immune antagonism.},
}
@article {pmid42126429,
year = {2026},
author = {Huang, RS and Phung, SK and Sumstad, D and Weis, AJ and Kile, QM and Bendzick, L and Khaw, MJ and Vue, YY and McKenna, DH and Kennedy, PR and Miller, JS and Felices, M},
title = {Reprogramming endogenous NK circuits by highly efficient nonviral genome editing.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {7},
pages = {},
doi = {10.1084/jem.20260192},
pmid = {42126429},
issn = {1540-9538},
support = {R35 CA283892/NH/NIH HHS/United States ; P01 CA111412/NH/NIH HHS/United States ; P01 CA065493/NH/NIH HHS/United States ; P30 CA 77598/NH/NIH HHS/United States ; },
mesh = {*Killer Cells, Natural/immunology/metabolism ; *Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; Mice ; Gene Regulatory Networks ; Interleukin-12/genetics/metabolism ; Receptors, Chimeric Antigen/genetics/immunology ; },
abstract = {Natural killer (NK) cells are promising platforms for off-the-shelf immunotherapy, yet nonviral precision engineering remains limited by poor HDR efficiency, DNA toxicity, and manufacturing challenges. The aim of this study was to establish a high-yield, nonviral knock-in platform. Through extensive in-depth rational screens, we achieved ∼90% HDR insertion of therapeutic payloads while maintaining 100% postediting recovery. By hijacking endogenous transcriptional programs, we installed genetic circuits into defined genomic loci to tune transgene expression. To enable context-dependent therapeutic responses, we integrated a synthetic positive feedback circuit at the CISH locus, which enhanced NK cell persistence and drove strong expression of anti-CD22/19 dual CAR. A hypoxia-responsive IL-12 circuit gated by the PFKFB4 promoter restored cytotoxicity under environmental stress. Finally, we showed this platform is compatible with GMP manufacturing and supports clinical-scale expansion. These findings provide a scalable framework for programmable, nonviral editing of NK cell effector functions for therapeutic and research applications.},
}
@article {pmid42127676,
year = {2026},
author = {Cheng, M and Chen, X and Cheng, H and Gao, X and Ao, H and Bao, X and Song, X and Tai, Y and Jin, D and Zhang, L},
title = {An ultrasensitive CRISPR/Cas12a based electrochemical biosensor for detection of toxigenic Clostridioides difficile.},
journal = {Biosensors &amp; bioelectronics},
volume = {308},
number = {},
pages = {118779},
doi = {10.1016/j.bios.2026.118779},
pmid = {42127676},
issn = {1873-4235},
abstract = {Clostridioides difficile (C. difficile) infection (CDI) represents a formidable global healthcare challenge, necessitating the development of rapid, accurate, and cost-effective diagnostic platforms to mitigate nosocomial transmission and improve patient outcomes. Compared with the conventional methods, CRISPR/Cas systems featured by specific target reorganization by a single chain RNA, coupled with electrochemical technology enables highly sensitive detection of various biomarkers. However, their application to CDI has remained unexplored due to the lack of tailored crRNAs. Herein, we present the integration of CRISPR/Cas12a with electrochemical transduction for the direct detection of C. difficile. A novel crRNA was engineered to specifically recognize the toxin B gene (tcdB), activating the trans-cleavage activity of Cas12a upon target binding. This cascade triggers the cleavage of immobilized ssDNA reporters on the electrode surface, generating measurable amperometric signal changes. The developed biosensor demonstrates exceptional performance, achieving a detection limit of pM level for tcdB DNA within 40 min, while exhibiting high specificity against non-target pathogens and robust stability over 7 days. This work establishes a rapid and reliable CRISPR-electrochemical diagnostic platform, offering significant potential for point-of-care CDI management.},
}
@article {pmid42128325,
year = {2026},
author = {Cai, Y and Yang, J and Hou, M and Su, W and Liang, F and Zhu, M and Wu, T},
title = {Multi-omics precision diagnosis of brucellosis: Advances in biomarker discovery and clinical application.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {},
number = {},
pages = {121074},
doi = {10.1016/j.cca.2026.121074},
pmid = {42128325},
issn = {1873-3492},
abstract = {Brucellosis, a neglected zoonosis caused by intracellular Brucella bacteria, remains a formidable global public health challenge, especially in developing regions. The notorious ability of Brucella to evade host immunity and establish chronic focal infections limits the utility of traditional diagnostic methods like bacterial culture and serology for early detection, therapeutic monitoring, and disease staging. This review comprehensively synthesizes the ongoing paradigm shift from pathogen-centric detection toward multi-omics precision diagnosis. We critically evaluate advances in nucleic acid amplification technologies (NAATs), charting the progression from quantitative PCR to absolute quantification via droplet digital PCR (ddPCR) and examining the transformative potential of CRISPR-Cas biosensing for ultrasensitive, instrument-free detection. The discussion also encompasses the renaissance of serology through immunoproteomics, which has identified novel serodominant antigens and multi-epitope fusion proteins to address the persistent specificity problems arising from cross-reacting bacteria. Furthermore, we analyze the emerging landscape of host-response biomarkers, integrating transcriptomic, metabolomic, and single-cell RNA sequencing data to delineate distinct immune signatures of acute and chronic infection. Finally, we consider how artificial intelligence (AI) can integrate these multi-dimensional datasets to build predictive diagnostic models. This consolidated multi-omics framework charts a course for precision medicine in brucellosis, aiming to bridge the gap between biomarker discovery and point-of-care clinical application. SUMMARY: Multi-omics technologies (genomics, proteomics, metabolomics, transcriptomics) are advancing brucellosis diagnosis via sensitive detection and accurate biomarkers, and improving treatment through novel strategies like nano-delivery, vaccines, and AI integration.},
}
@article {pmid42128554,
year = {2026},
author = {Xu, W and Cheng, Y and Sun, K and Wu, Y and Xu, Y and Ye, J and Li, P and Wu, H},
title = {Inception-level signal amplification: Cascaded DNAzyme-Cas9 nickase achieves sub-nanomolar kanamycin tracking.},
journal = {Analytica chimica acta},
volume = {1408},
number = {},
pages = {345562},
doi = {10.1016/j.aca.2026.345562},
pmid = {42128554},
issn = {1873-4324},
mesh = {*Kanamycin/analysis/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *Biosensing Techniques/methods ; Milk/chemistry ; *Nucleic Acid Amplification Techniques ; *Deoxyribonuclease I/metabolism/chemistry ; Limit of Detection ; Animals ; *Anti-Bacterial Agents/analysis ; CRISPR-Cas Systems ; Water Pollutants, Chemical/analysis ; },
abstract = {BACKGROUND: Kanamycin's persistent contamination in agricultural products poses significant human health risks due to its nephrotoxicity and bioaccumulation via the food chain. Existing methods for on-site kanamycin monitoring lack sufficient sensitivity and portability, limiting their utility in field settings. The urgent need for rapid detection technologies remains unmet. This work addresses the critical gap in developing a field-deployable platform for ultrasensitive kanamycin residue screening.<br><br>RESULTS: We engineered a fluorescence biosensor integrating DNAzyme-assisted Cas9 nickase-based amplification reaction (Cas9nAR). Kanamycin binding induces aptamer conformational changes, triggering Cas9nAR-driven cascade amplification that continuously generates DNAzymes. These cleave reporter probes to enable quantitative detection. The system achieved a broad linear range (1&#x202f;nM - 5&#x202f;&#x3bc;M) with an ultralow detection limit (0.3&#x202f;nM), surpassing conventional methods. It exhibited high specificity against interfering antibiotics and delivered consistent recoveries (97% to 103%) in spiked water and milk samples. Following pretreatment, analysis can be completed within 120&#x202f;min, validating its operational simplicity and robustness for complex matrices.<br><br>SIGNIFICANCE AND NOVELTY: This work reports the first CRISPR-Cas9 nickase/DNAzyme cascade amplification platform for small-molecule detection, establishing a new paradigm that integrates programmable nucleic acid amplification with catalytic signal turnover. By using Cas9nAR-generated ssDNA as an in situ template for autonomous DNAzyme assembly, the biosensor achieves ultrasensitive, and homogeneous detection of kanamycin-addressing a critical gap in field-deployable antibiotic residue monitoring. The modular design offers a generalizable strategy for translating non-nucleic acid recognition events into amplified fluorescent outputs, with broad implications for point-of-need diagnostics in food safety and environmental analysis.},
}
@article {pmid42128971,
year = {2026},
author = {Xie, R and Zhu, C and Liang, X and Lu, K and Wu, J},
title = {Knockout of bsal/cel.2 results in growth retardation, reduced lipid digestion and altered energy metabolism in medaka larvae (oryzias latipes).},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {42128971},
issn = {1438-7948},
support = {2024YFD2401502//the National Key Research and Development Program of China/ ; },
mesh = {Animals ; *Oryzias/genetics/growth &amp; development/metabolism ; *Energy Metabolism/genetics ; *Lipid Metabolism/genetics ; *Fish Proteins/genetics/metabolism ; Larva/growth &amp; development/genetics/metabolism ; *Lipase/genetics/metabolism ; CRISPR-Cas Systems ; Lipolysis/genetics ; },
abstract = {To evaluate the significance of bile salt-activated lipase (BSAL) in lipid digestion and metabolism in fish, this study used CRISPR/Cas9 gene editing to generate medaka (Oryzias latipes) bsal mutant lines. Given that the bsal gene comprises three copies (bsal, cel.2, and bsal-like) in the medaka genome, bsal-like variants may lead to functional loss in lipid hydrolysis owing to amino acid residue alterations in the bile salt binding site. Consequently, two types of medaka mutants, bsal[-/-] and bsal[-/-]/cel.2[-/-], were generated for experimental exploration in this study. Compared to wild-type (WT) medaka, the bsal[-]/[-]/cel.2[-]/[-] group showed significant reductions in body length, expression of growth-related genes (gh and igf), total lipase and protease activities, and body composition (cholesterol, triglyceride, and protein levels). The bsal[-]/[-]/cel.2[-]/[-] group also exhibited upregulated expression of lipid synthesis genes (fas, acc1, scd) and downregulated expression of lipolysis-related genes (cpt1, acox1). Notably, key glycolysis genes (pk, gk) and gluconeogenesis-related genes (pck2) were significantly upregulated in the bsal[-]/[-]/cel.2[-]/[-] group. However, the bsal[-/-] group exhibited no significant differences from the WT group in all assays, except for notable reductions in protease activity and expression levels of the cpt1 and gk genes, as well as a significant increase in pck2 gene expression compared to the WT group. Remarkably, the expression level of the cel.2 gene was significantly elevated in the bsal[-/-] group compared to the WT group. In summary, this study demonstrates the pivotal role of the bsal gene in lipid digestion and metabolism in medaka. Furthermore, the presence of multiple copies of the bsal gene aids in fulfilling the demands of lipid digestion in medaka. This conclusion can provide insights into the research on lipid digestion and metabolism in other fish species.},
}
@article {pmid42128985,
year = {2026},
author = {Hou, M and Li, Y and Wu, X and Long, D and Sun, D and Chen, P and Huang, H},
title = {Recent Advances in the Development of CRISPR-Based Live-Cell Molecular Imaging and Sensing.},
journal = {Molecular imaging and biology},
volume = {},
number = {},
pages = {},
pmid = {42128985},
issn = {1860-2002},
support = {2026JJ90110//Natural Science Foundation of Hunan Province Joint Fund for Universities/ ; S202512034126//Hunan Province Undergraduate Innovation Training Program/ ; },
abstract = {Visualizing genome organization and transcriptional dynamics with spatial and temporal precision in living cells is essential for elucidating gene regulation and chromatin-associated disease mechanisms, yet conventional methods confront a fundamental tension between endogenous-sequence targeting and live-cell compatibility. Operator-repressor systems require prior insertion of repetitive arrays at engineered loci, whereas fluorescence in situ hybridization mandates cell fixation and thereby precludes temporal analysis. CRISPR-Cas technologies, originally developed for genome editing, have been re-engineered into a versatile molecular-imaging toolkit capable of interrogating native sequences in living cells. Here, we systematically review CRISPR-based live-cell imaging and sensing platforms, critically evaluating their design principles, mechanistic foundations, and performance limitations. We examine dCas9-based DNA labeling, dCas12a systems for non-repetitive loci, Cas13- and Csm-mediated RNA imaging, novel fluorescent reporters, engineered ribonucleoproteins, and delivery innovations including reagent-based Oligo-LiveFISH. To organize this diverse literature, we distinguish three operationally distinct modalities-live-cell imaging, intracellular sensing, and diagnostic biosensing-and assess each platform through three unifying design trade-offs: sensitivity versus cellular perturbation, multiplexing capacity versus system complexity, and detection threshold versus biological fidelity. Building on this framework, we evaluate the integration of CRISPR imaging with super-resolution microscopy, artificial-intelligence-driven computational analysis, and multimodal spatial omics. Collectively, this synthesis clarifies current capabilities, delineates unresolved constraints, and charts a coherent path toward clinically relevant applications of CRISPR-based live-cell molecular imaging.},
}
@article {pmid42129030,
year = {2026},
author = {Watanabe, K and Ishikawa, M and Ishibashi, K},
title = {Development of Tobamovirus-Resistant Tomato Plants by CRISPR-Cas9-Mediated Knockout of Susceptibility Genes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3039},
number = {},
pages = {1-10},
pmid = {42129030},
issn = {1940-6029},
mesh = {*Solanum lycopersicum/genetics/virology ; *CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/virology/genetics ; *Tobamovirus/pathogenicity/physiology ; Gene Editing/methods ; *Gene Knockout Techniques/methods ; Host-Pathogen Interactions/genetics ; Plants, Genetically Modified/genetics/virology ; },
abstract = {The advent of genome editing technologies such as CRISPR-Cas9 has revolutionized the development of disease-resistant crops, offering precision and efficiency in targeting specific genetic loci responsible for susceptibility. In this protocol, we harness the CRISPR-Cas9 system to disrupt key susceptibility genes in tomato, aiming to fortify resistance against tobamoviruses, particularly the aggressive tomato brown rugose fruit virus (ToBRFV). By systematically knocking out four TOM1 homologs, genes essential for tobamoviral replication, tomato lines with robust and heritable resistance can be developed while minimizing adverse developmental effects. The approach not only underscores the significance of basic research on host-pathogen interactions in modern crop protection but also lays the groundwork for sustainable, gene-driven resistance strategies in commercial tomato breeding.},
}
@article {pmid42129032,
year = {2026},
author = {Yoshida, T and Ishibashi, K},
title = {Heritable Tissue-Culture-Free Gene Editing in Nicotiana benthamiana Using a Meristem-Invading Virus Vector.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3039},
number = {},
pages = {21-27},
pmid = {42129032},
issn = {1940-6029},
mesh = {*Nicotiana/genetics/virology ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Meristem/genetics/virology ; CRISPR-Cas Systems ; *Potyvirus/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Gene editing can be achieved using sequence-specific nucleases. This protocol describes a plant gene editing method that eliminates the need for tissue culture by employing a virus-based delivery system. Tobacco ringspot virus (TRSV) can access meristematic tissues in infected plants, enabling the introduction of site-directed mutations into germline cells. This allows for heritable gene modification in the model plant Nicotiana benthamiana without tissue culture.},
}
@article {pmid42129103,
year = {2026},
author = {Ash, S and Attianese, GMPG and Kosti, P and Semilietof, A and Stefanidis, E and Triboulet, M and Irving, M},
title = {Generation and Characterization of CAR-T Cells.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2998},
number = {},
pages = {439-504},
pmid = {42129103},
issn = {1940-6029},
mesh = {Humans ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Animals ; Mice ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; *Receptors, Antigen, T-Cell/genetics/immunology/metabolism ; Neoplasms/therapy/immunology ; Lymphocyte Activation ; Lentivirus/genetics ; Retroviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {CARs are synthetic receptors that link antigen binding to T-cell activation. Most CARs used in the clinic for treating cancer are second generation (2G) and comprise (i) a single chain variable fragment (scFv) that binds the target tumor antigen, (ii) a linker/hinge region, (iii) a transmembrane domain, (iv) a costimulatory endodomain, and (v) the endodomain of CD3 zeta. Our lab is focused on the development of function and safety-enhanced, next-generation CAR-T cells for the treatment of solid tumors. For example, we have designed switchable CARs that can be remotely turned on or off upon small molecule administration in order to mitigate toxicity or exhaustion. To address barriers to CAR-T cells in the solid tumor microenvironment, we are further developing rational coengineering strategies to support their function. While we have implemented non-viral tools like CRISPR/Cas9 knockout and knockin, adenine base editing, and transposon-based systems for T cell engineering in the lab, currently we mostly use lentivirus and retrovirus for our pre-clinical studies. Here, we present our most frequently used protocols, improved over many years in the lab, for the production and titration of lentivirus and retrovirus, as well as the purification, activation, transduction and expansion of both mouse and human CAR-T cells. In addition, we share protocols for our most commonly run in vitro assays for characterizing CAR-T cells, including for evaluating transduction efficiency, proliferation, phenotype, cytokine/chemokine production, cytotoxicity, and resistance to stress. Most of these protocols can also be applied to the production and characterization of T cell receptor (TCR)-engineered T cells. Finally, we explain how to set up and perform CAR-T cell transfer studies in subcutaneous tumor-bearing mice, both for syngeneic and xenograft models, and perform ex vivo analysis on tumor tissues post-treatment.},
}
@article {pmid42129496,
year = {2026},
author = {Duchêne, C and Craig, RJ and Martinho, C and Luthringer, R and Agullo, F and Hipp, K and Escudeiro, P and Alva, V and Haas, FB and Coelho, SM},
title = {Latent endogenous giant viruses drive active infection and inheritance in a multicellular algal host.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {42129496},
issn = {2058-5276},
support = {101109906//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 Marie Sk&#x142;odowska-Curie Actions (H2020 Excellent Science - Marie Sk&#x142;odowska-Curie Actions)/ ; },
abstract = {Endogenous viral elements inserted in host genomes are often regarded as inert relics of past infections. Whether they can retain infective potential and contribute to active viral cycles has remained largely unresolved. Here we demonstrate that giant viral elements in the multicellular alga Ectocarpus can reactivate and drive productive viral infections. Using long-read sequencing and transcriptomics, we identify full-length, transcriptionally active phaeoviruses integrated within the host genome, and we use classical genetics and CRISPR-Cas to demonstrate that these elements are stably inherited through the germline, while their reactivation is precisely regulated by developmental and environmental cues including temperature. We resolve the genomic integration sites and propose a mechanism for phaeovirus integration and replication. Our work provides direct evidence and uncovers the mechanisms by which giant viral elements can reactivate, replicate and transmit both horizontally and vertically in a multicellular eukaryote, establishing a new model of latency, inheritance and evolutionary impact of giant dsDNA viruses.},
}
@article {pmid35851300,
year = {2022},
author = {Droogers, WJ and Willems, J and MacGillavry, HD and de Jong, APH},
title = {Duplex Labeling and Manipulation of Neuronal Proteins Using Sequential CRISPR/Cas9 Gene Editing.},
journal = {eNeuro},
volume = {9},
number = {4},
pages = {},
pmid = {35851300},
issn = {2373-2822},
mesh = {Animals ; *CRISPR-Cas Systems ; *Neurons/metabolism ; *Gene Editing/methods ; Rats ; *Gene Knock-In Techniques/methods ; *Nerve Tissue Proteins/metabolism/genetics ; Synapses/metabolism ; Cells, Cultured ; },
abstract = {CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable in rat neurons to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization correlates with synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons.},
}
@article {pmid41062702,
year = {2026},
author = {Casagrande Raffi, G and Kuiken, HJ and Lieftink, C and Bernards, R and Beijersbergen, RL and Wang, L},
title = {Inducible CRISPR-Cas9 screening platform to interrogate non-proliferative cellular states.},
journal = {Nature protocols},
volume = {21},
number = {5},
pages = {1896-1926},
pmid = {41062702},
issn = {1750-2799},
support = {19-051-ASP//Bergmark Foundation/ ; KWF-12539//KWF Kankerbestrijding (Dutch Cancer Society)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Proliferation ; Flow Cytometry/methods ; Cellular Senescence/genetics ; },
abstract = {CRISPR screens have revolutionized the study of diverse biological processes, particularly in cancer research. Both pooled and arrayed CRISPR screens have facilitated the identification of essential genes for cell survival and proliferation, drivers of drug resistance and synthetic lethal interactions. However, applying loss-of-function CRISPR screening to non-proliferative states remains challenging, largely because of slower editing and the poor sensitivity of identifying guide RNAs that 'drop out' in a population of non-dividing cells. Here, we present a detailed protocol to accomplish this, using an inducible Cas9 system that offers precise temporal control over Cas9 expression. This inducible system allows gene editing to occur only after the non-proliferative state is fully established. We describe the complete procedure for generating an inducible Cas9-expressing model and for measuring editing efficiency by using flow cytometry. In addition, we discuss how to optimize key parameters for performing successful CRISPR screens in various non-proliferative states. We describe a detailed workflow for performing a screen in senescent cells to identify senolytic targets. This protocol is accessible to researchers with experience in molecular biology techniques and can be completed in 8-12 weeks, from the generation of an inducible Cas9 cell line clone to the analysis of a CRISPR screen for hit identification. These techniques can be applied by researchers across different fields, including stem cell differentiation, immune cell development, aging and cancer research.},
}
@article {pmid41258015,
year = {2026},
author = {Longo, GMC and Sayols, S and Roukos, V},
title = {Multilevel characterization of genome editor nuclease activity with BreakTag.},
journal = {Nature protocols},
volume = {21},
number = {5},
pages = {2043-2082},
pmid = {41258015},
issn = {1750-2799},
support = {393547839-SFB 1361//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 402733153-SPP 2202//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 455784893//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; INST 247/845-1 FUGG//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *High-Throughput Nucleotide Sequencing/methods ; DNA Breaks, Double-Stranded ; Humans ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {BreakTag is a scalable next-generation sequencing-based method for the unbiased characterization of programmable nucleases and guide RNAs at multiple levels. BreakTag allows off-target nomination, nuclease activity assessment and the characterization of scission profile, that, in Cas9-based gene editing, is mechanistically linked with the indel repair outcome. The method relies on digestion of genomic DNA by Cas9 and guide RNAs in ribonucleoprotein format, followed by enrichment of blunt and staggered DNA double-strand breaks generated by CRISPR nucleases at on- and off-target sequences. Next-generation sequencing and data analysis with BreakInspectoR allows high-throughput characterization of Cas nuclease activity, specificity, protospacer adjacent motif frequency and scission profile. Here we first describe a detailed BreakTag protocol for the nomination of CRISPR off-targets and multilevel characterization of engineered Cas variants and second, we describe a step-by-step protocol for data analysis using BreakInspectoR. Third, we provide a web interface for XGScission, a machine learning model amenable to training with scission-aware BreakTag data to predict the relative frequency of blunt and staggered double-strand breaks at new sequences unseen by the model. XGScission allows a preselection of target sequences predicted to be cut in staggered configuration that are preferably repaired as single-nucleotide templated insertions. Furthermore, XGScisson can be used to assess sequence determinants of blunt and staggered cleavage by SpCas9 and engineered nuclease variants. As a companion strategy, we describe HiPlex for the generation of hundreds to thousands of single guide RNAs in pooled format for the production of robust BreakTag datasets. The BreakTag library preparation takes ~6 h, and the entire protocol can be completed in ~3 d, including sequencing, data analysis with BreakInspectoR and XGScission model training.},
}
@article {pmid41833894,
year = {2026},
author = {Ma, Y and Liao, Y},
title = {CRISPR-mediated cancer therapies: Approaches to direct tumor targeting.},
journal = {Critical reviews in oncology/hematology},
volume = {222},
number = {},
pages = {105277},
doi = {10.1016/j.critrevonc.2026.105277},
pmid = {41833894},
issn = {1879-0461},
mesh = {Humans ; *Neoplasms/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Tumor Microenvironment/genetics ; Animals ; },
abstract = {CRISPR-Cas9 technologies have opened new possibilities for precision cancer treatment, addressing limitations inherent in conventional therapies such as chemotherapy and radiation. This review examines CRISPR-based strategies for direct tumor targeting, including oncogene inactivation, tumor suppressor gene reactivation, and tumor microenvironment (TME) modification. Key advances include KRAS[G12D] inactivation via base editing, in which engineered deaminases introduce precise single-nucleotide changes without generating double-strand breaks; TP53 correction through homologous recombination, which uses a donor DNA template to repair mutant sequences at the targeted locus; and CDKN2A epigenetic remodeling using CRISPR-dCas9-TET1 demethylation, where catalytically inactive Cas9 guides the TET1 demethylase to hypermethylated promoters to restore gene expression. CRISPR screening has identified synthetic lethal interactions, such as PARP1 dependency in BRCA1[-/-] tumors. TME editing strategies, including modification of cancer-associated fibroblasts, demonstrate enhanced antitumor responses. Delivery challenges are being addressed through viral vectors, including adenovirus, AAV, and lentivirus. Non-viral approaches include lipid nanoparticles, gold nanoparticles, exosomes, and stimuli-responsive systems such as MMP-cleavable and hypoxia-responsive nanoparticles. Clinical trials with CRISPR-engineered T-cells (e.g., CTX130) have demonstrated remission rates in hematologic malignancies. However, significant challenges remain, including cytokine release syndrome, immunotoxicity, tumor heterogeneity, and limited delivery efficiency in solid tumors. Overcoming these barriers requires interdisciplinary innovation, ethical oversight, and technological refinement to support the safe and effective integration of CRISPR-based strategies into precision oncology.},
}
@article {pmid42046341,
year = {2026},
author = {Wang, Z and Chen, Y and Wang, Y and Li, W and Zhang, X and Zhang, S},
title = {One-Pot Ligation-Recombinase Polymerase Amplification-Clustered Regularly Interspaced Short Palindromic Repeats/Cas12a-Powered Trimode Lateral Flow Assay for Sensitive MicroRNA Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {18},
pages = {13365-13376},
doi = {10.1021/acs.analchem.5c07208},
pmid = {42046341},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Gold/chemistry ; Humans ; *Recombinases/metabolism ; Limit of Detection ; Platinum/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of microRNAs (miRNAs) holds significant importance for the early diagnosis of cancer. Since current sensitive nucleic acid detection methods like recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats (RPA-CRISPR)/Cas12a are not suitable for detecting short-stranded miRNAs, we introduced a T4 ligase-based ligation process to the RPA-CRISPR/Cas12a system and developed a novel miRNA detection method termed ligation-RPA-CRISPR/Cas12a (LRCC). This assay utilizes a glycerol-enhanced one-pot reaction strategy combined with a lateral flow assay (LFA) to streamline the operation, minimize aerosol contamination, and improve point-of-care testing performance. Kinetic studies have shown that the catalytic efficiency of the glycerol-enhanced one-pot reaction is 3.11 and 2.09 times higher than that of the direct one-pot and stepwise methods, respectively. By synthesizing "three-in-one" Au-Pt nanostars (Au@Pt NSs) as probes and stabilizing them via "click" chemistry modification, this work enabled a trimode detection approach (colorimetric, photothermal, and surface-enhanced Raman spectroscopy (SERS)) with improved accuracy. In the experiment, tetrahedron DNAs were immobilized on the test line of the strip to enhance the capture efficiency of probes, thereby improving the detection sensitivity. The entire detection process was completed in 70 min with detection limits of 23.6 fM for colorimetric (C-LFA), 2.19 fM for photothermal (P-LFA), and 72.29 aM for SERS (S-LFA). The results demonstrate the strong practical applicability of the LRCC strategy, which plays a crucial role in miRNA-based early disease diagnosis.},
}
@article {pmid42065492,
year = {2026},
author = {Han, R and Xiao, N and Wu, Z and Zhao, Y and Wang, X and Tang, X},
title = {Sensitive and Robust One-Pot RPA-CRISPR/Cas12a Assay with Elimination of cis-Cleavage.},
journal = {Analytical chemistry},
volume = {98},
number = {18},
pages = {13307-13318},
doi = {10.1021/acs.analchem.5c06674},
pmid = {42065492},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; Limit of Detection ; DNA, Single-Stranded/genetics/chemistry ; *Replication Protein A/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The rapid detection technology utilizing CRISPR/Cas12a is characterized by high sensitivity, portability, and efficiency, making it a prominent focus in the field of point-of-care testing (POCT). However, it still has limitations in one-pot detection systems. This study developed a one-pot assay based on CRISPR/Cas12a and RPA (11bp+9ss one-pot assay), which nearly eliminates the cis-cleavage activity of Cas12a while retaining its trans-cleavage activity. Specifically, cis-cleavage was abolished by shortening the complementary length between the crRNA and the target to maintain the double-stranded conformation at the cis-cleavage site, whereas trans-cleavage activity was preserved by using an ssDNA complementary to the remaining region. The trans-cleavage activity was applicable to targets within a 200 bp range and under suboptimal PAM conditions. Moreover, rational design of the ssDNA enables effective discrimination of single-base mutations. The 11bp+9ss one-pot assay achieved a limit of detection (LOD) of 1 × 10[0] copies/μL for various targets, demonstrating robust performance even in suboptimal RPA systems. Furthermore, the assay was successfully applied to the detection of Salmonella and Avian Leukosis Virus subgroup J (ALV-J) samples. Overall, the 11bp+9ss one-pot assay exhibits superior sensitivity and robustness, showing great potential for POCT of bacteria and viruses.},
}
@article {pmid42115922,
year = {2026},
author = {Bhowmik, PK and Williams, JT and Polley, B and Chen, N and Kavuri, NR and Zang, W and Barakate, A and Yang, H and Narra, MK and Beattie, AD and Starker, C and Voytas, DF and Baysal, C},
title = {BSMV-mediated genome editing exhibits host-specific heritability: germline transmission in barley and somatic edits in Nicotiana benthamiana.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-08866-3},
pmid = {42115922},
issn = {1471-2229},
abstract = {BACKGROUND: Plant RNA virus-mediated guide RNA (gRNA) delivery represents a transformative advance in genome editing technologies. Unlike conventional transformation methods that rely on labor-intensive tissue culture and regeneration for each individual gRNA delivery, viral vectors can rapidly and systemically transmit gRNAs into pre-established Cas-expressing plants, providing an accelerated route for functional genomics and trait discovery directly in planta. However, key design parameters, including subgenomic promoter choice, transcript architecture, and their effects on viral fitness and editing outcomes, remain to be elucidated for most viral platforms.<br><br>RESULTS: We developed five Barley stripe mosaic virus (BSMV) vectors, each with distinct subgenomic promoter elements to drive single gRNA expression. These were initially evaluated in Cas9-expressing transgenic Nicotiana benthamiana plants targeting the Phytoene desaturase (PDS) gene to compare their editing efficiencies. Single gRNAs expressed under the duplicated &#x3b3;b subgenomic promoter or when fused directly to the &#x3b3;b genome achieved the highest mutation frequencies (up to 90% at 60 days post-inoculation), whereas &#x3b2;1- and &#x3b2;2-driven sgRNAs produced delayed and reduced editing. Thus, promoter selection critically determines gRNA accumulation and the efficacy of BSMV-mediated genome editing. The top-performing design was then applied to Cas9-expressing barley (Hordeum vulgare) targeting HvCMF7 (conferring green-white variegation) and HvGW2.1 (impacts grain width and weight). BSMV spread systemically throughout barley, inducing somatic and heritable mutations at frequencies up to 100%, with virus-free edited progeny. In contrast, despite robust somatic editing in N. benthamiana, no heritable mutations were detected indicating species-dependent limitations in germline transmission.<br><br>CONCLUSION: Our systematic comparison of subgenomic promoter architectures establishes clear design principles for optimizing viral vector-mediated delivery. Promoter choice and transcript structure critically shape editing efficiency and viral stability. The host-specific boundary for germline editing, defined by efficient heritable editing in barley but not N. benthamiana, highlights where BSMV offers advantages and where alternative vectors or hybrid strategies are required, guiding rational platform selection for diverse crop species and applications. Collectively, these findings establish BSMV as a promising next-generation vector for rapid, tissue culture-free, and transformation-independent genome editing in cereals and other recalcitrant monocots.},
}
@article {pmid42116436,
year = {2026},
author = {Sun, L and Yan, J and Xie, J and Wang, XY},
title = {From interactions to applications: the role of microbial communities in shaping the physicochemical, safety, and quality attributes of aquatic products.},
journal = {Food research international (Ottawa, Ont.)},
volume = {236},
number = {},
pages = {119126},
doi = {10.1016/j.foodres.2026.119126},
pmid = {42116436},
issn = {1873-7145},
mesh = {*Food Safety ; *Food Microbiology ; *Microbiota ; *Seafood/microbiology ; *Microbial Interactions ; Bacteria/growth &amp; development ; Animals ; Food Handling ; Food Quality ; Quality Control ; },
abstract = {Aquatic products are regarded as important dietary resources due to their abundant high-quality proteins, while their physicochemical properties, safety, and quality attributes during storage and processing are strongly influenced by microbial communities. Microbial interactions in aquatic products can exacerbate spoilage and pathogenic contamination, thereby intensifying food safety issues. However, previous studies primarily focused on identifying and characterizing individual microbial species in aquatic products, whereas current research increasingly emphasizes the critical role of multi-species interactions and applications in quality and safety evaluation of aquatic products. This review focuses on the interactions, characteristics, and growth models of spoilage and pathogenic microorganisms in aquatic products. The regulation mechanisms underlying microbial interactions and their neutral, negative, and positive effects on aquatic products' quality were summarized. Additionally, the paper analyzes targeted strategies for regulating microbial interactions in aquatic product preservation, providing theoretical and practical support for quality control. The microbiota primarily consists of spoilage and pathogenic bacteria, whose growth dynamics and interactions can be quantitatively modeled using models such as the Baranyi and P-model to predict their behavior. These microorganisms directly regulate the spoilage process and health risks via physical structures (e.g., extracellular polymeric substances, intercellular nanotubes) and chemical signaling networks (e.g., metabolic cross-feeding, quorum sensing). Conventional strategies (e.g., modified atmosphere packaging, ultra-high pressure processing) and precision approaches (e.g., digital twin technology and CRISPR/Cas system) have been applied to regulate the microbial interactions, thereby significantly enhancing the quality control in aquatic products.},
}
@article {pmid42117889,
year = {2026},
author = {Wu, W and Jin, F and Xu, H and Liao, R and Fang, Z},
title = {Negative Regulators of Rice Agronomic Traits: Functional Insights and Applications in Genome Editing-Based Breeding.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70684},
pmid = {42117889},
issn = {1467-7652},
support = {32572249//National Natural Science Foundation of China/ ; qiankehepingtairencai-YQK (2023) 002//Guizhou Provincial Excellent Young Talents Project of Science and Technology/ ; qiankehejichu-ZD(2026)068//Science and Technology Program of Guizhou Province/ ; qiankehechengguo (2024) general 116//Science and Technology Program of Guizhou Province/ ; Qiankehepingtairencai-BQW (2024) 001//Science and Technology Program of Guizhou Province/ ; Qiankehepingtai ZSYS (2025) 037//Guizhou Key Laboratory of High Quality, High Efficiency, and Yield Enhancement in Grain and Oil Crops/ ; Qianjiaoji (2023) 007//Key Laboratory of Functional Agriculture of Guizhou Provincial Higher Education Institution/ ; GZSDCYJSTX-202602//Guizhou Provincial Modern Agricultural Industry Technology System Construction Special Program/ ; },
abstract = {Rice is the staple crop for more than half of the global population, and improving grain yield, grain quality, and stress resistance remain central goals of modern rice breeding. Among current precision breeding strategies, genome editing has created new opportunities for crop improvement, but its success depends heavily on the selection of effective target genes. In this context, negative regulators of agronomic traits are particularly valuable because their disruption or attenuation can relieve constraints on desirable phenotypes and generate beneficial variation. In this review, we summarize recent progress in the identification and functional characterization of negative regulatory genes associated with rice grain yield, grain quality and stress resistance. We further integrate the current knowledge of their molecular functions, regulatory mechanisms, and genetic networks and discuss their potential applications in genome editing-assisted breeding. This review provides a target-oriented framework for understanding negative regulation in rice and facilitating the development of improved varieties with increased productivity, quality and stress resistance.},
}
@article {pmid42119611,
year = {2026},
author = {Ghonse, K and Dandekar, S and Koratkar, S},
title = {Multidrug-resistant Acinetobacter baumannii: Molecular insights, clinical challenges, and therapeutic approaches.},
journal = {Journal of microbiological methods},
volume = {},
number = {},
pages = {107544},
doi = {10.1016/j.mimet.2026.107544},
pmid = {42119611},
issn = {1872-8359},
abstract = {Acinetobacter baumannii has emerged as an agent of potentially life-threatening nosocomial infections, particularly among immunocompromised patients. Its ability to rapidly acquire resistance genes has made traditional antibiotic therapies progressively ineffective. The spread of bacterial contamination in hospital facilities increased due to lack of awareness among healthcare workers and improper management/handling of infectious samples. The persistence of pathogen in the hospital environment is increased with its ability to form biofilms, quorum sensing, and virulence factors. The infections caused by these agents are increasing in incidence and severity; necessitating efficient and timely management. This review highlights the epidemiological trends and molecular mechanisms involved in the pathogenesis and resistance of A. baumannii. The key resistance mechanisms that the organism possess include the activity of efflux pumps, beta-lactamase enzymes, and mobile genetic elements. This review discusses emerging treatment strategies - such as phage therapy - antimicrobial peptides, CRISPR-Cas-based technologies, and nanotechnology-enabled drug delivery- highlighting their respective benefits and limitations, with special emphasis on innovations like phage-antibiotic synergy and precision genome editing approaches. Despite promising advances, challenges remain, including the emergence of resistance pathogen, limited clinical scalability, and concern regarding the safety and toxicity of novel treatment options. Addressing these issues require focus on molecular insights of resistance mechanisms, the development of effective alternative therapies, and implementation of preventive strategies such as vaccines. Furthermore, execution of global antimicrobial stewardship program and robust surveillance systems are critical for effectively control and manage the threat posed by A. baumannii.},
}
@article {pmid41668390,
year = {2026},
author = {Gao, J and Hao, Y and Du, T and Li, K and Qiao, S and Xu, M and Guo, J and Hu, G and Ren, F and Fan, X and Dong, Y},
title = {Establishment of an RT-LAMP-CRISPR/Cas12a detection system for grapevine fabavirus and improvement of grapevine leaf crude extract with alkaline resin for on-site naked-eye detection.},
journal = {Pest management science},
volume = {82},
number = {6},
pages = {5245-5254},
doi = {10.1002/ps.70633},
pmid = {41668390},
issn = {1526-4998},
support = {2024BBF01002//Key Research and Development Project of Ningxia Hui Autonomous Region/ ; CARS-29-bc-1//China Agriculture Research System of MOF and MARA/ ; CAAS-ASTIP-RIP//Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences/ ; },
mesh = {*Vitis/virology ; Plant Leaves/virology/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Plant Diseases/virology ; *Molecular Diagnostic Techniques/methods ; *Plant Viruses/isolation &amp; purification/genetics ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: Grapevine fabavirus (GFabV) induces severe chlorosis and malformation in grapevine leaves, and its infection can substantially compromise both fruit yield and quality. Despite its growing impact on viticulture, no rapid and accurate on-site molecular diagnostic tool is currently available for the detection of GFabV. Thus, developing a reliable and field-deployable detection method is urgently needed.<br><br>RESULTS: In this study, we designed six sets of primers targeting conserved regions of GFabV for loop-mediated isothermal amplification (LAMP), and identified one optimal primer set capable of detecting all three GFabV variants. To enhance assay specificity and enable instrument-free visual readout, LAMP and CRISPR/Cas12a were integrated to establish a real-time (RT)-LAMP-CRISPR/Cas12a single-tube assay. The established method demonstrated exceptional specificity and remarkable sensitivity, and it is &#x2264;10&#x2009;000 times more sensitive than conventional RT-PCR. We streamlined the RNA preparation process by replacing traditional RNA extraction with optimized crude extract protocols. For grapevine leaves, we developed a novel 'Alkaline Resin method', utilizing a newly engineered alkaline resin material that effectively neutralizes the acidic lysate while simultaneously adsorbing chlorophyll, polyphenols and polysaccharides. For grapevine branches, we introduced an extremely simple 'branch-washing method', wherein crude nucleic acids are obtained simply by rinsing the phloem tissue with water. By coupling crude extraction strategies with the RT-LAMP-CRISPR/Cas12a single-tube assay, we achieved on-site detection of GFabV within 50&#x2009;min.<br><br>CONCLUSION: We developed an RT-LAMP-CRISPR/Cas12a single-tube method, along with corresponding crude extraction procedures for grapevine branches and leaves, enabling simple, rapid, accurate and on-site detection of GFabV. &#xa9; 2026 Society of Chemical Industry.},
}
@article {pmid41674090,
year = {2026},
author = {Han, K and Xie, B and Luo, C and Luo, Y and Yang, M and Lei, L and Jia, MA and Xu, T},
title = {Identification of a novel BBWV2 isolate and a sensitive and rapid RT-RPA-CRISPR/Cas12a-LFD detection method development.},
journal = {Pest management science},
volume = {82},
number = {6},
pages = {5255-5265},
doi = {10.1002/ps.70635},
pmid = {41674090},
issn = {1526-4998},
support = {//Guizhou Provincial Innovation Talents Team/ ; //the Guizhou Province Key Core Technology Research Project for Mountain Agriculture/ ; //the Plateau Characteristic Vegetable Industry Technology System Program of Guizhou Province/ ; //Special Program of China National Tobacco Corporation/ ; //National Natural Science Foundation of China/ ; //Guizhou Provincial Science and Technology Foundation/ ; //Provincial College Students' innovation and entrepreneurship training program/ ; },
mesh = {*Plant Diseases/virology ; *CRISPR-Cas Systems ; *Capsicum/virology ; *Nucleic Acid Amplification Techniques/methods ; China ; Sensitivity and Specificity ; },
abstract = {BACKGROUND: Broad bean wilt virus 2 (BBWV2) is a major viral pathogen causing significant economic losses in vegetable production. Existing detection methods often lack the speed, sensitivity, or simplicity required for effective on-site field diagnosis. This study aimed to identify a novel BBWV2 isolate and develop a rapid, equipment-free visual detection method suitable for field applications.<br><br>RESULTS: A novel BBWV2 isolate (BBWV2-GZCa) was identified and characterized from pepper in Guizhou Province, China. We developed a one-step visual detection assay by integrating reverse transcription-recombinase polymerase amplification (RT-RPA) with a CRISPR/Cas12a system, enabling readout via fluorescence or lateral flow dipstick (LFD). The assay demonstrated a detection limit of 7.5 copies/&#x3bc;L, which is 10[5] times more sensitive than conventional RT-PCR. It showed no cross-reactivity with other common pepper viruses and achieved 100% accuracy when validated using 20 field-collected samples.<br><br>CONCLUSION: The entire detection process can be completed within one hour without specialized equipment, requiring only visual interpretation. This RT-RPA-CRISPR/Cas12a-LFD method provides a rapid, highly sensitive, specific, and user-friendly platform for on-site detection of BBWV2, offering a practical tool for early diagnosis and disease management in agricultural settings. &#xa9; 2026 Society of Chemical Industry.},
}
@article {pmid41854410,
year = {2026},
author = {Dias, RG and Freitas, FPM and de Almeida, ELM and Fietto, LG and Zsögön, A and Silveira, WBD},
title = {CRISPR/Cas9 Genome Engineering in Non-Conventional Oleaginous Yeasts: Applications, Challenges, and Prospects.},
journal = {Yeast (Chichester, England)},
volume = {43},
number = {3},
pages = {77-88},
doi = {10.1002/yea.70015},
pmid = {41854410},
issn = {1097-0061},
mesh = {*CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Gene Editing/methods ; *Yeasts/genetics/metabolism ; *Genome, Fungal ; Yarrowia/genetics ; Lipid Metabolism/genetics ; Fatty Acids/biosynthesis ; *Genetic Engineering/methods ; },
abstract = {Given the biotechnological potential of yeast-derived oils for oleochemical production, genes encoding lipid metabolism enzymes are key targets for metabolic engineering. Genetic engineering tools such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, Transcription Activator-Like Effector Nucleases (TALENs), Zinc-Finger Nucleases (ZFNs), RNA interference (RNAi), and integrative plasmids can be used to modulate fatty acid biosynthesis and optimize lipid production. Among them, the CRISPR/Cas9 system, recognized for its simplicity and efficiency, has been deployed as a tool to create oleaginous yeast strains with high lipid productivity and features suitable for application in biorefineries. Species such as Cutaneotrichosporon oleaginosus, Rhodotorula toruloides, Candida spp., and Yarrowia lipolytica have already been engineered using CRISPR/Cas9 to enhance the production of fatty acids and their derivatives. However, designing and constructing an efficient CRISPR/Cas9 platform for oleaginous yeasts faces several hurdles, including low transformation efficiency, difficulties in expressing Cas9 and sgRNAs efficiently and consistently, the lack of well-characterized promoters, limited availability of PAM sequences, and poorly understood DNA repair mechanisms. Here, we address the application of the CRISPR/Cas9 system in oleaginous yeasts, laying out the challenges to developing efficient platforms and highlighting key trends in the field. We compare and discuss alternative CRISPR-Cas9 expression strategies to provide an overview of the current landscape and support the development of new approaches.},
}
@article {pmid41930821,
year = {2026},
author = {Zheng, P and Zheng, S},
title = {Xenotransplantation: Promise, progress, and risks.},
journal = {Veterinary immunology and immunopathology},
volume = {296},
number = {},
pages = {111110},
doi = {10.1016/j.vetimm.2026.111110},
pmid = {41930821},
issn = {1873-2534},
mesh = {*Transplantation, Heterologous/veterinary/adverse effects/trends ; Animals ; Humans ; Gene Editing ; CRISPR-Cas Systems ; Graft Rejection/prevention &amp; control/immunology ; },
abstract = {Xenotransplantation has seen remarkable progress in recent years, largely driven by the advent of CRISPR-Cas9 gene editing technology. Several patients have received genetically modified kidney and heart transplants, achieving graft and patient survival ranging from days to months. This review offers a brief history of xenotransplantation and highlights advancements in gene editing that represent breakthroughs in minimizing rejection. It also presents recent cases of xenotransplantation and their clinical outcomes. Finally, the review addresses the ethical considerations and risks associated with this emerging field.},
}
@article {pmid42024428,
year = {2026},
author = {Jia, H and Zhao, P and Pei, J and Tian, P},
title = {Rewiring the morphology and metabolism of Escherichia coli with an engineered CRISPRi system.},
journal = {Journal of applied microbiology},
volume = {137},
number = {5},
pages = {},
doi = {10.1093/jambio/lxag104},
pmid = {42024428},
issn = {1365-2672},
support = {22278022//National Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/metabolism/cytology ; *Metabolic Engineering/methods ; DNA Replication/genetics ; Escherichia coli Proteins/genetics ; *CRISPR-Cas Systems ; Lactic Acid/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation, Bacterial ; },
abstract = {AIMS: Microbial morphology is an increasingly leveraged target in metabolic engineering. To causally investigate the coupling between DNA replication, morphology, and metabolism, we constructed a CRISPR interference (CRISPRi) system in Escherichia coli BL21(DE3) to repress essential replication genes (dnaN, dnaG, polA, and ssb).<br><br>METHODS AND RESULTS: Transcriptional repression disrupted cell division and yielded distinct, quantifiable shape changes: knockdown of dnaG (encoding primase) and ssb (encoding single-stranded DNA-binding protein), in particular, induced significant cellular elongation and widening. This morphological reprogramming concurrently reallocated metabolic flux, as evidenced by a substantial increase in lactic acid titer from 0.89&#xa0;g L&#x207b;&#xb9; to 6.01&#xa0;g L&#x207b;&#xb9;. RNA-seq and subsequent analyses (differential expression, GO/KEGG enrichment, GSEA, and PPI) reveal that replication inhibition drives extensive metabolic reprogramming and cell envelope remodeling, with notable perturbations in peptidoglycan biosynthesis.<br><br>CONCLUSION: Our results suggest that targeted replication stress can coordinately reshape bacterial morphology and alter metabolic output, providing a controllable strategy for engineering microbial cell factories.},
}
@article {pmid42024433,
year = {2026},
author = {Sato, R and Maruyama, K and Ara, S and Shibata, M and Shida, Y and Ogasawara, W and Yamazaki, H and Takaku, H},
title = {A CRISPR/Cas9-based genome-editing platform enabling efficient and precise gene replacement in Lipomyces starkeyi.},
journal = {FEMS yeast research},
volume = {26},
number = {},
pages = {},
doi = {10.1093/femsyr/foag014},
pmid = {42024433},
issn = {1567-1364},
support = {JPJS00420240017//JSPS/ ; 25K08907//JSPS/ ; 25K18164//JSPS/ ; JPMJPF2211//Japan Science and Technology Agency/ ; //New Energy and Industrial Technology Development Organization/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Lipomyces/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Lipomyces starkeyi is a promising oleaginous yeast with industrial potential. However, its genome engineering remains constrained by low gene-targeting efficiency and the requirement for long homologous regions. Herein, we established a CRISPR/Cas9 genome-editing platform for L. starkeyi by expressing codon-optimized Streptococcus pyogenes Cas9 fused to an SV40 nuclear localization signal. Furthermore, in vitro-transcribed single-guide RNAs (sgRNAs) were directly delivered into the host, eliminating the need for endogenous RNA polymerase III-dependent sgRNA expression. CRISPR/Cas9 activity was validated using a codon-optimized Aequorea coerulescens GFP reporter. Cas9-induced frameshift mutations caused GFP disruption, leading to fluorescence loss. Gene replacement at the LsURA3 locus was evaluated using donor constructs with homologous regions ranging from 50-3000 bp. In a Cas9-expressing wild-type background, precise gene replacement was dependent on homology arm length, increasing from 36% with 50-bp arms to 80% with 3000-bp arms. Notably, in a Cas9-expressing Δlslig4 strain with suppressed non-homologous end joining (NHEJ), precise gene replacement was achieved with 100% accuracy using 50-bp homology arms under CRISPR/Cas9-dependent conditions. Together, these results demonstrate that a Pol III-independent CRISPR/Cas9 system combined with NHEJ suppression enables precise genome editing in L. starkeyi, providing a foundation for functional genomics and metabolic engineering.},
}
@article {pmid42108472,
year = {2026},
author = {Maxim, DS and Sostena, J and Johnson, NS and Wu, DW and Charu, V and Carter, JN and Anand, S and Church, GM and Bhalla, V},
title = {Designing genome editing experiments with EditABLE.},
journal = {Genome biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s13059-026-04095-x},
pmid = {42108472},
issn = {1474-760X},
support = {1R41DK138689/DK/NIDDK NIH HHS/United States ; },
abstract = {While many computational tools exist for designing CRISPR-Cas experiments, there is a need for a centralized resource that combines individual tools to predict the most efficient genome editing strategy for a given application. To fill this gap, we develop EditABLE (EditABLE-app.stanford.edu), an online resource that provides optimal CRISPR editors and guide RNAs based on user provided sequence data with functionalities for base editing, prime editing, and integrase-mediated editing. We demonstrate the utility of EditABLE by applying it to one of the most common monogenic disorders, autosomal dominant polycystic kidney disease (ADPKD), identifying specific editing tools across the ADPKD mutation landscape.},
}
@article {pmid42109826,
year = {2026},
author = {Sun, A and Jin, SL and Liu, JG},
title = {A practical guide for characterization of novel CRISPR-Cas systems with Pro-CRISPR factors.},
journal = {Biophysics reports},
volume = {12},
number = {2},
pages = {85-99},
pmid = {42109826},
issn = {2364-3420},
abstract = {The emergence of advanced genome editing technologies has revolutionized research in life sciences, offering an unprecedented way to uncover unknown biological functions and innovative therapeutic strategies. Among all genome editing tools, CRISPR-Cas-based technologies play a pivotal role in this revolution, particularly Class 2 effectors such as Cas9 and Cas12, owing to their high efficacy and ease of programmability. With the advancements in genome sequencing and metagenomics, an increasing number of novel CRISPR-Cas systems have been discovered, including those found in extreme environments and viruses. Furthermore, recent studies have revealed an unexpected role of non-Cas accessory genes, such as the Tn7-like transposon and Pro-CRISPR factors (Pcr), in conferring additional functionalities to the CRISPR system, providing new insights into the understanding of CRISPR-mediated bacterial immunity and advancing the development of genome editing technologies. Therefore, it is essential to develop comprehensive methods for characterizing the Cas proteins and Pro-CRISPR factors with a growing diversity. In this protocol, we provide a method encompassing protein purification, biochemical characterization, validation of protein-protein interactions, and preliminary in vivo functional assays in bacteria for Cas nuclease and its associated Pro-CRISPR factor. We hope this protocol will not only assist in the characterization of the CRISPR-Cas system, but also provide valuable guidance for the characterization of other nucleases or nucleic acid modification systems.},
}
@article {pmid42112301,
year = {2026},
author = {Saedi, S and Nezhadi, J and Feizi, H and Memar, MY and Arefi, V and Kadkhoda, H},
title = {A comparative analysis of CRISPR systems, virulence factors, and antibiotic resistance genes in carbapenem-sensitive and carbapenem-resistant Klebsiella pneumoniae.},
journal = {Iranian journal of microbiology},
volume = {18},
number = {1},
pages = {1-13},
pmid = {42112301},
issn = {2008-3289},
abstract = {BACKGROUND AND OBJECTIVES: Klebsiella pneumoniae is a major cause of healthcare-associated infections, particularly in immunocompromised patients. This study compares the CRISPR systems, virulence factors, and antibiotic resistance genes in carbapenem-sensitive (CSKP) and carbapenem-resistant (CRKP) clinical isolates.<br><br>MATERIALS AND METHODS: Carbapenemase-producing isolates were identified by mCIM/eCIM. PCR and RT-qPCR detected key genes, including cas3, involved in CRISPR-Cas function. In silico analyses included STRING for protein interactions, CRISPRCasdb for CRISPR subtype distribution, and Phyre2/AlphaFold for cas3 structure prediction.<br><br>RESULTS: Among the isolates, 35.2% were resistant to carbapenems. Among CRKP strains, high prevalence of bla-NDM-1 (82%) and bla-OXA-48 (64%) was observed. The cas3 expression was significantly upregulated in resistant isolates (P = 0.002). CRISPR subtype I-E was identified in 16% of CRKP and 36% of CSKP isolates. Structural-functional analysis supported the integrity of Cas3 and revealed interactions with regulatory and iron acquisition proteins. Statistically significant differences in virulence and resistance gene profiles were found between CRKP and CSKP groups (P < 0.05).<br><br>CONCLUSION: This study highlights key differences between CRKP and CSKP isolates, particularly in CRISPR-Cas systems, resistance, and virulence. The findings suggest that cas3 plays a critical role in genomic adaptation and resistance mechanisms in K. pneumoniae, offering insights for future therapeutic strategies.},
}
@article {pmid42113695,
year = {2026},
author = {Yao, Y and Li, J and Duan, L and Chen, J and Li, G and Hu, Z},
title = {One-step Overlapping PCR for Rapid Synthesis of Single-guide RNA DNA Templates for the CRISPR System.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {230},
pages = {},
doi = {10.3791/70369},
pmid = {42113695},
issn = {1940-087X},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/biosynthesis/chemical synthesis ; *CRISPR-Cas Systems/genetics ; *Polymerase Chain Reaction/methods ; *DNA/genetics/chemical synthesis ; Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; },
abstract = {The CRISPR-Cas system has revolutionized genome editing; however, conventional methods for generating single-guide RNA (sgRNA) often involve time-consuming cloning steps or expensive commercial synthesis kits. An optimized one-step overlapping PCR strategy is presented for the rapid, cost-effective synthesis of DNA templates for in vitro sgRNA transcription. Using four partially overlapping primers spanning the T7 promoter, target-specific guide sequence, and sgRNA scaffold, full-length templates are assembled in a single PCR reaction without cloning. Systematic experimental optimization established an optimal primer ratio (AF1:AF2:AF3:Tracr-R = 50:5:1:50), minimizing non-specific byproducts while maximizing full-length product yield, as confirmed by agarose gel electrophoresis. This approach was successfully extended to generate templates for Staphylococcus aureus Cas9 (saCas9) sgRNA, demonstrating cross-system applicability beyond Streptococcus pyogenes Cas9 (SpCas9). Although direct chemical synthesis of sgRNAs offers advantages such as high purity, chemical modifications to enhance stability, and reduced off-target effects, it remains prohibitively expensive for high-throughput applications or large-scale screens that require numerous sgRNAs. In vitro cleavage assays demonstrated that guide RNAs generated using this method achieve editing efficiencies comparable to those obtained via conventional plasmid-based cloning. Furthermore, ribonucleoprotein complexes assembled with these sgRNAs and delivered into HEK293T cells via electroporation resulted in detectable indel formation at the target locus, confirming functionality in vivo. Cost analysis indicates that this method substantially reduces template preparation costs compared to commercial synthesis kits while reducing turnaround time from days to hours, thereby providing an accessible and scalable approach for laboratories engaged in genetic research.},
}
@article {pmid42113764,
year = {2026},
author = {Iwata, S and Miura, Y and Iwamoto, T},
title = {Non-viral in vivo electroporation-based chromosomal engineering and repair assessment in the murine uterine epithelium.},
journal = {PloS one},
volume = {21},
number = {5},
pages = {e0348797},
doi = {10.1371/journal.pone.0348797},
pmid = {42113764},
issn = {1932-6203},
mesh = {Animals ; Female ; *Electroporation/methods ; *Uterus/metabolism ; Mice ; CRISPR-Cas Systems ; Epithelium/metabolism ; *DNA Repair ; *Genetic Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Translocation, Genetic ; },
abstract = {Chromosomal rearrangements generated by CRISPR/Cas systems are valuable for studying genomic architecture and repair mechanisms. However, most in vivo approaches rely on viral vectors, which require specialised production, prolonged nuclease expression, and elevated biosafety containment. Here, we applied Cas9 ribonucleoprotein (RNP) electroporation to the murine uterine epithelium as a simple, non-viral strategy for somatic chromosomal engineering. This method successfully induced defined interchromosomal translocations at multiple loci and enabled the molecular assessment of large-scale inversion repair (57.8 Mb) using paired gRNAs with an ssODN donor. While rearranged alleles were detected at low apparent frequencies in bulk uterine DNA-consistent with epithelial-restricted delivery and somatic mosaicism-high-depth whole-genome sequencing (WGS) and PCR provided nucleotide-resolution confirmation of precise junction formation. Our findings demonstrate that uterine electroporation of CRISPR RNPs is a feasible, rapid approach for evaluating engineered chromosomal rearrangements in vivo, providing a controlled platform for analyzing somatic DNA repair outcomes without viral confounds.},
}
@article {pmid42115498,
year = {2026},
author = {Valiñas, MA and Cerrudo, I and Marchetti, F and Villarreal, F and Pagnussat, G and Zabaleta, E},
title = {CRISPR/Cas9-mediated disruption of the gamma carbonic anhydrase 2 gene leads to reduced mitochondrial complex I and growth alterations in tomato.},
journal = {Planta},
volume = {263},
number = {6},
pages = {},
pmid = {42115498},
issn = {1432-2048},
support = {PICT 2020 00013//ANPCyT/ ; },
mesh = {*Solanum lycopersicum/genetics/growth &amp; development/enzymology/metabolism ; *CRISPR-Cas Systems/genetics ; *Electron Transport Complex I/metabolism/genetics ; Mitochondria/metabolism ; *Carbonic Anhydrases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Germination ; },
abstract = {Despite similar complex I reduction, γCA2 disruption in tomato, unlike in arabidopsis, triggers hormonal and developmental changes, challenging assumptions of conserved mitochondrial responses across plant species. NADH-ubiquinone oxidoreductase [complex I (CI)] is the main entry point of electrons to OXPHOS being essential for metabolism and redox balance. In most organisms, except animals and fungi, CI contains an additional domain composed of gamma carbonic anhydrase (γCA) subunits, termed the CA module. In Arabidopsis thaliana, this module includes AtɣCA1/3, AtɣCA2, and AtɣCAL1/2. AtɣCA2 is critical for CI biogenesis, yet its role in other species remains unclear. In tomato, the γCA family comprises SlɣCA1a, SlɣCA1b, SlɣCA2, and SlɣCAL. Here, we report the inactivation of the tomato SlɣCA2 using CRISPR/Cas9 technology. As in arabidopsis, SlɣCA2-KO tomato plants show comparable reduction in CI levels and activity and a similar decrease in oxygen consumption, yet display increased ATP levels in seeds. However, unlike arabidopsis, mutant tomato plants exhibit delayed seed germination and retarded growth and development. Our results further suggest that abscisic acid and gibberellin homeostasis is altered in SlɣCA2-KO plants. Together, these findings support a connection between mitochondrial respiration and hormonal regulation, by which plants adjust developmental processes to mitochondrial electron transport chain functionality, thereby preventing energy depletion during early growth stages.},
}
@article {pmid41932456,
year = {2026},
author = {Silva, CS and Nascimento, GR and Cruz, PEO and Arancibia, RH and Andrade Belitardo, EMM and Castro, TLP and Villar, LM and Pacheco, LGC},
title = {Design principles for LAMP-CRISPR molecular diagnostics.},
journal = {Methods (San Diego, Calif.)},
volume = {251},
number = {},
pages = {1-22},
doi = {10.1016/j.ymeth.2026.03.014},
pmid = {41932456},
issn = {1095-9130},
mesh = {*Nucleic Acid Amplification Techniques/methods/instrumentation ; *Molecular Diagnostic Techniques/methods/instrumentation ; Humans ; *CRISPR-Cas Systems/genetics ; Point-of-Care Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Pathology, Molecular/methods ; },
abstract = {Nucleic acid detection methods leveraging Cas9, Cas12, and Cas13 enzymes have recently been widely integrated with isothermal amplification techniques, particularly Loop-Mediated Isothermal Amplification (LAMP), to develop CRISPR-based diagnostic assays for a broad range of pathogens. Coupling these systems with portable result-readout platforms such as lateral flow devices, microfluidics, and smartphones offers a promising pathway for deploying LAMP-CRISPR diagnostics at the point-of-care (PoC), especially in settings where conventional, resource-intensive methods like real-time PCR are not feasible. However, the development of LAMP-CRISPR assays presents unique challenges not typically encountered in real-time PCR workflows. These include the need for a larger number of oligonucleotides, the complexity of integrating multiple biochemical conditions, and a heightened risk of false-positive results. Despite the growing number of bioinformatics tools designed to aid assay development, establishing a robust and reproducible workflow for LAMP-CRISPR remains a significant hurdle. In this review, we critically examine current strategies for designing LAMP-CRISPR assays and offer a detailed, step-by-step guide to achieving high-performance diagnostic tools using this approach. We cover key aspects of target sequence selection, oligonucleotide and CRISPR system design, and the strategic choice of readout methods. We further discuss available tools for LAMP primer and CRISPR guide RNA design, providing practical recommendations for optimizing sequence selection. Various probe formats for Cas-mediated trans-cleavage detection are summarized, and we present best practices for assay standardization and minimizing false-positive signals. Finally, we highlight the current limitations and outline future directions for LAMP-CRISPR diagnostics in decentralized and PoC testing environments.},
}
@article {pmid42019303,
year = {2026},
author = {Sun, X and Qian, L and Jin, D and Guo, B and Tao, S and Fang, J and Wang, S and Chen, H and Tian, T and Lei, H},
title = {Bifunctional DNA multivalent structure integrating stable capture of urothelial carcinoma cells with CRISPR/Cas12a signal amplification for bladder cancer detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {306},
number = {},
pages = {118710},
doi = {10.1016/j.bios.2026.118710},
pmid = {42019303},
issn = {1873-4235},
mesh = {Humans ; *Urinary Bladder Neoplasms/diagnosis/urine/genetics/pathology ; *Biosensing Techniques/methods ; CRISPR-Cas Systems/genetics ; Aptamers, Nucleotide/chemistry/genetics ; *DNA/chemistry ; Nanostructures/chemistry ; Cell Line, Tumor ; Nucleic Acid Hybridization ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Noninvasive detection of bladder cancer remains challenging due to the limited analytical performance of current urine-based assays under realistic detection conditions. Here, we report a linear programmable DNA nanostructure that integrates multivalent aptamer recognition with CRISPR/Cas12a signal transduction for detecting tumor-derived urinary exfoliated cells. Assembled via hybridization chain reaction, the multivalent scaffold was shown to enhance ligand-cell binding stability under mechanically perturbed detection processes, thereby supporting mechanically stable biological recognition and yielding an approximately 14-fold increase in cellular binding affinity compared with monovalent aptamers. The optimized architecture (MAP12) enables detection with a LOD of 1.1 cells/mL in model systems and achieves high diagnostic performance in clinical urine samples (92% sensitivity, 88% specificity; AUC = 0.9424), supporting dual signal readouts via fluorescence and lateral flow devices (LFD). This work establishes a DNA nanostructural strategy for reliable, rapid, and noninvasive cancer cell detection under realistic operational conditions.},
}
@article {pmid42025056,
year = {2026},
author = {Ren, Y and Du, W},
title = {Integrated single-tube detection of miRNAs in subpopulation-specific extracellular vesicles via spatially colocalized dual-module DNA scaffold.},
journal = {Biosensors &amp; bioelectronics},
volume = {306},
number = {},
pages = {118711},
doi = {10.1016/j.bios.2026.118711},
pmid = {42025056},
issn = {1873-4235},
mesh = {*MicroRNAs/isolation &amp; purification/genetics/analysis ; *Extracellular Vesicles/chemistry/genetics ; Humans ; *Biosensing Techniques/methods ; Aptamers, Nucleotide/chemistry ; Limit of Detection ; CRISPR-Cas Systems ; *DNA/chemistry ; Biomarkers, Tumor/genetics ; },
abstract = {Extracellular vesicles (EVs)-derived miRNAs are valuable non-invasive biomarkers for early cancer diagnosis. However, most current methods detect either total EVs-miRNAs or those from a single subpopulation, overlooking subpopulation heterogeneity. Moreover, EVs isolation and miRNA analysis are often conducted in separate workflows, increasing sample handling and compromising reproducibility. Herein, we presented an integrated, single-tube platform for subpopulation-specific miRNA profiling, in which multivalent aptamer-based EVs capture and CRISPR/Cas12a-mediated signal amplification were co-localized on a rationally designed DNA scaffold. The multivalent aptamer system achieved a capture efficiency of 84.3%, significantly outperforming monovalent aptamers (56.4%). Upon in situ lysis of captured EVs subpopulation, the released miRNAs preferentially encountered adjacent recognition probes due to the increased local concentration, thereby efficiently initiating catalytic hairpin assembly (CHA). The resulting abundant duplex subsequently activated Cas12a, achieving a limit of detection (LOD) as low as 1.42 × 10[4] particles/μL. Critically, the modularity of the platform allowed for straightforward reconfiguration to target distinct EVs subpopulations by exchanging the capture aptamer. We demonstrated this flexibility by profiling miR-21 and miR-155 across two specific subpopulations (CD63[+] and MUC1[+]). The results revealed both cell-line-specific expression patterns and marked heterogeneity across CD63/MUC1-captured EVs fractions. Meanwhile, MUC1[+] subpopulation outperformed CD63[+] in clinical diagnosis, with higher AUC values for both miR-21 (0.85 vs 0.75) and miR-155 (0.83 vs 0.73). Overall, our study highlighted the critical importance of EVs subpopulation heterogeneity in cancer diagnostics and provided more informative biomarker readouts.},
}
@article {pmid42051315,
year = {2026},
author = {Nolan, M and Aryal, S and Ndayambaje, IS and Cao, M and Lee, P and Hovde, M and Yun, S and Wlaschin, J and Held, A and Beaussant, H and Wymann, B and Zong-Lee, C and Lim, SM and Jiang, X and Ramesh, N and Agra Almeida Quadros, AR and Boulos, A and Zinter, N and Salem, S and El-Tayar, L and Beccari, M and Presa, M and Jourdan Ferreras Reyes, C and Ruan, YY and Griesman, G and Aguilar, C and Hawrot, J and Wheeler, H and Melamed, Z and Kleinstiver, BP and Albers, M and Cleveland, DW and Tanzi, RE and Lutz, CM and Hubbard, RD and Kobayashi, D and Ward, M and R R Alves, C and Wainger, B and Pichon, CL and Lagier-Tourenne, C},
title = {Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {42051315},
issn = {2692-8205},
abstract = {Loss of neuronal regenerative capacity is a common feature of neurodegenerative disease and axonal injury, yet the transcriptional programs governing this state remain poorly defined. Stathmin-2 (STMN2), a tubulin-binding protein essential for axon maintenance and repair, is profoundly depleted following loss of nuclear TDP-43 in neurodegenerative disease. Here, we identify statins as potent inducers of STMN2 expression. Pharmacological and genetic suppression of the mevalonate pathway, and subsequent prevention of protein geranylgeranylation, restored STMN2 levels in TDP-43 deficient cells and promoted neurite growth. STMN2 induction was abrogated when using a statin analogue unable to interact with HMG-CoA reductase, and through co-administration of mevalonate or geranylgeranyl diphosphate substrates. RNA-seq revealed that statins induce a coordinated pro-regenerative transcriptional response, including activation of the AP-1 transcription factor complex gene, ATF3 . Loss of ATF3 attenuated STMN2 induction in vitro , and diminished injury-induced Stmn2 upregulation in spinal motor neurons in vivo . These results demonstrate statins as modulators of ATF3 and STMN2 expression and highlight their therapeutic potential in neurodegenerative disease.},
}
@article {pmid42061344,
year = {2026},
author = {Han, X and Chen, H and Chang, Y and Zha, J and Lam, CYK and Yang, M and Wong, SHD and Yin, B},
title = {Surface-confined CRISPR-Cas12a biosensor with metal-enhanced fluorescence for rapid and ultrasensitive detection of SARS-CoV-2 nucleocapsid protein.},
journal = {Biosensors &amp; bioelectronics},
volume = {306},
number = {},
pages = {118649},
doi = {10.1016/j.bios.2026.118649},
pmid = {42061344},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods/instrumentation ; SARS-CoV-2/isolation &amp; purification ; *CRISPR-Cas Systems ; Gold/chemistry ; Humans ; COVID-19/diagnosis/virology ; Limit of Detection ; *Coronavirus Nucleocapsid Proteins/analysis ; Metal Nanoparticles/chemistry ; Silicon Dioxide/chemistry ; Aptamers, Nucleotide/chemistry ; Fluorescence ; *Phosphoproteins/analysis ; *Coronavirus Infections/diagnosis/virology ; *Nucleocapsid Proteins/analysis ; *Pneumonia, Viral/diagnosis/virology ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-Cas12a integrated with nanomaterials has formulated powerful biosensors for viral protein detection, addressing the urgent need for point-of-care diagnostics. However, existing platforms are hindered by either multi-step separation procedures or insufficient signal amplification, limiting their sensitivity and practicality. Here, we report a one-pot "on-off" biosensor that combines metal-enhanced fluorescence (MEF) and nanoscale spatial confinement by co-localizing both reporter substrates and the CRISPR-Cas12a system on gold-silica core-shell nanoparticles (Au@SiO2 NPs), enabling rapid and ultrasensitive protein detection. Using SARS-CoV-2 nucleocapsid (N) protein as a model analyte, Au@SiO2 NPs are co-functionalized with (i) ssDNA activators blocked by N protein-specific aptamers, (ii) light-up hairpin DNA (DAP) complexed with auramine O (AO) as reporters, and (iii) short polyethylene glycol (PEG) spacers to mitigate steric hindrance. The nanoplatform displays an ultrabright "on-state" fluorescence, with an intensity >860-fold higher than that of free AO, enabled by the interaction with DAP and optimized fluorophore-metal spacing (∼20 nm). Upon target binding, aptamer displacement exposes the activator to locally initiate Cas12a trans-cleavage, disrupting proximal DAP structure and its interaction with AO, thereby producing a distinct "off-state" signal. Within the linear detection range, the system demonstrates up to ∼85% signal reduction relative to the initial signal and a signal-to-noise ratio (SNR) of 83.89, corresponding to a ∼2.5-fold higher SNR than the solution-phase system. The platform attains a limit of detection at 67.2 fg/mL within 30 min, with excellent sensitivity, selectivity, stability, and recovery in bronchoalveolar lavage fluid. By combining MEF-driven signal amplification with surface-confined CRISPR-Cas12a trans-cleavage, this platform establishes an efficient strategy for sensitive N protein detection.},
}
@article {pmid42106593,
year = {2026},
author = {Enciso-Rodríguez, F and Barrero, LS and Garzón-Martínez, GA and Kim, JH and Kumam, Y and Pagliai, FA and Jiang, T and Huo, H and Munoz, P},
title = {Overcoming breeding barriers with genome editing in autopolyploid crops.},
journal = {BMC plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12870-026-08862-7},
pmid = {42106593},
issn = {1471-2229},
abstract = {Autopolyploid crops play a central role in global agriculture, yet their complex genomes pose significant barriers to genetic improvement. High allelic diversity, extensive redundancy, and polysomic inheritance impede both conventional breeding and the implementation of modern biotechnological tools. Genome editing offers a powerful alternative by enabling precise, multi-allelic modification of traits associated with yield, quality, and stress resilience. However, progress across autopolyploid crops remains uneven due to low transformation and regeneration efficiencies, limited genomic resources, and challenges in achieving complete allele disruption. This review focuses on recent advances in genome editing across four economically important autopolyploid crops-potato (Solanum tuberosum), alfalfa (Medicago sativa), sugarcane (Saccharum spp.), and blueberry (Vaccinium corymbosum). We highlight the diversity of traits targeted through CRISPR/Cas systems, including reporter and selectable marker validation, tuber and forage quality, biomass composition, stress tolerance, flowering modulation, and plant regeneration. We also describe technical constraints affecting genome editing in autopolyploids including genotype-dependent recalcitrance, low transformation and editing efficiency, multiallelic targeting and chimerism, outlining emerging solutions such as multiplexed designs, endogenous promoters, morphogenic regulators and virus-based approaches, among others. Together, these developments provide a path toward efficient and heritable genome editing in complex polyploid genomes, setting the stage for next-generation precision breeding in crops vital to food, forage, and bioenergy security.},
}
@article {pmid42106633,
year = {2026},
author = {Hannan, MN and Khan, S and Siddique, N and Hossain, MN and Islam, R and Nahar, K and Rahman, GKMM and Shozib, HB and Molla, AH and Hoque, MN and Haque, MM},
title = {Draft genome sequence of Priestia megaterium MHES4, a biofertilizer candidate isolated from tomato rhizosphere in Bangladesh.},
journal = {BMC genomic data},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12863-026-01431-9},
pmid = {42106633},
issn = {2730-6844},
support = {Project number: 002, Financial year: 2024 - 2027//This research was supported by Research Management Wing, Gazipur Agricultural University, Bangladesh./ ; },
abstract = {OBJECTIVE: The genus Priestia has recently gained attention for its plant growth-promoting potential. To examine the genomic traits and biosafety profile for potential field application as a native, climate-smart bioinoculant, we sequenced, assembled and annotated the genome of Priestia megaterium strain MHES4, isolated from the rhizosphere of tomato plant grown in drought-prone ecosystem of Rajshahi, Bangladesh.<br><br>DATA DESCRIPTION: Genome assembly data from the shotgun whole genome sequencing (WGS) of the P. megaterium MHES4 revealed 60 contigs with a total length of 5,267,048&#xa0;bp, an N50 of 446,003&#xa0;bp and 37.9% G&#x2009;+&#x2009;C content. The mean sequencing depth was 127.58&#xd7;, with 100% breadth of coverage. Genome completeness assessed was 97.43% with 3.5% contamination, confirming high assembly quality. In total, 5,484 protein-coding genes were annotated. Additionally, 5,445 protein-coding sequences, 28 tRNAs, and 5 rRNAs were identified. Functional analysis identified gene clusters involved in the synthesis of secondary metabolites, such as phytoene synthase and alpha-amylase, and a Type I CRISPR-Cas system. Biosafety assessment using in silico tools detected no virulence factors or transmissible antibiotic resistance genes, indicating its potential safe use in agriculture. Overall, this genomic resource provides valuable insights into the genetic potential of P. megaterium MHES4 for nutrient cycling and adaptation to the rhizosphere environment.},
}
@article {pmid42106677,
year = {2026},
author = {Peng, Z and Duan, W and Fan, Y and Yang, Q and Ye, Y and Xing, Y},
title = {PAM-flexible SpCas9 variants expand the targeting scope for porcine genome editing and cellular disease modeling.},
journal = {BMC biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12896-026-01164-8},
pmid = {42106677},
issn = {1472-6750},
support = {2023YFC3404302//National Key Reaearch and Development Program of China/ ; 32260825//National Natural Science Foundation of China/ ; },
abstract = {BACKGROUND: CRISPR-Cas-mediated gene editing has revolutionized life sciences, yet the targeting scope of the widely used SpCas9 is limited by its strict requirement for the NGG protospacer adjacent motif (PAM). To overcome this limitation, PAM-flexible SpCas9 variants have been developed and characterized in multiple species; however, their potential in pigs (an important biomedical model for humans) remains unexplored. Here, we systematically evaluated the editing performance of three PAM-flexible SpCas9 variants (SpRY, SpG, and SpCas9-NG) and their derived base editors in porcine fetal fibroblasts (PFFs).<br><br>RESULTS: Profiling across 228 target sites revealed that SpRY exhibits nearly PAM-less activity, with significantly higher editing efficiency at NRN (15.82%, R&#x2009;=&#x2009;A/G) than at NYN PAMs (5.75%, Y&#x2009;=&#x2009;C/T). SpG and SpCas9-NG preferentially targeted NGN PAMs, achieving mean efficiencies of 14.81% and 16.33%, respectively. PAM&#x2011;flexible cytosine base editors (CBEs) mediated efficient C:G&#x2011;to&#x2011;T:A conversion, with mean efficiencies of 12.01% for SpRY&#x2011;BE4max (NNN PAMs), 15.43% for SpG&#x2011;BE4max (NGN PAMs), and 18.39% for SpCas9&#x2011;NG&#x2011;BE4max (NGN PAMs). Similarly, PAM&#x2011;flexible adenine base editors (ABEs) mediated efficient A:T&#x2011;to&#x2011;G:C conversion, with mean efficiencies of 15.66% for SpRY&#x2011;ABE8e (NNN PAMs), 24.16% for SpG&#x2011;ABE8e (NGN PAMs), and 20.50% for SpCas9&#x2011;NG&#x2011;ABE8e (NGN PAMs). By exploiting this expanded targeting scope, we successfully introduced 16 pathogenic single&#x2011;nucleotide variants (SNVs) at NRN PAM sites in the porcine genome, with editing efficiencies reaching up to 40.68% for CBEs and 61.76% for ABEs.<br><br>CONCLUSIONS: PAM-flexible SpCas9 variants and their derived base editors greatly expand the targeting scope for porcine genome engineering, thereby substantially broadening the applicability potential of CRISPR-Cas-mediated genome editing tools in porcine genetic improvement and disease model generation.},
}
@article {pmid42107032,
year = {2026},
author = {Singh, V and Fetoh, MEA and Fetoh, IEA},
title = {Degradation dynamics: an insight into microbial interactions with explosive compounds.},
journal = {Biodegradation},
volume = {37},
number = {3},
pages = {},
pmid = {42107032},
issn = {1572-9729},
mesh = {Biodegradation, Environmental ; *Explosive Agents/metabolism ; *Bacteria/metabolism ; *Fungi/metabolism ; *Microbial Interactions ; },
abstract = {Degradation dynamics is an essential aspect in the field of environmental science and is crucial in understanding the interaction between microbes and explosive compounds. Explosive compounds and their residues, such as nitramines, nitro-substituted aromatics, picric acid, TETRYL, and HEXYL), and aliphatic, RDX, etc.are highly persistent in the environment. These compounds are toxic to many life forms at high concentrations, specific microbial species have evolved resistance and degradation capabilities, though their growth can still be inhibited beyond certain thresholds, The results of microbial biodegradation can range from complete mineralization to only the biotransformation into less toxic or more resistant metabolites. Research using pure cultures of bacteria and fungi has provided insight into the degradation pathways of certain nitro-organic compounds, and some key enzymes (laccases and lignin peroxidases) have been identified and studied. This review mainly aims to provide an overview of the current state of research on the degradation dynamics of explosive compounds Recent advancements have pivoted toward 'Bio-omics' and synthetic biology tools, such as CRISPR/Cas systems, to engineer high-activity microbial strains.},
}
@article {pmid42108387,
year = {2026},
author = {Pandya, K and Jaisinghani, LS and Tripathi, A and Kumar, D and Saraf, SK and Sahoo, J and Kumar, D},
title = {Rational Design and Optimisation of CRISPR-Cas9 Delivery Systems for Targeted Genomic Transformation.},
journal = {The journal of gene medicine},
volume = {28},
number = {5},
pages = {e70095},
doi = {10.1002/jgm.70095},
pmid = {42108387},
issn = {1521-2254},
support = {EMDR/SG/11/2023-5582//Indian Council of Medical Research/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Genetic Vectors/genetics ; *Gene Editing/methods ; Animals ; Genetic Therapy/methods ; *Gene Transfer Techniques ; Blood-Brain Barrier/metabolism ; Dependovirus/genetics ; Alzheimer Disease/genetics/therapy ; },
abstract = {The CRISPR-Cas9 genome-editing technique offers a promising therapeutic strategy for genetic disorders, including neurodegenerative diseases like Alzheimer's disease (AD), characterised by inherited susceptibility and progressive cognitive decline, as well as other hallmarks such as amyloid beta (Aβ1-42) plaques and neurofibril tangles (NFTs). However, the blood-brain barrier (BBB) poses a significant challenge to the effectiveness of gene editing components in the affected brain region and impedes clinical translation. This comprehensive review compares various CRISPR-Cas9 delivery vectors, viral, nonviral and physical, with a focus on their efficacy in neurological diseases such as AD. Viral vectors viz., adeno-associated viruses (AAVs) and lentiviruses (LVs) demonstrate high transduction efficiency and BBB permeability. AAVs are preferred for their low immunogenicity, minimal toxicity, high neuronal tropism and episomal persistence, enabling sustained expression without insertional mutagenesis. LVs offer larger genetic payloads but raise concerns about genomic integration and potential oncogenesis, though integration-defective variants mitigate these risks. Nonviral vectors, including peptide and polymer-based nanoparticles, lipid nanoparticles (LNPs) and Inorganic carriers such as gold and silver nanoparticles, are less immunogenic and easier to handle but require further optimisation for in vivo BBB crossing and endosomal escape. Physical methods such as electroporation and microinjection are suitable for in vitro/ex vivo use, while novel CNS-targeted strategies, such as RVG-tagged particles, TfR-directed LNPs and engineered AAV variants, enhance brain penetration via receptor-mediated transcytosis. These preclinical studies show that these technologies can successfully edit genes and provide therapeutic benefits, including amyloid reduction and cognitive improvement in AD models. Yet off-target effects, immune responses and regulatory hurdles persist. Overall, continuous innovation in delivery vector design and safety profile-targeting strategies is crucial for advancing CRISPR-Cas9 towards clinical therapies for AD-based therapies and related neurological disorders.},
}
@article {pmid41146557,
year = {2026},
author = {Saputra, E and Vellarikkal, SK and Li, L and Sun, H and Nguyen, K and Montano, A and Natarajan, S and Piccioni, F and Tamburino, AM and Yu, X and Olow, AK},
title = {In vitro models of cancer-associated fibroblast heterogeneity uncover subtype-specific effects of CRISPR perturbations.},
journal = {Molecular oncology},
volume = {20},
number = {5},
pages = {1253-1269},
doi = {10.1002/1878-0261.70153},
pmid = {41146557},
issn = {1878-0261},
support = {//Merck/ ; },
mesh = {Humans ; *Cancer-Associated Fibroblasts/pathology/metabolism ; Cell Line, Tumor ; *Pancreatic Neoplasms/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Genetic Heterogeneity ; *Models, Biological ; *CRISPR-Cas Systems ; Single-Cell Analysis ; Tumor Microenvironment ; Coculture Techniques ; },
abstract = {Cancer-associated fibroblasts (CAFs) are sought after as potential therapeutic targets due to their pro- and antitumorigenic functions, which are attributed to specializations in CAF subtypes. A precise targeting of specific subtypes would be required to design therapies that effectively modulate CAF phenotypes, necessitating translatable model systems to support target discovery efforts. However, not only is our knowledge of CAF heterogeneity in solid tumors lacking, particularly in pancreatic tumors, but the translatability of CAF models has also not been rigorously evaluated. Here, we develop a coculturing model with primary CAFs and immortalized tumor cell lines that can reliably represent CAF phenotypes observed in tumors, with correlations to immuno-resistant and immunomodulatory phenotypes. Using single-cell transcriptomics, we characterize the CAF subtype heterogeneity in the in vitro CAF cell lines isolated from pancreatic cancer patients and investigate the impact of perturbing potential stromal genes on different CAF subtypes. We also infer the continuum of state changes underlying the interconvertibility of CAF subtypes. Finally, we use immortalized CAF cell lines to perform single-cell CRISPR perturbations of stromal targets, revealing the subtype-specific effects of perturbations and the impact of model-type selection on the translatability of insights.},
}
@article {pmid41958313,
year = {2026},
author = {Wong, E and Souza-Fonseca-Guimaraes, F},
title = {Gain-of-function enhancers optimize CAR-NK cell-based anti-cancer immunotherapy.},
journal = {Immunology and cell biology},
volume = {104},
number = {5},
pages = {494-498},
doi = {10.1111/imcb.70113},
pmid = {41958313},
issn = {1440-1711},
mesh = {*Killer Cells, Natural/immunology/transplantation/metabolism ; Animals ; Humans ; *Immunotherapy, Adoptive/methods ; Mice ; *Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems/genetics ; HT29 Cells ; *Immunotherapy/methods ; },
abstract = {Schematic overview of the two-stage screening approach used to identify NK cell fitness genes. (A) CRISPRa mechanism, showing dCas9-VP64-mediated upregulation of target genes. (B) Whole-genome CRISPRa screening in HER2-CAR-NK92 cells transduced with a CRISPR sgRNA library and transferred into mice bearing HT29 tumours, followed by tumour collection and next-generation sequencing (NGS). (C) Barcoded ORF mini-screen in primary peripheral blood NK (PBNK) cells transduced with HER2-CAR and an ORF library, transferred into HT29 tumour-bearing mice, with subsequent tumour collection and NGS analysis.},
}
@article {pmid41967792,
year = {2026},
author = {Yuan, W and Jiang, Z and Li, F and Chen, H and Zhang, X and Fan, X},
title = {THOC6 deficiency leads to cardiomyopathy by reducing myocardial contractile proteins in cardiomyocytes.},
journal = {Experimental cell research},
volume = {459},
number = {2},
pages = {115025},
doi = {10.1016/j.yexcr.2026.115025},
pmid = {41967792},
issn = {1090-2422},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; *Cardiomyopathies/metabolism/genetics/pathology ; Humans ; Animals ; Rats ; Cell Proliferation/genetics ; CRISPR-Cas Systems ; Apoptosis/genetics ; Actins/metabolism/genetics ; *Myocardial Contraction ; Collagen Type I/metabolism/genetics ; Induced Pluripotent Stem Cells/metabolism ; },
abstract = {BACKGROUND: The THOC6 protein is an essential part of the THO complex. Biallelic loss-of-function variants in the THOC6 gene are linked to Beaulieu-Boycott-Innes syndrome (BBIS; OMIM #613680). Although research predominantly focuses on THOC6's involvement in neurodevelopmental disorders, approximately 80% of BBIS patients present with cardiac anomalies, including structural heart disease, cardiomyopathy, and arrhythmia. Despite this, the connection between THOC6 expression and cardiac development remains underexplored. This study firstly investigates THOC6's role in heart development.<br><br>METHODS AND RESULTS: This study we firstly utilized CRISPR/Cas9 to knock out THOC6 in H9C2 cardiomyocytes, revealing a reduction in cell proliferation and an increase in apoptosis. With RNA sequencing (RNA-seq) analysis we found abundant gene changes after THOC6 knockout (KO) in H9C2, which associated with hypertrophic cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, and dilated cardiomyopathy. Protein-protein interaction analysis and experimental validation indicated that THOC6 regulates the expression of type I collagen (COL1A1, COL1A2) and cytoskeletal protein (Cardiac &#x3b1; actin 1) in cardiomyocytes. Subsequently, we generated a THOC6 knockout cell lines in human induced pluripotent stem cells (hiPSCs) derived from a healthy individual using CRISPR/Cas9 technology. THOC6 knockout (KO) in hiPSCs-derived cardiomyocytes (hiPSC-CMs) led to the early manifestation of hypertrophic cardiomyopathy and dilated cardiomyopathy phenotypic characteristics, including disrupted sarcomeric organization. Notably, THOC6 KO hiPSC-CMs demonstrated a significant decreased in COL1A2 and &#x3b2;-tubulin expression levels.<br><br>CONCLUSION: THOC6 may influence cardiac development by regulating myocardial contractile proteins, primarily type I collagen, cardiac &#x3b1; actin 1 and &#x3b2;-tubulin.},
}
@article {pmid41997283,
year = {2026},
author = {Zhang, Y and Yu, M and Huang, J},
title = {Arginase 2 regulates cholesterol biosynthesis in endothelial cells.},
journal = {Experimental cell research},
volume = {459},
number = {2},
pages = {115029},
doi = {10.1016/j.yexcr.2026.115029},
pmid = {41997283},
issn = {1090-2422},
mesh = {Humans ; *Arginase/metabolism/genetics ; *Cholesterol/biosynthesis ; *Human Umbilical Vein Endothelial Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Arginase 2 (Arg 2) is a mitochondrial enzyme that hydrolyzes L-arginine to L-ornithine and urea, influencing endothelial nitric oxide (NO) bioavailability and vascular function. Although Arg 2 is implicated in endothelial dysfunction under hypercholesterolemic and oxidative stress conditions, its potential role in endothelial cholesterol metabolism remains unknown.<br><br>METHODS: Arg 2 was deleted in immortalized human umbilical vein endothelial cells (HUVECs) using CRISPR/Cas9, followed by transcriptomic analyses. Differential gene expression was validated by quantitative RT-PCR and immunoblotting. Overexpression of wild-type Arg 2 and the catalytically inactive Arg 2 (H160F) mutant was achieved using recombinant lentiviral transduction. Arginase activity was quantified by measuring urea production using a colorimetric assay. Cholesterol intermediates were quantified by LC-MS.<br><br>RESULTS: RNA sequencing revealed that Arg 2 deletion markedly downregulated genes involved in the mevalonate and steroid biosynthesis pathways, including HMGCS1, FDFT1, FDPS, SQLE, and DHCR7. These transcriptional changes were accompanied by reduced protein levels of key cholesterol biosynthetic enzymes and decreased cellular concentrations of sterols, lanosterol, desmosterol, and cholesterol. Conversely, either overexpression of wild-type Arg 2 or the catalytically inactive Arg 2 (H160F) mutant enhanced the expression of these enzymes.<br><br>CONCLUSIONS: These findings identify a previously unrecognized role of Arg 2 in promoting endothelial cholesterol biosynthesis. Beyond competing with endothelial NO synthase for L-arginine, Arg 2 may regulate vascular homeostasis through modulation the mevalonate pathway, independent of its enzymatic activity. This dual function may link amino acid and lipid metabolism in the endothelium and suggests new mechanisms by which Arg 2 contributes to endothelial dysfunction and atherosclerotic progression.},
}
@article {pmid42024482,
year = {2026},
author = {Liang, Q and Cao, Y and Zhang, X and Ye, R and Liu, M and Zhang, S and Wang, Y},
title = {A Rapid and Ultrasensitive Detection of Coxsackievirus A16 Using Reverse Transcription Multiple Cross Displacement Amplification Combined with the CRISPR-Cas12a-Based Biosensing System.},
journal = {ACS infectious diseases},
volume = {12},
number = {5},
pages = {1765-1775},
doi = {10.1021/acsinfecdis.6c00151},
pmid = {42024482},
issn = {2373-8227},
mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Enterovirus/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; *Hand, Foot and Mouth Disease/diagnosis/virology ; Sensitivity and Specificity ; Reverse Transcription ; Limit of Detection ; RNA, Viral/genetics ; Child, Preschool ; },
abstract = {Coxsackievirus A16 (CVA16) is one of the primary viral etiological agents of hand, foot, and mouth disease (HFMD) in infants and children under five years of age. Prompt and reliable detection of CVA16 is crucial for guiding immediate therapeutic interventions and for implementing effective epidemic prevention and control strategies, particularly in settings with limited resources. Herein, a diagnostic platform for CVA16 (CVA16-RT-MCDA-CRISPR) was developed by combining reverse transcription multiple cross displacement amplification (RT-MCDA) with CRISPR-Cas12a-based detection. In this system, the CVA16 VP1 gene was preamplified using RT-MCDA technology. The resulting amplicons were then specifically recognized and cleaved by the CRISPR-Cas12a-based detection system. MCDA primers, an engineered CP1 primer, and a specific guide RNA (gRNA) were designed to target the VP1 gene of CVA16. The assay achieved a limit of detection of 2.8 × 10[-1] copies per microliter for CVA16 RNA standard templates and showed no cross-reactivity against non-CVA16 pathogens. Furthermore, the CVA16-RT-MCDA-CRISPR assay's feasibility was validated using 96 clinical samples. Taken together, these results demonstrate that the CVA16-RT-MCDA-CRISPR assay is a reliable diagnostic tool for rapidly and sensitively detecting CVA16.},
}
@article {pmid42100374,
year = {2026},
author = {Schrage, PR and Afonina, U and Wörtz, J and Marchfelder, A and Martens, KJA and Sáenz, JP and Endesfelder, U},
title = {A novel expression system for imaging single-molecule fluorescence in Haloferax volcanii WR806 enables visualization of altered Cas1 dynamics during UV-induced DNA damage response.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqag014},
pmid = {42100374},
issn = {2633-6693},
abstract = {Fluorescence microscopy has become an indispensable tool in biological research, offering powerful approaches to study protein dynamics and cellular processes in vivo. Among archaea, Haloferax volcanii has emerged as a particularly well-suited model organism for imaging studies, with a growing toolkit of established fluorescent markers, plasmids, and promoter systems. Recent advances in single-molecule imaging techniques have created new opportunities through WR806, a carotenoid-free H. volcanii strain providing reduced autofluorescence background. However, existing plasmid-based expression systems in WR806 show critical limitations in protein expression control and challenges with protein aggregation. To address these limitations, we developed pUE001, a novel plasmid system specifically designed for WR806. This system achieves precise expression control by decoupling selection and induction through strategic implementation of the trpA selection marker. Through comprehensive characterization, we demonstrate that pUE001 provides superior control over protein expression compared to the previously established pTA962 system. It enables linear, titratable expression of diverse proteins-from the highly regulated CRISPR-Cas component Cas1 to the abundant structural protein FtsZ1-while preventing protein aggregation that could compromise native cellular functions. Additionally, we performed a comprehensive analysis of WR806 to show that carotenoid depletion does not affect native cellular physiology. Finally, to demonstrate the system's utility, we investigated the role of Cas1 in UV-induced DNA repair using single-particle tracking photoactivated localization microscopy (sptPALM). Our findings reveal Cas1 colocalizing with DNA-dense cellular regions and significant, dose-dependent changes in Cas1 mobility following UV-light-induced damage, providing evidence for its possible involvement in DNA damage response processes and offering new insights into the expanding roles of CRISPR-Cas systems beyond adaptive immunity.},
}
@article {pmid42100688,
year = {2026},
author = {Yadav, J and Gehlot, P and Soni, P and Jain, T},
title = {Plant microbiome engineering: from inoculation to genome editing.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1781381},
pmid = {42100688},
issn = {1664-302X},
abstract = {Plant-associated microbiomes are central to crop productivity, nutrient efficiency, and stress resilience, yet conventional microbiome manipulation strategies, largely based on microbial inoculation and agronomic management, often suffer from inconsistent field performance and limited persistence. Although several recent reviews have discussed CRISPR-mediated plant-microbe engineering and synthetic microbial community (SynCom) design separately, few reviews integrate genome editing, ecological stability of microbiomes, and climate-resilient agricultural applications within a unified conceptual framework. Recent advances in molecular biotechnology are transforming this landscape by enabling precision engineering of plant-microbe interactions at genetic, metabolic, and community levels. In particular, synthetic biology tools including CRISPR/Cas genome editing, RNA interference, and synthetic microbial communities (SynComs), now allow targeted modification of plant traits governing microbial recruitment, microbial pathways underpinning nutrient cycling and stress tolerance, and community-level functional complementarity. This review integrates molecular genetics, microbial ecology, and systems-level microbiome design to frame the plant and its microbiome as an engineerable holobiont. We integrate insights from genome editing in plants and microbes, omics-guided SynCom design, climate-resilience mechanisms, and emerging AI-assisted decision frameworks, including machine learning and ecological modeling approaches used to analyze multi-omics datasets, and predict plant-microbiome interactions across experimental and field-based studies. Importantly, we critically assess limitations related to ecological stability, trait trade-offs, biosafety, and regulatory challenges that constrain large-scale deployment. By bridging genome-enabled microbiome manipulation with ecological design principles, this review proposes an integrative framework for climate-smart microbiome engineering and identifies key research priorities required to transition from empirical inoculation toward predictive, sustainable, and socially responsible agricultural biotechnology.},
}
@article {pmid42100873,
year = {2026},
author = {Kavanagh, EW and Joynt, AT and Pion, AR and Eastman, AC and Parr, AI and Starego, KL and Jain, M and Shannon, SR and Yoo, EJ and Newby, GA and Tzeng, SY and Sharma, N and Green, JJ and Cutting, GR},
title = {Base editing and nanoparticle transfection of airway cell types essential for treatment of cystic fibrosis.},
journal = {JCI insight},
volume = {11},
number = {9},
pages = {},
doi = {10.1172/jci.insight.198563},
pmid = {42100873},
issn = {2379-3708},
mesh = {*Cystic Fibrosis/genetics/therapy ; Humans ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; *Nanoparticles/administration &amp; dosage/chemistry ; *Gene Editing/methods ; *Transfection/methods ; Epithelial Cells/metabolism ; Genetic Therapy/methods ; Respiratory Mucosa/metabolism ; Animals ; CRISPR-Cas Systems ; Cells, Cultured ; },
abstract = {Cystic fibrosis (CF) is a life-limiting genetic disorder caused by deleterious variants in the CFTR gene that results in altered mucus impairing the airway epithelia. Durable correction of these variants in airway cells remains a therapeutic challenge for about 10% of individuals unresponsive to CFTR modulators. A common disease-causing CFTR splice site variant, 3120+1G>A, was corrected in primary CF airway cells using base editor RNAs. Single-cell RNA sequencing revealed a remarkable increase in detectable CFTR transcript in most CF airway epithelial cell types resulting in notable enrichment of CFTR-expressing ionocytes and secretory goblet cells. Progenitor basal cell subtypes were edited, but they decreased as a fraction of total cells and CFTR-expressing cells compared with unedited cells. CRISPR base editors delivered by polymeric nanoparticles (PNPs) facilitated functional rescue of CFTR to clinically meaningful levels in immortalized and primary airway cells. PNPs delivered GFP-encoding RNA to progenitor airway cells in fully differentiated airway cultures. Vitronectin was a major component of the PNP corona that formed in vivo, but preincubation with vitronectin did not enhance delivery. Together, these findings validate a scalable, nonviral platform with compelling translational promise for treating CF and other respiratory diseases involving respiratory epithelial cell dysfunction.},
}
@article {pmid42101329,
year = {2026},
author = {Moss, O and Li, X and Kanagarajan, S and Wang, ES and Ivarson, E and Zhu, LH},
title = {Evaluation of Bna.SCT and Bna.REF1 as Target Genes to Reduce Sinapine in Rapeseed Using a Protoplast-Based CRISPR RNP Approach.},
journal = {Physiologia plantarum},
volume = {178},
number = {3},
pages = {e70905},
doi = {10.1111/ppl.70905},
pmid = {42101329},
issn = {1399-3054},
support = {//SLU Grogrund- Centre for Breeding of Food Crops/ ; //Trees and Crops for the Future (TC4F)/ ; //The Royal Physiographic Society of Lund/ ; },
mesh = {Gene Editing/methods ; *Brassica napus/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Protoplasts/metabolism ; *Brassica rapa/genetics/metabolism ; Mutation ; Choline/analogs &amp; derivatives ; },
abstract = {Rapeseed is a major oil crop worldwide, producing both oil and a high amount of protein. However, the use of its seed meal as a protein source for animal feed is limited by antinutritional factors, such as sinapine, which reduces nutrient absorption and affects the palatability. Efforts to reduce sinapine levels through conventional breeding have had limited success. Given the challenges of a changing climate and a growing global population, maximising crop utility, particularly the value of seed meal as a byproduct, is increasingly important. Genetic modification has been successfully used to reduce sinapine in rapeseed, but regulatory restrictions limit its commercial adoption in some regions. CRISPR-Cas gene editing, which is gaining broader global acceptance, offers a promising alternative to directly produce transgene-free mutants. In this study, we build on our previous work by generating transgene-free rapeseed mutants using protoplast-based CRISPR RNP gene editing. We successfully targeted the Bna.SCT and Bna.REF1 genes with editing efficiencies of 22%-63%, frequently achieving mutations in all four alleles of the target genes in T2 plants with a single sgRNA. Seed sinapine content was reduced by up to 38% in Bna.SCT mutants and 77% in Bna.REF1 mutants, with no observed effects on plant growth or development. These findings suggest that Bna.REF1 is the most effective target for sinapine reduction in transgene-free mutants among the genes tested in our lab.},
}
@article {pmid42101638,
year = {2026},
author = {Uc-Chuc, MA and Jiménez-Ramírez, IA and Guzmán-Marín, EDS and Chan-Pérez, JI and Acosta-Viana, KY},
title = {Beyond the catalysis of Trypanosoma cruzi trans-sialidases: structure, function, post-translational modifications, intrinsically disordered regions and use of CRISPR/Cas9.},
journal = {Archives of microbiology},
volume = {208},
number = {8},
pages = {},
pmid = {42101638},
issn = {1432-072X},
mesh = {*Trypanosoma cruzi/enzymology/genetics ; *Neuraminidase/metabolism/genetics/chemistry ; Protein Processing, Post-Translational ; *Glycoproteins/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Protozoan Proteins/genetics/metabolism/chemistry ; Chagas Disease/parasitology ; Intrinsically Disordered Proteins/metabolism/genetics/chemistry ; Phylogeny ; Humans ; Gene Editing ; },
abstract = {Chagas disease, caused by the protozoan Trypanosoma cruzi, is a global health problem with limited treatment options. The parasite's trans-sialidase (TS) protein family has been widely described as a key component in its life cycle. In this work, we present a comprehensive review of the TS family, including its molecular structure, phylogenetic relationships, and known functions. Based on in silico analyses, we propose that intrinsically disordered regions (IDRs) present in the TS family may play a role in the spatial organization of these proteins. We also hypothesize that these IDRs could contribute to the formation of biomolecular condensates through liquid-liquid phase separation, providing a potential dynamic platform that is not fully explained by conventional structural models. In addition, we discuss recent advances in the application of the CRISPR/Cas9 gene-editing system to T. cruzi TS proteins. The available evidence indicates the multifunctional nature of these proteins, including enzymatic and non-enzymatic isoforms, as well as the presence of conserved motifs associated with host interactions and post-translational modifications. Taken together, this review integrates previous findings and proposes new hypotheses regarding additional functions of the TS family. The need for experimental studies to validate these proposals and clarify their relevance to the parasite's biology is emphasized. This approach could contribute to evaluating the potential of TS as a therapeutic target in Chagas disease.},
}
@article {pmid42103069,
year = {2026},
author = {Vikal, A and Maurya, R and Kumar, P and Verma, N and Mishra, AK},
title = {Next-generation gene editing strategies in cancer: Integrating CRISPR, PROTACs, and advanced molecular technologies.},
journal = {Life sciences},
volume = {},
number = {},
pages = {124434},
doi = {10.1016/j.lfs.2026.124434},
pmid = {42103069},
issn = {1879-0631},
abstract = {Cancer is a significant therapeutic problem as tumors are heterogeneous, multidrug-resistant, and oncogenic drivers are undruggable. Genome editing and targeted protein degradation are emerging approaches that are transforming precision oncology by allowing genetic and proteomic interventions. Such technologies as CRISPR-based systems and proteolysis-targeting chimeras (PROTACs) are alternative methods of correcting genes and the selective removal of a protein. Their combination provides them with new possibilities regarding long-lasting and specific cancer treatment and discovery of new therapeutic targets. Although this has good news, there are delivery, off-target effects and safety challenges. The continued advancements in nanotechnology, artificial intelligence (AI), and personalized medicine will be likely to improve clinical translation. Generally, the integration of these technologies is an inducing trend in the treatment of cancer in the next generation.},
}
@article {pmid40231503,
year = {2026},
author = {Shams, F and Sharif, E and Abbasi-Kenarsari, H and Hashemi, N and Hosseini, MS and Heidari, N and Noori, E and Amini, AH and Bazrgar, M and Rouhani, M and Teng, Y},
title = {CRISPR/Cas9 Technology for Modifying Immune Checkpoint in CAR-T Cell Therapy for Hematopoietic Malignancies.},
journal = {Current gene therapy},
volume = {26},
number = {2},
pages = {1-29},
pmid = {40231503},
issn = {1875-5631},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Hematologic Neoplasms/therapy/immunology/genetics ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics/immunology ; Gene Editing ; Immune Checkpoint Inhibitors/therapeutic use ; T-Lymphocytes/immunology ; Programmed Cell Death 1 Receptor/genetics/immunology ; B7-H1 Antigen/genetics/immunology ; CTLA-4 Antigen/genetics/immunology ; },
abstract = {Hematologic malignancies, which arise from dysregulation of hematopoiesis, are a group of cancers originating in cells with diminished capacity to differentiate into mature progeny and accumulating immature cells in blood-forming tissues such as lymph nodes and bone marrow. Immune- targeted therapies, such as Immune Checkpoint Blockade (ICB), chimeric antigen receptor T (CAR-T) cell therapy, and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system, a precise, popular, and versatile genome engineering tool, has opened new avenues for the treatment of malignancies. Targeting immune checkpoints has revolutionized FDA approval in cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), PD-1 (programmed death-1), and PDL1. According to the ICB and CAR techniques, the production of efficient CAR-T cells depends on the successful genetic modification of T cells, making them less susceptible to immune escape and suppression by cancer cells, which results in reduced off-target toxicity. Therefore, CRISPR/Cas9 has revolutionized the immune checkpoint-based approach for CAR-T cell therapy of hematologic malignancy. Continued research and clinical trials will undoubtedly pave the way for further advances in this field, ultimately benefiting patients and improving outcomes.},
}
@article {pmid41109795,
year = {2026},
author = {Ghataora, JS and Ellis, T},
title = {Rewiring holobiont systems with synthetic biology.},
journal = {Trends in biotechnology},
volume = {44},
number = {5},
pages = {1312-1329},
doi = {10.1016/j.tibtech.2025.09.017},
pmid = {41109795},
issn = {1879-3096},
mesh = {*Synthetic Biology/methods ; *Microbiota/genetics ; Bacteria/genetics ; Biotechnology ; CRISPR-Cas Systems ; Biosensing Techniques ; },
abstract = {Holobionts are complex communities comprising a host and its diverse microbiota. Their intricate relationships are crucial for biological processes like nutrient uptake, immune function, and environmental adaptation. However, understanding these complex interactions remains challenging. We review how synthetic biology can help address these challenges. We highlight advances in bacterial biosensor design, engineering interkingdom communication, surface display, and clustered regularly interspaced short palindromic repeats (CRISPR) systems to both understand and manipulate holobiont interactions. We also highlight progress in engineering non-model microbiota members and provide rationale for a new field at the intersection of holobiont research and synthetic biology, which we term de novo holobiont design. The integration of synthetic biology with holobiont research promises to deepen our understanding of host-microbiota relationships and open new frontiers in biotechnology.},
}
@article {pmid41635086,
year = {2026},
author = {Castelli, JMP and Poljakov, K and Jwa, Y and Cunningham, R and Cassidy, ME and Gray, MD and Sanchez Gaytan, JN and Enstrom, MR and Gastelum, G and Wang, Z and Linton, JD and Rongvaux, A and Taylor, JJ and Adair, JE},
title = {In vivo production of an anti-HIV antibody in mice by non-viral gene knockin in primate hematopoietic stem and progenitor cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {5},
pages = {2754-2769},
pmid = {41635086},
issn = {1525-0024},
support = {D43 TW013052/TW/FIC NIH HHS/United States ; P30 AI027757/AI/NIAID NIH HHS/United States ; R01 AI158728/AI/NIAID NIH HHS/United States ; R01 AI167009/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Hematopoietic Stem Cells/metabolism/cytology ; Mice ; *HIV Antibodies/immunology/genetics/biosynthesis ; *Gene Knock-In Techniques ; CRISPR-Cas Systems ; Gene Editing/methods ; Humans ; *HIV Infections/immunology/therapy ; Hematopoietic Stem Cell Transplantation ; Primates ; Transgenes ; },
abstract = {Gene editing strategies that do not rely on viral vectors are being explored for their potential to support durable biologics production. While clinical trials have shown that adeno-associated virus encoding broadly neutralizing antibodies can protect against HIV, these interventions often yield limited, short-lived responses. The development of non-viral gene editing approaches in hematopoietic stem and progenitor cells holds promise for long-term antibody production. In this study, we evaluated CRISPR-Cas9 and CRISPR-Cas12a for gene knockin at the immunoglobulin heavy chain locus in non-human primate (NHP) hematopoietic stem and progenitor cells (HSPCs). Delivering the nuclease as a protein alongside a custom DNA template, we optimized editing with Cas12a and demonstrated higher knockin efficiency and fewer non-specific edits than with Cas9. Transplantation of edited NHP HSPCs into MISTRG mice led to engraftment, B cell differentiation, and transgene expression of a reporter transgene or anti-HIV antibody after gp120 antigen immunization with detectable titers in circulation. These findings demonstrate the feasibility of using non-viral knockin in HSPCs as a potential strategy for sustained biologics production in the treatment of chronic diseases. Future work will assess the efficacy of this approach in an NHP model of HIV infection.},
}
@article {pmid41856885,
year = {2026},
author = {Ahmar, S and Zhang, R and Pouramini, P and Janeczko, A and Shafique, MS and Rapacz, M and Reis, RS and Zhu, Q and Hensel, G and Pociecha, E},
title = {Precision harvest: path to genetically modified organism-free crops with CRISPR by 2035.},
journal = {Trends in plant science},
volume = {31},
number = {5},
pages = {719-730},
doi = {10.1016/j.tplants.2025.12.014},
pmid = {41856885},
issn = {1878-4372},
mesh = {*Crops, Agricultural/genetics ; *Plants, Genetically Modified/genetics ; *Gene Editing ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR) technology enable precise genetic modifications and produce genetically modified organism -free crops that match consumer preferences. By 2035, we will be able to consume CRISPR-edited crops, addressing food security issues and boosting economies for individual countries. This review highlights the progress of genetically modified crops and the regulatory challenges involved in bringing CRISPR-edited crops to market based on product- and process-based approaches across different regions. We also examine public preferences regarding these technologies and the current status of CRISPR-edited crops in terms of market availability. Furthermore, we stress the importance of establishing clear safety standards, effective patent management, and guidance on regulatory pathways for crop approval, as well as exploring future directions for integrating these technologies with artificial intelligence.},
}
@article {pmid41872289,
year = {2026},
author = {You, HJ and Kim, GY and Kang, MJ},
title = {CRISPR/Cas9-mediated targeted knock-in of human erythropoietin at the β-casein locus results in lactogenic hormone-responsive expression in HC11 mammary epithelial cells.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41872289},
issn = {2045-2322},
support = {RS-2024-00343478//National Research Foundation of Korea (NRF) grant awarded by the Korean government (MSIT)/ ; },
mesh = {*Caseins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Epithelial Cells/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; *Erythropoietin/genetics/metabolism ; Female ; *Mammary Glands, Animal/cytology/metabolism ; Cell Line ; Genetic Loci ; Gene Expression Regulation ; },
abstract = {Precise genomic integration strategies are essential for achieving stable and regulated transgene expression. In this study, we established a CRISPR/Cas9-mediated targeted knock-in system to integrate the human erythropoietin (hEPO) gene into the endogenous regulatory region of the mouse β-casein locus in HC11 mammary epithelial cells. A donor vector carrying hEPO was designed for homology-directed repair and successfully introduced into the β-casein locus. A heterozygous single-cell knock-in clone was isolated and validated by genomic analysis. Upon stimulation with lactogenic hormones, the integrated hEPO transgene exhibited hormone-responsive expression, resulting in an approximately 20-fold increase in protein levels compared to non-induced conditions. To enable downstream purification and activity assessment, a GST-tagged hEPO construct was employed. The fusion protein was purified using glutathione affinity chromatography, followed by proteolytic cleavage to obtain recombinant hEPO. The purified protein displayed measurable biological activity with a specific activity of 53.4 mIU/μg. These findings demonstrate that targeted integration at the β-casein locus results in lactogenic hormone-responsive expression driven by endogenous regulatory elements in mammary epithelial cells. This approach provides a controlled gene expression platform that may be applicable to hormone-inducible expression systems for therapeutic protein studies.},
}
@article {pmid41896732,
year = {2026},
author = {Herrera-Cardoso, ED and Tapia-Cervantes, KA and Cepeda-Negrete, J and Gutiérrez-Vargas, S and León-Galván, MF},
title = {Genome sequencing analysis reveals probiotic potential of Lactiplantibacillus plantarum IGMA4EH isolated from the gut of the white maguey worm (Aegiale hesperiaris).},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41896732},
issn = {1471-2164},
support = {CIIC 147/2023//Direcci&#xf3;n de Apoyo a la Investigaci&#xf3;n y al Posgrado of the Universidad de Guanajuato (DAIP UG)/ ; },
abstract = {BACKGROUND: The use of probiotics is increasingly popular for health applications, and Lactiplantibacillus plantarum strains are among the most widely studied for their potential in promoting gut health. In this study, we present the first genomic characterization of L. plantarum IGMA4EH, isolated from the gut of the maguey worm (Aegiale hesperiaris), an edible insect with traditional significance in Mexico.<br><br>METHODOLOGY: Whole-genome sequencing was performed on the L. plantarum IGMA4EH strain. Bioinformatics analyses were conducted to identify probiotic-related genes, antimicrobial potential, and safety features. Genes associated with resistance to environmental stressors, adhesion, and immunomodulation were screened, and safety assessment included the detection of antibiotic resistance genes, virulence factors, and plasmids.<br><br>RESULTS: The genomic analysis revealed the presence of multiple probiotic-related genes, including those associated with resistance to temperature, acidity, oxidative stress, and bile. Additionally, gene clusters related to bacteriocins, and secondary metabolites were identified, suggesting strong antimicrobial potential. Safety evaluations showed no evidence of acquired antibiotic resistance genes, virulence factors, or plasmids. Furthermore, elements related to genetic stability, such as CRISPR-Cas systems and prophage regions, were also detected.<br><br>CONCLUSIONS: The findings highlight the potential of L. plantarum IGMA4EH as a novel probiotic strain with promising applications.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-026-12798-5.},
}
@article {pmid42019854,
year = {2026},
author = {Farheen, J and Iqbal, MZ and Mustaq, A and Kong, X},
title = {A synergistic CRISPR-nano-immunotherapeutic system for targeted Bcl-2 silencing in breast tumour.},
journal = {International journal of biological macromolecules},
volume = {363},
number = {},
pages = {152114},
doi = {10.1016/j.ijbiomac.2026.152114},
pmid = {42019854},
issn = {1879-0003},
mesh = {*Breast Neoplasms/genetics/therapy/pathology/immunology ; Humans ; Female ; Animals ; *Proto-Oncogene Proteins c-bcl-2/genetics ; Cell Line, Tumor ; Mice ; *Immunotherapy/methods ; *Gene Silencing ; Apoptosis/genetics ; *CRISPR-Cas Systems ; Nanoparticles/chemistry ; Metal Nanoparticles/chemistry ; Mice, Inbred BALB C ; Gold/chemistry ; },
abstract = {B-cell lymphoma-2 (Bcl-2) protein has an extensive role in anti-cell death regulation and immuno-response modulation. It seizes apoptosis when Bcl-2 interacts and binds to Bax via its BH domain. Breast tumour (BT) was found to have overexpression of Bcl-2 coupled with mitochondrial membrane deprivation and stumpy immune response. Here, we developed a phyto-nanomedicine (HRP-MET)-based immunotherapeutic system integrating gold di‑manganese tri-oxide nanoparticles (GMNPs) with a BT-directed gene knockdown strategy. The nanoformulation is designed to accumulate in tumour tissue through enhanced permeability and retention (EPR)-mediated passive targeting, followed by activation within the tumour microenvironment. Initially, the expression of the Bcl-2 gene was selectively silenced in BT cells using a one-step advanced GenCRISPR™ Ultra NLS-Cas9 ribonucleoprotein (RNP) system with four designed effective sgRNAs (gene-CRISPR). Subsequently, transfected BT cells were treated with GMNP@HRP-MET phyto-nanomedicine in vitro and in vivo. As a result, this combinatorial strategy significantly induced tumour cell death via a mitochondria-mediated apoptotic signalling cascade (mitochondria → Bh3-only → Bax/Bak → Cycs → Apoptosome → Casp-9/Casp-3 → apoptosis) in various BT cells. Notably, Bcl-2 gene expression was prominently blocked in BALB/c female mice, accompanied by enhanced T-cell activation and sustained immune responses at both proteomic and transcriptomic levels. Furthermore, the gene-CRISPR and phyto-nanomedicine combination significantly inhibited tumour growth, migration, and distant organ metastasis in xenograft and syngeneic mice models. Collectively, this study demonstrates a practical and durable therapeutic strategy based on gene-CRISPR-enhanced apoptosis integrated with microenvironment-responsive phyto-nanomedicine for BT treatment.},
}
@article {pmid42094414,
year = {2026},
author = {Yoon, PH and Loi, K and Zhang, Z and Docter, TA and Lopez, SC and Langeberg, CJ and Ur-Rehman, MM and Vohra, K and Zhou, Z and Shi, H and Boger, R and Wang, PY and Adler, BA and Brohawn, SG and Doudna, JA},
title = {A Noncontiguous Code for RNA-Guided DNA Recognition Preceded CRISPR.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.04.26.720920},
pmid = {42094414},
issn = {2692-8205},
abstract = {CRISPR-Cas systems use RNA-guided proteins for adaptive immunity through a mechanism whose origin is unknown. Here we report the discovery of Viral Interference Programmable Repeat (VIPR) systems consisting of a Vipr protein more ancient than CRISPR-Cas and vrRNAs comprising alternating GGY/NN motifs. Unlike canonical guide RNAs that base pair with nucleic acid targets using an uninterrupted sequence, vrRNAs recognize double-stranded DNA through a noncontiguous code in which the variable NNs of each repeat collectively specify a target that itself contains a gapped recognition sequence. Analysis of natural vrRNA targets suggests VIPR acts against competing phages. We demonstrate programmable phage defense by redirecting the complex for transcriptional repression. These results suggest that the roots of adaptive immunity lie in ancient warfare between viruses, and reveal a new logic for programmable genetic control.},
}
@article {pmid42094579,
year = {2026},
author = {Malaluan, RPP and Dy, RLV},
title = {Acquisition of novel arrays via horizontal gene transfer rewire CRISPR-mediated defense in Pseudomonas aeruginosa.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.04.27.721218},
pmid = {42094579},
issn = {2692-8205},
abstract = {CRISPR-Cas systems form the adaptive immunity of prokaryotes, conferring sequence-specific protection against genetic parasites. Here, we functionally characterized the CRISPR-Cas system of Pseudomonas aeruginosa ATCC 10145 (PA10145), which led us to discover the existence of an isolated CRISPR array, unique to this system. PA10145 possesses a type I-F CRISPR-Cas composed of a cas operon flanked by two divergently organized CRISPRs. The isolated CRISPR array, CRISPR3, is located ∼1.3 million bp away from the cas loci. The cas and three CRISPR arrays are active. Plasmids with an engineered protospacer matching any of the three arrays were targeted and stimulated hyperactive adaptation in all CRISPR arrays of PA10145 if the plasmids possessed an intact protospacer adjacent motif (PAM), whereas minimal to no adaptation was observed when PAM was mutated. Spacer acquisition via interference-driven adaptation proceeds through strand-biased priming in PA10145. Interestingly, the isolated CRISPR3 and the cas -adjacent CRISPR2 have nearly identical leader sequences with only 3 bp mismatches. From a survey of CRISPR loci in 1,198 P. aeruginosa genomes, isolated arrays only occur as type I-F with similarly matching leaders to CRISPR2. Highly-transmissible mobile genetic elements (MGEs) associate with CRISPR2 and CRISPR3, suggesting that isolated arrays might have originated from recombination events involving CRISPR2 as facilitated by these MGEs. Tracing evolutionary trajectories of the isolated CRISPR3 relative to cas -adjacent arrays revealed that CRISPR3 is laterally transferred across P. aeruginosa genomes. Taken together, these results implicate the role of horizontally-acquired isolated arrays in CRISPR-mediated pan-immunity as gateways to mobilize genetic memories.},
}
@article {pmid42096148,
year = {2026},
author = {Zhu, D and Wang, S and Sun, X and Britton, RA},
title = {CRISPR-AsCas12a and dAsCas12a-Mediated Gene Knockout and Knockdown in Clostridioides difficile.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3046},
number = {},
pages = {47-55},
pmid = {42096148},
issn = {1940-6029},
mesh = {*Clostridioides difficile/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Gene Knockdown Techniques/methods ; Bacterial Proteins/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {Clostridioides difficile (C. difficile) is a leading cause of antibiotic-associated diarrhea and severe colitis, yet its genetic manipulation has long been constrained by low DNA transfer efficiency and limited recombination systems. Recent advances in CRISPR-based technologies have revolutionized the genetic toolkit for this pathogen, enabling precise genome editing and transcriptional regulation. Among CRISPR nucleases, Cas12a offers distinct advantages over Cas9 for bacterial applications, including a smaller size, T-rich PAM recognition, single-crRNA requirement, and reduced toxicity, which enhances conjugation efficiency in genetically recalcitrant organisms. AsCas12a-based platforms have enabled large fragment deletions, multiplex editing, and rapid generation of marker-free mutants in C. difficile. Complementing these nuclease-active systems, nuclease-deactivated variants (dCas9 or dAsCas12a) support CRISPR interference (CRISPRi)-a reversible, tunable approach for transcriptional repression without altering genomic sequences. Compared to traditional mutagenesis, CRISPRi greatly accelerates functional genomics by enabling high-throughput screening and drug target discovery. Together, our lab has independently developed CRISPR-AsCas12a-mediated genome editing and dAsCas12a-based CRISPRi tools, providing complementary strategies to overcome longstanding genetic barriers in C. difficile. These tools open new avenues for system-level interrogation of virulence, antibiotic resistance, and host-pathogen interactions.},
}
@article {pmid42096830,
year = {2026},
author = {Yin, Y and Wang, M and Sun, Y and Zhao, P and Ye, J},
title = {Sugarcane viral diseases: Epidemiology, detection, and advanced breeding methods for resistance.},
journal = {Virology},
volume = {621},
number = {},
pages = {110940},
doi = {10.1016/j.virol.2026.110940},
pmid = {42096830},
issn = {1096-0341},
abstract = {Sugarcane viruses threaten yield and sucrose content, imposing substantial global economic losses. This review provides current knowledge on the major sugarcane pathogens, with a focus on virus biology, transmission, and virus-host interactions, and uses this foundation to frame practical control strategies. We first describe the epidemiology and economic impact of sugarcane viral diseases to establish the urgency of robust surveillance and management. Building on these insights, we then evaluate detection and surveillance technologies, ranging from traditional immunoassays and nucleic acid hybridization to molecular diagnostics, isothermal amplification, and CRISPR-based approaches, and discuss how high-throughput sequencing accelerates virus discovery. Next, we assess management strategies that include virus-free planting material, vector control, sanitation, and modern breeding technologies, like RNA interference (RNAi), CRISPR/Cas genome editing, marker-assisted selection (MAS), and genome wide association studies (GWAS) within genomic selection (GS) frameworks. We also consider supplementary strategies such as plant-derived antivirals and environmental management, and discuss how they complement core approaches. Crucially, we identify key knowledge gaps in sugarcane virus-host interactions and resistance breeding, and propose data-driven, multi-omics and AI-assisted approaches to precision breeding and integrated disease management. The review concludes with a practical roadmap for advancing sugarcane virus control and promoting sustainable production.},
}
@article {pmid42097051,
year = {2026},
author = {Liao, J and Rima, J and Sharma, A and Tsade, J and Jiang, F},
title = {Machine learning-enabled smartphone CRISPR-Cas12a lateral flow platform for sensitive detection of circulating HPV DNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {307},
number = {},
pages = {118765},
doi = {10.1016/j.bios.2026.118765},
pmid = {42097051},
issn = {1873-4235},
abstract = {Persistent infection with high-risk human papillomavirus (HPV) is a major cause of cervical cancer, and improved point-of-care (POC) detection is critical for early intervention. Although PCR-based assays are highly sensitive, their reliance on centralized laboratory infrastructure limits accessibility in decentralized settings. CRISPR-Cas diagnostics combined with lateral flow assays (LFA) offer a rapid alternative; however, visual interpretation of faint test bands remains subjective and variable. Here, we developed a smartphone-based CRISPR-Cas12a LFA platform integrated with an interpretable machine learning (ML) framework for quantitative detection of circulating HPV DNA in plasma. Standardized image acquisition was implemented using a light-controlled enclosure, and radiomics-inspired features were analyzed using a multivariable logistic regression model. The system was trained on 150 plasma samples and validated in an independent cohort of 60 samples. The optimized model achieved 96.7% sensitivity and 100% specificity, outperforming visual interpretation, particularly for low-signal samples. Performance remained stable across different smartphone models, lighting conditions, and operators, with rapid on-device inference enabling consistent and reliable operation. This integrated CRISPR-LFA platform demonstrates accurate and reproducible detection of circulating HPV DNA and supports feasibility for POC applications, pending further validation in broader clinical settings.},
}
@article {pmid42097949,
year = {2026},
author = {Fuller, MGA and Foley, M and Barrangou, R},
title = {CRISPR-based technologies for large DNA insertions.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.04.004},
pmid = {42097949},
issn = {1879-3096},
abstract = {While the advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based technologies has democratized the genesis of precise mutations, there is a need for more sophisticated tools to enable large-scale DNA manipulations, advancing genome editing across medicine, biotechnology, and agriculture. The success of Cas9 and Cas12 has hinged on the generation of precise DNA nicks and double-stranded breaks (DSBs), enabling local sequence mutagenesis, albeit of a limited size range. Emerging effectors combining Cas with other enzymatic functions, such as CRISPR-associated transposons and site-specific recombinases, enable larger integrations. Sophisticated combinations such as programmable addition via site-specific targeting element (PASTE), prime-editing-assisted site-specific integrase gene editing (PASSIGE), and prime-editing-mediated recombination of opportune target (PrimeRoot) expand payload options and DSB-free editing modalities, with translational potential for next-generation crop breeding in sustainable agriculture and the development of gene and cell therapies in personalized medicine.},
}
@article {pmid42098118,
year = {2026},
author = {Gierisch, ME and Barchi, E and Marogna, M and Wallnöfer, MH and Ankarcrona, M and Naia, L and Salomons, FA and Dantuma, NP},
title = {Mitochondria serve as a holdout compartment for aggregation-prone proteins hindering efficient degradation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42098118},
issn = {2041-1723},
support = {2021-02562//Vetenskapsr&#xe5;det (Swedish Research Council)/ ; 211653Pj//Cancerfonden (Swedish Cancer Society)/ ; FO2022-0271//Hj&#xe4;rnfonden (Swedish Brain Foundation)/ ; FO2023-0376//Hj&#xe4;rnfonden (Swedish Brain Foundation)/ ; KID grant//Karolinska Institutet (Karolinska Institute)/ ; GI-1329/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Mitochondria/metabolism ; Humans ; Huntingtin Protein/metabolism/genetics ; Proteolysis ; alpha-Synuclein/metabolism/genetics ; Proteasome Endopeptidase Complex/metabolism ; HEK293 Cells ; *Protein Aggregates ; Neurodegenerative Diseases/metabolism/genetics ; CRISPR-Cas Systems ; Protein Aggregation, Pathological/metabolism ; },
abstract = {The accumulation of protein aggregates has been causatively linked to the pathogenesis of neurodegenerative diseases. Here, we conduct a genome-wide CRISPR-Cas9 screen to identify cellular factors that regulate the degradation of an aggregation-prone reporter. Genes encoding proteins involved in mitochondrial homeostasis, including the translation factor eIF5A, are enriched among suppressors of the degradation of the reporter. Genetic or chemical inhibition of eIF5A leads to dissociation of the aggregation-prone substrate from mitochondria, which is accompanied by enhanced ubiquitin-dependent proteasomal degradation. The presence of an aggregation-prone, amphipathic helix that localizes the reporter to mitochondria is crucial for the stimulatory effect of eIF5A inhibition on proteasomal degradation. Additionally, inhibition of eIF5A also enhances degradation of mutant huntingtin and α-synuclein, two disease-associated proteins that contain amphipathic helices and mislocalize to mitochondria. We propose that mitochondria serve as a holdout compartment for aggregation-prone proteins. Therefore, preventing mitochondrial localization of aggregation-prone proteins may offer a viable therapeutic strategy for reducing disease-associated proteins in neurodegenerative disorders.},
}
@article {pmid42098301,
year = {2026},
author = {He, L and Na, H and Zhang, Y and Gong, P and Zhou, H and Wan, F},
title = {Ultrasensitive Detection and Its Potential Applications in the Diagnosis of Brucellosis.},
journal = {Current microbiology},
volume = {83},
number = {6},
pages = {},
pmid = {42098301},
issn = {1432-0991},
support = {2024LHMS08055//Natural Science Foundation of Inner Mongolia Autonomous Region/ ; 2025MS08058//Natural Science Foundation of Inner Mongolia Autonomous Region/ ; 2025---14//Hohhot Basic and Applied Basic Research Science and Technology Plan Project/ ; DC2000000754//the Prairie Talent Leadership Program/ ; BR22-14-02//the Basic Scientific Research Funds for Inner Mongolia Autonomous Region Government/ ; },
mesh = {*Brucellosis/diagnosis/microbiology ; Humans ; Animals ; *Brucella/isolation &amp; purification/genetics ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Biosensing Techniques/methods ; Zoonoses/diagnosis/microbiology ; Sensitivity and Specificity ; },
abstract = {Brucellosis is a major zoonotic disease caused by Brucella spp., notably B. melitensis, B. abortus, B. canis, and B. suis. It continues to impose a significant public health and economic burden in endemic regions, including parts of Asia, Africa, the Middle East, and Latin America. The clinical presentation of the disease is often nonspecific, and early, accurate diagnosis is further complicated by the susceptibility of conventional serological assays to cross-reactivity. This narrative review covers literature published between 2000 and 2025 regarding the detection of Brucella, indexed in PubMed, Web of Science, and China National Knowledge Infrastructure (CNKI). The review focuses on the limitations of traditional diagnostic methods and the performance characteristics and latest developments of emerging ultrasensitive detection technologies, including nucleic acid amplification (LAMP, RPA, SRCA), droplet digital PCR, CRISPR-Cas-based integrated systems, nanomaterial-enhanced assays, and protein/antigen-based biosensors. Their potential for integration into the One Health framework for zoonotic disease surveillance is also discussed.},
}
@article {pmid41447548,
year = {2026},
author = {Hefferon, K and Venkataraman, S and Alok, A and Moiketsi, BN and Malik, S and Masisi, K and Rantong, G and Kwape, T and Gaobotse, G and Makhzoum, A},
title = {CRISPR-Cas9 editing of agricultural crops and medicinal plants: toward a cornucopia of natural products.},
journal = {Critical reviews in biochemistry and molecular biology},
volume = {61},
number = {1-3},
pages = {1-16},
doi = {10.1080/10409238.2025.2577956},
pmid = {41447548},
issn = {1549-7798},
mesh = {*Plants, Medicinal/genetics/metabolism ; *Gene Editing/methods ; *Crops, Agricultural/genetics/metabolism ; *CRISPR-Cas Systems ; *Biological Products/metabolism ; Humans ; Plants, Genetically Modified/genetics/metabolism ; },
abstract = {Plants have been a part of human health since our very beginnings, and many of our modern pharmaceuticals claim their origins from medicinal plants. The range of specialized metabolites synthesized by plants is highly diverse, and metabolic functions have developed over the millennia to cover roles such as defense, adaptation to environmental stress, and even reproduction. These metabolites subsequently play roles in human health and diseases that are both significant and profound. The importance of plant natural products for the pharmaceutical, cosmetic and nutraceutical industries cannot be overstated. However, the fact that these specialized metabolites may be available only in low quantities from plants that are slow growing, endangered, or from fragile environments due to certain biotic and abiotic stresses makes their commercial use challenging despite the scenario that some stresses can enhance the production of secondary metabolites. Genome editing is a technique or technology that comprises of tools like CRISPR/Cas9, TALEN, ZFN. The following review describes the successful use of CRISPR/Cas9 genome editing in engineering medicinal plants, food crops and commercial crops to modulate metabolic pathways involved in the biosynthesis of valuable compounds to improve natural product identification, development and ultimately, commercial viability.},
}
@article {pmid41712409,
year = {2026},
author = {Nenad, WC and Kuhlers, PC and Sturgill, IR and Biju, I and Bucklan, M and Hernandez, L and Zhu, LC and Hoadley, KA and Raab, JR},
title = {Hepatocyte-targeted Bap1 reduction in the liver primes an inflammatory transcriptional response.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {5},
pages = {},
doi = {10.1093/g3journal/jkag047},
pmid = {41712409},
issn = {2160-1836},
support = {/GM/NIGMS NIH HHS/United States ; },
mesh = {*Ubiquitin Thiolesterase/genetics/metabolism ; Animals ; *Hepatocytes/metabolism ; *Tumor Suppressor Proteins/genetics/metabolism ; Mice ; *Liver/metabolism/pathology ; *Inflammation/genetics/metabolism/pathology ; Mice, Knockout ; Transcriptome ; CRISPR-Cas Systems ; *Transcription, Genetic ; Gene Expression Profiling ; },
abstract = {BRCA1-associated protein 1 (BAP1) is a deubiquitinase, frequently altered in cancers including hepatocellular carcinoma and cholangiocarcinoma. While Bap1 has been shown to play key roles in metabolism, maintenance of tissue homeostasis, and immune cell development, little is known about its normal functions in the liver in vivo. Using AAV8-mediated CRISPR/CAS9 genome editing, we generated a mouse hepatocyte-specific model of Bap1 knockout to define the changes that occur in liver biology in an in vivo system and characterize how loss of Bap1 alters the liver's response to injury. Single-cell resolution spatial transcriptomics were performed in conjunction with immunohistochemistry to analyze cell-type composition and immune cell recruitment changes. Bulk RNA-sequencing was performed to further assess the impact of Bap1 loss on transcription. Hepatocyte-specific depletion of Bap1-induced transcriptional changes shared with acute injury. We observed a strong dysregulation of inflammatory pathways associated with Bap1 loss. Moreover, the transcriptional response of Bap1 depletion in hepatocytes to damage was markedly different than in control liver, with Bap1-depleted livers showing a decreased hepatocyte identity based on gene expression. Spatial transcriptomics and quantitative texture analysis of immunohistochemistry revealed an altered immune environment prior to damage and an impaired recruitment of immune cells in Bap1-depleted livers after damage. Our data suggest Bap1 is a critical modulator in the liver's immune cell response and its loss leads to an inflammatory environment prior to damage and disrupts the recruitment immune cells. Our quantitative spatial analysis highlights the power of such approaches to characterize the spatial distribution of different cell types in a tissue.},
}
@article {pmid41742729,
year = {2026},
author = {Krupa, LS and Villamayor, PR and Bandara, S and Zhang, Y and Palmiotti, A and von Lintig, J and Bendesky, A},
title = {Retinoic acid production via the ray-finned fish gene bco1l is essential for juvenile development.},
journal = {Genetics},
volume = {233},
number = {1},
pages = {},
pmid = {41742729},
issn = {1943-2631},
support = {R35 GM143051/GM/NIGMS NIH HHS/United States ; R35GM143051/NH/NIH HHS/United States ; EY020551/GF/NIH HHS/United States ; EY028121/GF/NIH HHS/United States ; //Columbia University SURF program/ ; },
mesh = {Animals ; *beta-Carotene 15,15'-Monooxygenase/genetics/metabolism ; *Tretinoin/metabolism ; *Fish Proteins/genetics/metabolism ; beta Carotene/metabolism ; Vitamin A/metabolism ; *Fishes/genetics/growth &amp; development/metabolism ; CRISPR-Cas Systems ; },
abstract = {In vertebrates, vitamin A (VA) is crucial for development, tissue homeostasis, vision, and immunity. Retinal, a form of VA, can be produced via enzymatic cleavage of β-carotene by beta-carotene oxygenase 1 (bco1) and bco1-like (bco1l), but the developmental and tissue-specific functions of these genes are poorly understood. While bco1 is found across vertebrate taxa, bco1l is a paralog of bco1 that we discover to have evolved in the ray-finned fishes, the most abundant, speciose, and commercially important group of fishes. We investigated the function of bco1l in ray-finned Siamese fighting fish, commonly known as betta, an emerging model for genetics and development. Using CRISPR/Cas9 knockouts, we find that lack of bco1l results in reduced VA and elevated β-carotene in larvae, starting when animals have exhausted their yolk supply of retinal, followed by stunted growth and death during juvenile development. Exogenous retinoic acid largely rescues the mutation, demonstrating its deficiency causes these defects. bco1l is 7× more abundant than bco1 in the intestine. This, coupled with the inability of bco1 to sustain VA production in the bco1l mutant, indicates that bco1l is the primary enzyme for dietary carotenoid conversion into retinal. Our results show that VA production by bco1l is required for post-embryonic development, and that bco1l became essential after evolving via duplication of bco1.},
}
@article {pmid41856683,
year = {2026},
author = {Wang, Y and Guo, Y and Lu, Q and Liu, X and Xu, H and Chen, J and Pi, R and Yuan, S and Yang, Z and Lu, R and Meng, FL and Gan, T and Hu, J},
title = {Restoring the potency of a neutralizing antibody via guided hypermutation with hyper-antibody editor HAE1.},
journal = {Genome research},
volume = {36},
number = {5},
pages = {1029-1039},
doi = {10.1101/gr.281396.125},
pmid = {41856683},
issn = {1549-5469},
mesh = {Humans ; HEK293 Cells ; *Antibodies, Neutralizing/genetics/immunology ; *Somatic Hypermutation, Immunoglobulin ; *SARS-CoV-2/immunology ; COVID-19/immunology/virology ; CRISPR-Cas Systems ; *Antibodies, Viral/genetics/immunology ; Cytidine Deaminase/genetics ; Mutation ; },
abstract = {Somatic hypermutation (SHM) drives antibody affinity maturation in B cells. By mimicking this process, guided hypermutation (GHM) tools employing CRISPR systems and activation-induced cytidine deaminase (AID) have advanced antibody development. However, GHM-induced mutations in cultured cells exhibit mutation patterns distinct from those observed in natural antibody diversification following in vivo affinity selection. To address this, we engineer a hyper-antibody editor, HAE1, by integrating cytidine and adenine deaminases with a nicked, PAMless Cas9 variant, SpRY, to closely resemble the mutation spectrum of natural SHM. Moreover, to streamline mutation, selection, and validation within the same cells, we develop a dual-expression system in HEK293F cells that allows simultaneous expression of both transmembrane and secreted full-length antibodies. Using this system, we apply HAE1 to the SARS-CoV-2 neutralizing antibody CV07-209 and restore the antibody's binding affinity and neutralization potency against Omicron variants, specifically BA.1, including at least one mutation beyond the reach of current GHM tools. HAE1 thus provides a versatile, high-throughput strategy for expediting antibody evolution, presenting significant potential for therapeutic antibody development and protein engineering.},
}
@article {pmid41865104,
year = {2026},
author = {Yuasa, T and Nakagawa, T and Honda, T and Nishiuchi, G and Sato, M and Tokunaga, A and Nakahara, M and Tourtas, T and Schlötzer-Schrehardt, U and Kruse, F and Padmanabhan, P and Chatterjee, A and Sathe, G and Ghose, V and Janakiraman, N and Koizumi, N and Elchuri, SV and Okumura, N},
title = {TCF4 trinucleotide repeat expansion drives distinct proteomic signatures in Fuchs endothelial corneal dystrophy.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41865104},
issn = {2045-2322},
support = {EMR/2015/000607//Science and Engineering Research Board/ ; KAKENHI 18K09464//Japan Society for the Promotion of Science/ ; },
mesh = {Humans ; *Fuchs' Endothelial Dystrophy/genetics/metabolism/pathology ; *Transcription Factor 4/genetics/metabolism ; *Trinucleotide Repeat Expansion ; Proteomics/methods ; *Proteome/genetics ; Protein Interaction Maps ; CRISPR-Cas Systems ; },
abstract = {The aims of this study were to use an isogenic cell model system to investigate the proteomic consequences of TCF4 trinucleotide repeat expansion in Fuchs endothelial corneal dystrophy (FECD) and to identify potential molecular pathways contributing to disease pathogenesis. We used our previously established immortalized FECD cells (iFECD) that harbor CTG repeat expansion in TCF4 and CRISPR/Cas9 genome editing to generate an isogenic counterpart (iFECD TCF4ΔCTG) in which the expansion was deleted. Comprehensive proteomic analysis was then performed using tandem mass tag (TMT)-labeled quantitative LC-MS/MS. The resulting data were subjected to differential expression analysis, functional enrichment analysis, and protein-protein interaction network construction to elucidate the molecular impact of the CTG repeat expansion. Deletion of the CTG repeat expansion significantly altered the corneal endothelial proteome, with 90 upregulated and 111 downregulated proteins (|log2 fold change| ≥ 0.5, P-value < 0.05). Functional enrichment analysis revealed that the downregulated proteins were predominantly associated with extracellular matrix organization and cell-substrate adhesion pathways, while the upregulated proteins were enriched in interferon signaling and antigen-processing pathways. The most significantly upregulated proteins included neuropilin-1, Cip1-interacting zinc finger protein, and protein-glutamine gamma-glutamyltransferase 2, while protein phosphatase 1 regulatory subunit 14 C, alpha-crystallin B chain, and 14-3-3 protein sigma showed the greatest downregulation. Our findings demonstrate that TCF4 CTG repeat expansion significantly impacts the corneal endothelial proteome, particularly affecting the extracellular matrix and cell adhesion proteins that likely contribute to guttae formation. These proteomic alterations provide mechanistic insights connecting trinucleotide repeat expansion to endothelial dysfunction and suggest potential therapeutic targets for FECD.},
}
@article {pmid41871879,
year = {2026},
author = {Lutz, S and Lawler, M and Amidon, S and Albert, FW},
title = {High-resolution genotype-free mapping of genetic variation with CRI-SPA-Map.},
journal = {Genome research},
volume = {36},
number = {5},
pages = {1016-1028},
doi = {10.1101/gr.281514.125},
pmid = {41871879},
issn = {1549-5469},
support = {R01 HG014395/HG/NHGRI NIH HHS/United States ; R35 GM124676/GM/NIGMS NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *Chromosome Mapping/methods ; *Genetic Variation ; CRISPR-Cas Systems ; Genotype ; Genome, Fungal ; Phenotype ; Chromosomes, Fungal ; },
abstract = {Genetic variation within species shapes phenotypes, but identifying the specific genes and variants that cause phenotypic differences is costly and challenging. Here, we introduce CRI-SPA-Map, a genetic mapping strategy combining CRISPR-Cas9 genome engineering, selective ploidy ablation (SPA), and high-throughput phenotyping for precise genetic mapping with or without genotyping in the yeast Saccharomyces cerevisiae In CRI-SPA-Map, a donor strain carrying SPA machinery is mated to a genetically different recipient strain harboring a genome-integrated selectable cassette. In the resulting diploid, CRISPR-Cas9 cuts the cassette for replacement with DNA from the homologous donor chromosome. Donor chromosomes are then removed using SPA to yield haploid recombinant strains. To establish CRI-SPA-Map, we mate a W303 SPA strain to 92 strains from the BY4742 yeast knockout collection that carry gene deletion cassettes on the left arm of Chromosome XIV and create 1451 recombinant isolates. Whole-genome sequencing verifies that deletion cassette replacement introduces short donor DNA tracts of variable length, resulting in a finely recombined mapping population. Using only the known locations of the gene deletions, which mark where donor DNA is introduced, we identify a 6.5 kb region shaping yeast growth. We further dissect this region and identify two causal variants in two genes, MKT1 and SAL1 Engineering these variants alone and in combination reveals gene-by-environment interactions at both genes, as well as epistatic interactions between them that are dependent on the environment. CRI-SPA-Map is a cost-effective, meiosis-free strategy for creating high-resolution recombinant panels of yeast strains for identifying the genetic basis of phenotypic variation.},
}
@article {pmid41931965,
year = {2026},
author = {Li, X and Zong, X and Yuan, P and Yan, X and Yang, C and Chen, X and Wei, S and Wen, Y and Du, J and Liu, X and Liu, F and Dai, J},
title = {An epigenetically enhanced whole-cell vaccine in a stimulatory hydrogel for robust antitumor immunity.},
journal = {Biomaterials},
volume = {333},
number = {},
pages = {124173},
doi = {10.1016/j.biomaterials.2026.124173},
pmid = {41931965},
issn = {1878-5905},
mesh = {Animals ; *Hydrogels/chemistry ; *Epigenesis, Genetic ; *Cancer Vaccines/immunology ; Mice ; Mice, Inbred C57BL ; Humans ; *Neoplasms/immunology/therapy ; Antigen Presentation ; Cell Line, Tumor ; Immunotherapy ; Female ; CRISPR-Cas Systems ; Histocompatibility Antigens Class I/genetics/immunology ; Antigens, Neoplasm/immunology ; },
abstract = {Inadequate antigen presentation, driven by epigenetic repression of major histocompatibility complex class I (MHC-I), represents a fundamental barrier to effective cancer immunotherapy. Here, we identify polycomb group ring finger 1 (PCGF1) as a tumor cell-intrinsic epigenetic repressor of MHC-I through a genome-wide CRISPR screen. Genetic ablation of PCGF1 alone is sufficient to relieve repressive histone modifications (H2AK119ub and H3K27me3) at both the MHC-I gene cluster and its master regulator NLRC5, thereby restoring cell-surface antigen presentation and immunotherapy sensitivity. Building on this epigenetic foundation, we introduce modular engineering strategies to enhance translational robustness against tumor antigen heterogeneity. Specifically, CRISPR activation (CRISPRa) is used to broaden the repertoire of endogenous tumor antigens without altering the restored antigen presentation machinery. These epigenetically reprogrammed cells are subsequently cryoinactivated and formulated into an injectable thermosensitive chitosan-based hydrogel, in which manganese-mediated STING activation serves as an immunostimulatory adjuvant to amplify antigen capture and systemic T-cell priming.Collectively, this study establishes epigenetic reprogramming of antigen presentation as a foundational principle for whole-cell vaccine design and demonstrates how modular antigen and innate immune augmentation can enhance therapeutic robustness without obscuring the core mechanism. This platform offers a rational and adaptable framework for overcoming immune resistance in next-generation cancer immunotherapy.},
}
@article {pmid41980379,
year = {2026},
author = {Liu, X and Wang, C and Wang, W and Chen, Z and Luo, M and Yang, R and Deng, H},
title = {In situ thermosensitive mRNA-loaded hydrogel modulates post-surgery tumor immune microenvironment to prevent recurrence and metastasis.},
journal = {Biomaterials},
volume = {333},
number = {},
pages = {124188},
doi = {10.1016/j.biomaterials.2026.124188},
pmid = {41980379},
issn = {1878-5905},
mesh = {*Tumor Microenvironment/immunology/drug effects ; *Hydrogels/chemistry ; Animals ; Mice ; Nanoparticles/chemistry ; Macrophages/immunology ; Reactive Oxygen Species/metabolism ; *RNA, Messenger/administration &amp; dosage/chemistry ; Cell Line, Tumor ; Female ; *Neoplasm Recurrence, Local/prevention &amp; control/immunology ; Humans ; Neoplasm Metastasis/prevention &amp; control ; Temperature ; CRISPR-Cas Systems ; },
abstract = {Surgery remains the primary cancer treatment, but postoperative trauma disrupts the local immune microenvironment by altering the critical balance between M1 and M2 macrophages while simultaneously elevating reactive oxygen species (ROS) levels at the surgical site. The critical clinical dilemma in postoperative tumor management lies in achieving re-balancing over macrophage polarization within the post-operative niche - specifically maintaining tumor-suppressing M1 phenotype while permitting necessary wound-healing M2 function. Here, we developed an in situ thermosensitive hydrogel platform capable of co-delivering two nanoparticle systems (BC12D NPs and PPS NPs) to alleviate the immunosuppressive microenvironment. Specifically, CRISPR/Cas9-loaded nanoparticles (BC12D@CRISPR NPs) were incorporated into the hydrogel for addressing the high proportion of M2-type macrophages at the resection site, reprogramming the macrophages with an effective M1/M2 ratio to exert potent antitumor functions. Meanwhile, the PPS nanoparticles were employed for the clearance of ROS at the surgical site, thereby ensuring that the normal wound healing process remained unimpeded. Using an in situ tumor resection model, the synergistic effects of ROS clearance and macrophage repolarization at the postoperative site were leveraged to achieve efficient immune microenvironment modulation.},
}
@article {pmid42067223,
year = {2026},
author = {Andres-Lopez, Y and Santambrogio, A and Kafetzopoulos, I and Todd, CD and El Khouri-Gonzalez, C and Gonzalez-Alvarez, JE and Alda-Catalinas, C and Clark, SJ and Reik, W and Hernando-Herraez, I},
title = {Using CRISPR barcoding as a molecular clock to capture dynamic processes at single-cell resolution.},
journal = {Genome research},
volume = {36},
number = {5},
pages = {1005-1015},
doi = {10.1101/gr.280915.125},
pmid = {42067223},
issn = {1549-5469},
mesh = {Animals ; *Single-Cell Analysis/methods ; Mice ; *CRISPR-Cas Systems ; Mouse Embryonic Stem Cells/metabolism/cytology ; *DNA Barcoding, Taxonomic/methods ; Mutation ; Transcriptome ; },
abstract = {Biological processes are inherently dynamic, yet current methods for capturing temporal changes remain limited. Here, we present scDynaBar, a novel approach that combines CRISPR-Cas9 dynamic barcoding with single-cell sequencing. In this system, genetic barcodes gradually accumulate mutations over time; these barcodes are sequenced alongside the transcriptome of individual cells. We propose that the divergence of these barcodes from the original sequence can serve as a record of the timing of cellular events. To demonstrate the potential of this method, we track the transition from a pluripotent state to a two-cell (2C)-like state in mouse embryonic stem cells (mESCs), providing evidence for the transient nature of the 2C-like state. Additionally, our system shows consistent mutation rates across diverse cell types in a mouse gastruloid model, highlighting its applicability to other biological systems. This approach not only improves our ability to study single-cell dynamics but also opens up new possibilities for recording other temporal signals-in other words, using dynamic barcoding as a molecular clock in individual cells.},
}
@article {pmid42087786,
year = {2026},
author = {Ramongolalaina, C and Pastor-Pareja, JC and Zhang, E and Jia, Y},
title = {Optimized optogenetic anti-CRISPR for endogenous gene regulation in Drosophila.},
journal = {Nucleic acids research},
volume = {54},
number = {9},
pages = {},
doi = {10.1093/nar/gkag244},
pmid = {42087786},
issn = {1362-4962},
support = {YB202212280503//Tsinghua-Peking Center of Life Sciences/ ; 100401473//Tsinghua-Peking Center of Life Sciences/ ; 32150710524//J. C. Pastor-Pareja/ ; PID2021-122119NB-I00//National Natural Science Foundation of China/ ; //Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; CEX2021-001165-S//"Severo Ochoa" Program for Centers of Excellence/ ; 2025-I-ZD-004//"Severo Ochoa" Program for Centers of Excellence/ ; 2025-O-ZD-004//"Severo Ochoa" Program for Centers of Excellence/ ; //State Key Laboratory of Complex, Severe, and Rare Diseases/ ; 2025B-07-08//Changping Laboratory/ ; //SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; },
mesh = {Animals ; *Optogenetics/methods ; *Gene Expression Regulation/radiation effects ; Light ; *CRISPR-Cas Systems ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics ; *Drosophila/genetics ; },
abstract = {Optogenetic tools-light-responsive proteins that enable to regulate specific cellular activities, study biological processes, and develop new therapies-are attractive approaches for achieving endogenous gene regulation under minimally invasive conditions. Our first step in constructing an optogenetic system to regulate endogenous Drosophila gene expression was to identify inhibitory anti-CRISPR (Acr) proteins that block CRISPRa-mediated activation. Next, we inserted optogenetic protein LOV2 into these Acrs, tested for their ability to optogenetically modulate endogenous gene upregulation through the CRISPRa-based flySAM system in Drosophila, and found that the photoswitchability of these prototypes was weak. We therefore engineered an optimized Acr-LOV2 fusion module by refining length of intrinsically disordered and ordered regions (IDR and IOR) of Acrs. This optimization yielded a variant with significantly greater sensitivity to blue-light-induced endogenous gene upregulation than the prototypes, leading to new in vivo discoveries. In addition, this work provides insights for in vivo functional characterization of the IDR and the IOR of these small-sized proteins. Together, these findings establish a robust optogenetic toolbox for precise, light-controlled endogenous gene regulation in Drosophila.},
}
@article {pmid42088606,
year = {2026},
author = {Fujii, T and Sakoda, Y and Yoshimi, K and Takeshita, K and Watanabe, S and Iida, R and Obo, T and Yokoyama, K and Tamada, K and Mashimo, T},
title = {Efficient gene disruption with CRISPR-Cas3 in human T cells.},
journal = {NAR cancer},
volume = {8},
number = {2},
pages = {zcag009},
pmid = {42088606},
issn = {2632-8674},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *T-Lymphocytes/immunology/metabolism ; *Gene Editing/methods ; *beta 2-Microglobulin/genetics ; Graft vs Host Disease/genetics/immunology ; Immunotherapy, Adoptive/methods ; Gene Deletion ; },
abstract = {The CRISPR-Cas9 system has been widely adopted as a genome editing tool due to its high efficiency and versatility, contributing to the development of various therapeutic strategies. However, its clinical application remains limited by safety concerns, including off-target effects and large-scale chromosomal rearrangements such as translocations and inversions. Recently, the CRISPR-Cas3 system, a Class 1 CRISPR effector complex with unidirectional DNA degradation activity, has gained attention as a potential alternative, offering reduced off-target activity. In this study, we applied the CRISPR-Cas3 system to human T cells and successfully disrupted two clinically relevant genes, T cell receptor alpha constant (TRAC) and beta-2 microglobulin (B2M). These gene deletions were associated with a reduction in both graft-versus-host disease risk and host immune rejection. Importantly, no off-target mutations were detected in CRISPR-Cas3-edited cells, in contrast to the off-target effects observed with CRISPR-Cas9. Furthermore, CAR-T cells generated by deleting TRAC or B2M using CRISPR-Cas3 maintained their antigen-specific cytotoxicity against tumor cells, while exhibiting reduced alloreactivity. These results suggest that CRISPR-Cas3 provides a safer and promising platform for genome editing in T cell engineering, with potential applications in the development of next-generation allogeneic T cell therapies.},
}
@article {pmid42089305,
year = {2026},
author = {Kooistra, T and Sarraf, TR and Chen, M and Gally, CK and Medoff, BD},
title = {Efficient CRISPR-Cas RNP-based gene targeting of human AT2 cells.},
journal = {American journal of respiratory cell and molecular biology},
volume = {},
number = {},
pages = {},
doi = {10.1093/ajrcmb/aanag062},
pmid = {42089305},
issn = {1535-4989},
abstract = {Alveolar type 2 (AT2) cells play numerous roles in the alveolus related to stem cell, immunoregulatory, and secretory functions. Primary human AT2 cells can now be isolated and studied as organoids consisting of self-organizing epithelial tissues as pure populations without the need for stromal support cells. However, genetic manipulation of AT2 cells to investigate their biology has relied on expensive and time-consuming processes requiring the use of viral vectors or conducting gene editing with induced pluripotent stem cells (iPSCs)-derived AT2 cells. Here we describe a high-efficiency method of accomplishing highly effective gene editing in cultured primary human AT2 cells, which can be done rapidly and at significantly lower costs. Using an optimized CRISPR ribonucleoprotein (RNP) approach, we can achieve nearly complete genetic knockout while preserving AT2 identity and viability. Our results simplify the process of genetically manipulating human AT2 cells to better understand the role of the alveolar epithelium in human lung biology.},
}
@article {pmid42090038,
year = {2026},
author = {Jaffal, S and Jaffal, G},
title = {Genetic modulation of pain pathways: toward a new era in pain therapy- a systematic review.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42090038},
issn = {1573-4978},
mesh = {Humans ; *Pain Management/methods ; *Genetic Therapy/methods ; *Pain/genetics ; CRISPR-Cas Systems/genetics ; RNA Interference ; Animals ; Gene Transfer Techniques ; },
abstract = {Gene therapy represents a new strategy in pain management targeting the causes of pain rather than its symptoms. This review summarizes recent advances across RNA interference (RNAi), viral vector delivery systems, CRISPR/Cas9, gene replacement therapy, and endogenous opioid gene delivery, emphasizing efficacy, safety, and mechanisms of action in pain conditions. RNAi and gene replacement techniques remain powerful tools for reducing pain and improving the quality of life by modulating pain-associated genes. Long-term relief may also be achieved with CRISPR/Cas9 and site-directed delivery using nanoparticle systems. Safety profiles especially with CRISPR/Cas9 remains a concern. A systematic search of Web of Science, Medline, Scopus, and Google Scholar identified 512 records (January 2010-March 2023). Following PRISMA screening, 18 studies met inclusion criteria. These studies evaluated strategies of gene therapy in neuropathy, arthritis, fibromyalgia, and complex regional pain syndrome. The review outlines available options and highlights preclinical findings.},
}
@article {pmid42090053,
year = {2026},
author = {Dhara, C and Sah, H and Gantayat, S and Rajput, M and Mishra, S},
title = {CRISPR/Cas9 in perspective: evaluating efficacy, delivery methods, and ethical challenges in genome editing.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42090053},
issn = {1573-4978},
mesh = {*Gene Editing/ethics/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Gene Transfer Techniques/ethics ; Genetic Engineering/methods/ethics ; },
abstract = {The latest developments in DNA sequencing techniques have revealed genes that play a role in determining our vulnerability to diseases and have given us a deeper understanding of our genetic composition. A groundbreaking milestone in genetic engineering has transformed the capabilities of the scientific community in altering the genetic material of different organisms. Among recent innovations, the clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) has emerged as a powerful and precise tool for genome editing across diverse organisms. Its applications span immunotherapy, agriculture, poultry science, and human therapeutics, marking a transformative shift in biomedical and biotechnological research. However, the rapid progress and clinical translation of CRISPR/Cas9 have raised significant concerns regarding off-target effects, delivery challenges, long-term safety, and ethical implications. This review critically evaluates the CRISPR/Cas9 system by examining its molecular mechanism, editing efficiency, gene delivery approaches, and potential for inducing unintended mutations. A comparative analysis with other gene-editing tools is presented, emphasizing the advantages of CRISPR/Cas9 in programmability and editing efficiency. Furthermore, we discuss current advances including base editing, prime editing, and high-fidelity Cas variants, along with the ethical and societal dimensions of genome editing. This synthesis provides an updated perspective on the potential and limitations of CRISPR/Cas9 technology and highlights key areas for future research and responsible application.},
}
@article {pmid42090299,
year = {2026},
author = {Xiong, Y and Tsai, LK and Zhou, J and Chen, S and Xia, X and Zhang, J and Chen, YE and Xu, J and Huang, X},
title = {Fully computational design of PAM-relaxed Staphylococcus aureus Cas9 with expanded targeting capability using UniDesign.},
journal = {eLife},
volume = {15},
number = {},
pages = {},
doi = {10.7554/eLife.110906},
pmid = {42090299},
issn = {2050-084X},
support = {GM149016/NH/NIH HHS/United States ; HL164205/NH/NIH HHS/United States ; },
mesh = {*Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-Cas9 nucleases have transformed genome engineering, yet their application is often constrained by protospacer-adjacent motif (PAM) requirements. Staphylococcus aureus Cas9 (SaCas9) is particularly attractive for in vivo applications due to its compact size; however, its NNGRRT PAM limits targetable genomic sites. Here, we report KRH, a SaCas9 variant designed entirely from the wild-type enzyme through a fully computational point-mutation design workflow, UniDesign, without additional experimental optimization. As expected, KRH efficiently recognizes an expanded NNNRRT PAM and exhibits substantially enhanced editing efficiency at non-canonical PAM sites, with improvements of up to 116-fold over the wild type. KRH achieves genome- and base-editing efficiencies comparable to, or exceeding, those of the well-known evolution-derived KKH variant. Computational modeling by UniDesign provides a mechanistic explanation for the PAM relaxation observed in both KRH and KKH, with structural and energetic analyses revealing that KRH relaxes PAM specificity by fine-tuning the balance between sequence-specific interactions with PAM bases and nonspecific contacts with the DNA backbone. Beyond its practical utility, KRH demonstrates that computational design can identify a minimal set of mutations sufficient to remodel the PAM interface while preserving high nuclease activity. This approach recapitulates-and in some cases surpasses-the performance of evolution-derived variants, offering a scalable strategy for high-throughput Cas9 engineering. Overall, these results establish KRH as a blueprint for rationally engineered, PAM-relaxed nucleases and underscore the power of computational design to accelerate next-generation genome editing.},
}
@article {pmid42091816,
year = {2026},
author = {Malapaka, A and Meunier, FA and Gormal, RS},
title = {Single-Molecule Imaging of Endogenous Proteins.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3034},
number = {},
pages = {211-237},
pmid = {42091816},
issn = {1940-6029},
mesh = {*Single Molecule Imaging/methods ; Microscopy, Fluorescence/methods ; Humans ; *Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing/methods ; Single-Domain Antibodies ; Plasmids/genetics ; },
abstract = {Single-molecule imaging is a technique of choice to investigate the dynamic nanoscale organization of proteins of interest, revealing a wealth of new information on how proteins perform their biological function. However, overexpression, which is often used in this context, can alter biological functions. It is therefore useful to implement tools to visualize and track endogenous proteins in living cells and observe their dynamic clustering and motion within their native environment. Herein, we describe two approaches: Fluorescent intrabody Localization Microscopy (FiLM) and gene-editing (CRISPR/Cas9) and provide a workflow including the design of plasmid backbones used to track endogenous proteins by either i) introducing nanobodies that target endogenous proteins to perform single-particle tracking or ii) gene-editing cells in culture to express tagged endogenous proteins at biologically relevant expression levels.},
}
@article {pmid39976035,
year = {2026},
author = {Sen, A and Singh, V and Dwivedi, S and Agrawal, R and Bansal, A and Shekhar, S and Kushwaha, SS},
title = {Gene Therapy and Gene Editing: Current Trends and Future Prospects of Molecular Medicine.},
journal = {Current gene therapy},
volume = {26},
number = {1},
pages = {53-66},
pmid = {39976035},
issn = {1875-5631},
mesh = {Humans ; *Genetic Therapy/trends/methods ; *Gene Editing/trends/methods/ethics ; CRISPR-Cas Systems ; *Molecular Medicine/trends/methods ; Precision Medicine ; *Genetic Diseases, Inborn/therapy/genetics ; Neoplasms/therapy/genetics ; },
abstract = {Gene therapy and genome editing have emerged as transformative approaches in the management of a diverse range of genetic and acquired diseases. This evaluation offers a thorough examination of the present state and prospects of these innovative technologies. Gene therapy is a prospective approach to the treatment and prevention of a variety of conditions, including complex cancers and inherited genetic disorders, which entail the introduction, removal, or modification of genetic material within a patient's cells. Genome editing, particularly through techniques such as CRISPR-Cas9, enables targeted corrections of genetic defects and opens new possibilities for personalized medicine by allowing for precise modifications at the DNA level. The review addresses the ethical implications, clinical applications, and significant advancements of these technologies. This article endeavors to underscore the substantial influence of gene therapy and genome editing on contemporary medicine by assessing the most recent research and clinical trials, thereby emphasizing their potential to revolutionize disease treatment and management.},
}
@article {pmid40129147,
year = {2026},
author = {Khan, Z and Mumtaz, and Gupta, S and Mehan, S and Sharma, T and Kumar, M and Maurya, PK and Sharma, AK and Gupta, GD and Narula, AS},
title = {CRISPR-Cas9: Transforming Functional Genomics, Precision Medicine, and Drug Development - Opportunities, Challenges, and Future Directions.},
journal = {Current gene therapy},
volume = {26},
number = {1},
pages = {160-172},
pmid = {40129147},
issn = {1875-5631},
support = {CRG/2021/001009//Department of Science and Technology, Science and Engineering Board, DSTSERB/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Genomics/methods ; *Drug Development ; *Precision Medicine/methods ; *Genetic Therapy/methods ; Animals ; },
abstract = {CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) is a groundbreaking gene-editing technology that enables scientists to make precise changes to the DNA of living organisms. It was first discovered in Escherichia coli and emerged as a breakthrough tool in molecular biology. This technique is essential because of its adaptability, affordability, and ease of use. It uses the adaptive immune response of bacteria and archaea to repel viral invasions. It significantly influences drug discovery, functional genomics, disease models, and pharmaceutical research. CRISPR-Cas9 is a better and more accurate way to change genes than other methods, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). This technology promotes the generation of double-strand breaks in DNA, allowing for precise genetic alterations required for therapeutic target identification and confirmation. Functional genomics enables high-throughput screening (HTS) to identify gene functions, disease causes, and therapeutic targets. CRISPR-Cas9 increases drug development by enabling Cas9 to create novel antimicrobial drugs and cancer therapies. It has also helped to generate disease models, advance our understanding of neurodegenerative and other diseases, test a variety of chemicals, and facilitate precise genetic changes. Despite its promise, ethical considerations and the possibility of off-target effects require careful evaluation to ensure its safe and effective clinical application. This study investigates the current and future possibilities of CRISPR-Cas9 in drug development, focusing on its transformational influence and addressing the challenges and limitations of its therapeutic application.},
}
@article {pmid41427901,
year = {2026},
author = {Matsumoto, K and Yamamoto, W and Fukutomi, Y and Koshikawa, S},
title = {Functions of melanin synthesis genes, yellow and tan, in wing pigmentation revealed by CRISPR/Cas9-mediated mutagenesis in Drosophila guttifera.},
journal = {Insect molecular biology},
volume = {35},
number = {3},
pages = {246-256},
doi = {10.1111/imb.70024},
pmid = {41427901},
issn = {1365-2583},
support = {//Japan Society for the Promotion of Science/ ; JP17K19427//KAKENHI/ ; JP24K21982//KAKENHI/ ; JP25K02019//KAKENHI/ ; },
mesh = {Animals ; *Pigmentation/genetics ; CRISPR-Cas Systems ; *Melanins/biosynthesis/genetics ; Wings, Animal/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/metabolism/physiology ; Mutagenesis ; },
abstract = {Colour pattern formation is a key model for studying evolutionary and developmental mechanisms. In the fruit fly Drosophila guttifera, which exhibits distinctive polka-dot wing pigmentation, we investigated the roles of two putative melanin synthesis genes, yellow and tan, using CRISPR/Cas9-mediated genome editing. We established multiple mutant strains with lesions in either gene and found that both genes were essential for normal pigmentation intensity in wing spots, though the patterns themselves persisted. Double mutants showed further reduction in pigmentation, indicating additive effects but not complete loss of patterning. Ectopic expression of wingless failed to induce normal pigmentation in yellow or tan mutants, demonstrating that both genes act downstream of wingless and are required for its pigmentation-inducing function. Furthermore, mosaic phenotypes in G0 individuals revealed quasi-cell-autonomous functions of tan, suggesting that pigmentation in D. guttifera wings depends on local availability of precursors rather than solely on transport via wing veins. This study establishes D. guttifera as a genetically tractable system for functional analyses and contributes to understanding the molecular basis of insect colour pattern formation.},
}
@article {pmid41844662,
year = {2026},
author = {Zhang, J and Wang, Q and Cheng, Z and Liu, J and Liu, Q and Qi, S and Chen, Z and Duan, Y and Liu, Z and Jia, J and Li, C},
title = {Enhancer-mediated Etv4 activation stimulates osteogenic differentiation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41844662},
issn = {2041-1723},
support = {82072499, 32270610//National Natural Science Foundation of China (National Science Foundation of China)/ ; L242120//Natural Science Foundation of Beijing Municipality (Beijing Natural Science Foundation)/ ; },
mesh = {Animals ; *Osteogenesis/genetics ; Mice ; *Cell Differentiation/genetics ; Osteoblasts/metabolism/cytology ; *Enhancer Elements, Genetic/genetics ; STAT3 Transcription Factor/metabolism ; *Proto-Oncogene Proteins c-ets/genetics/metabolism ; Cell Line ; Humans ; Mice, Knockout ; CRISPR-Cas Systems ; Osteoporosis/genetics ; *Adenovirus E1A Proteins/genetics/metabolism ; },
abstract = {Enhancers, as cis-regulatory elements, play pivotal roles in transcriptional homeostasis. The abnormality in enhancers is highly associated with various diseases, including osteoporosis. However, the landscape of active enhancers underlying bone diseases remains incomplete. By conducting an integrative analysis of transcriptome and ChIP-seq data, we identify enh11 as an active enhancer during osteoblastogenesis. CRISPR/Cas9-mediated deletion of enh11 inhibits cell differentiation of pre-osteoblast MC3T3-E1 cells. The osteoblast-specific knockout of enh11 reduces bone formation and decreases bone mass in mice. In addition, Etv4 is identified as the downstream target of enh11. Functional experiments both in vitro and in vivo validate that Etv4 promotes osteogenesis and bone formation. Mechanistically, enh11 upregulates the expression of Etv4 to promote osteogenesis, probably via binding to the transcription factor Stat3. These findings not only deepen our comprehension of the molecular mechanisms of enh11 underlying bone formation but also highlight enh11 and Etv4 as promising therapeutic targets for osteoporosis.},
}
@article {pmid41882696,
year = {2026},
author = {Ren, W and Yang, M and Zhou, Y and Yang, Y and Li, H and Chen, Y and Li, S and Pang, Y},
title = {An ultra-sensitive cell-free DNA-based diagnostic assay for Tuberculous pleurisy utilizing the CRISPR-Cas13a system.},
journal = {Annals of clinical microbiology and antimicrobials},
volume = {25},
number = {1},
pages = {},
pmid = {41882696},
issn = {1476-0711},
support = {S2023050//Popularization and application of appropriate medical and health technology in Guangxi Province/ ; 20230484295//Beijing Nova Program/ ; KJ2024CX028//Beijing Tongzhou District Science and Technology Commission/ ; 2024-4-1042//Health Improvement and Research/ ; },
mesh = {Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Male ; Middle Aged ; Young Adult ; Cell-Free Nucleic Acids/analysis ; CRISPR-Cas Systems ; *DNA, Bacterial/analysis ; *Molecular Diagnostic Techniques/methods/statistics &amp; numerical data ; *Mycobacterium tuberculosis/isolation &amp; purification ; Pleural Effusion/microbiology ; Prospective Studies ; Sensitivity and Specificity ; *Tuberculosis, Pleural/diagnosis ; },
abstract = {BACKGROUND: Tuberculous pleurisy (TP), a predominant form of extrapulmonary tuberculosis, presents significant diagnostic challenges attributable to the paucibacillary nature of pleural effusion (PE) specimens. Cell-free Mycobacterium tuberculosis (MTB) DNA in PE represents a promising biomarker for TP diagnosis. This study aimed to develop and assess a novel cell-free DNA (cfDNA)-CRISPR assay targeting MTB DNA in PE supernatants.<br><br>METHODS: Patients with suspected TP were prospectively enrolled at Beijing Chest Hospital. PE samples underwent centrifugation, with sediments tested by MTB/RIF Xpert (Xpert) testing and mycobacterial culture, while supernatants were analyzed using the cfDNA-CRISPR assay. Diagnostic performance was evaluated using a composite reference standard (CRS).<br><br>RESULTS: Of 276 participants, 237 (85.9%) were included in the final analysis. Based on the CRS, cases were stratified as follows: 63 definite TP, 70probable TP, and 104 non-TP controls. The cfDNA-CRISPR assay in definite TP demonstrated superior sensitivity (81.0%) compared to mycobacterial culture (33.3%, P&#x2009;<&#x2009;0.001) and Xpert (42.9%, P&#x2009;<&#x2009;0.001). In probable TP, where both Culture and Xpert were negative, cfDNA-CRISPR maintained high sensitivity (80.0%), exceeding that of ADA testing (64.3%, P&#x2009;<&#x2009;0.05). Overall sensitivity of cfDNA-CRISPR for TP was 80.5%, markedly higher than Culture (15.8%) and Xpert (20.3%) (both P&#x2009;<&#x2009;0.001). The cfDNA-CRISPR assay exhibited a specificity of 94.2%, while both Culture and Xpert achieved 100% specificity.<br><br>CONCLUSIONS: The cfDNA-CRISPR assay based on the CRISPR-Cas13a system offers significantly improved sensitivity over conventional methods for detecting MTB in PE. It represents a promising, non-invasive diagnostic tool for enhancing TP detection in clinical practice.},
}
@article {pmid41963468,
year = {2026},
author = {Park, SB and Kim, JS and Ha, Y and Kim, MS and Kim, TW},
title = {Human pluripotent stem cell engineering with CRISPR-Cas9 for Parkinson's disease.},
journal = {Experimental &amp; molecular medicine},
volume = {58},
number = {4},
pages = {993-1009},
pmid = {41963468},
issn = {2092-6413},
support = {RS-2024-00351442//National Research Foundation of Korea (NRF)/ ; RS-2025-25437095//National Research Foundation of Korea (NRF)/ ; },
mesh = {Humans ; *Parkinson Disease/therapy/genetics/metabolism/pathology ; *CRISPR-Cas Systems ; Dopaminergic Neurons/metabolism ; *Pluripotent Stem Cells/metabolism/cytology ; Animals ; Gene Editing/methods ; Cell Differentiation ; *Cell Engineering/methods ; },
abstract = {Parkinson's disease (PD) entails loss of substantia nigra dopamine (DA) neurons and α-synuclein pathology. Currently, no effective disease-modifying therapies have been developed. Human pluripotent stem cells (hPS cells) can generate DA neurons on scale, enabling human genetic PD modeling of mitochondrial, lysosomal and synaptic connection failure that leads to DA neuron degeneration. Clustered regularly interspaced short palindromic repeats (CRISPR) extends this human model by providing causal, isogenic interrogation and transcriptional regulation of PD genes and reporter knock-ins that support purification and high-content screening. hPS cell-based DA cell grafts can restore motor function yet face >90% acute cell death and product heterogeneity in vivo post implantation. CRISPR enabled not only an in vivo cell survival screen to identify the cell death regulators but also a reporter-guided enrichment of DA neurons and chemogenetic control of grafted DA cell function in vivo. Here we summarize this progress and outline a practical road map to accelerate the development of precise human models and advanced hPS cell-based cell therapies for PD.},
}
@article {pmid42003707,
year = {2026},
author = {Wu, Z and Jin, F and Zhu, W and Zhong, W and Qi, T and Luo, S and Liu, Q and Cai, Z and Dai, C and Chai, Z and He, Y and Rui, Y and Miao, Y and Zheng, L and Fu, Q},
title = {Photoactivated Digital Recombinase Polymerase Amplification/CRISPR-Cas12a Assay for Point-of-Care of BK Polyomavirus Quantification.},
journal = {ACS nano},
volume = {20},
number = {17},
pages = {13301-13313},
doi = {10.1021/acsnano.6c02651},
pmid = {42003707},
issn = {1936-086X},
mesh = {*BK Virus/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems/genetics ; *Recombinases/metabolism/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Point-of-Care Systems ; DNA, Viral/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BK polyomavirus (BKV) serves as a critical biomarker for optimizing immunosuppressive therapy and preventing graft failure in kidney transplant recipients. Quantitative PCR (qPCR), the current gold standard for BKV load quantification, relies on batch-specific standard curves. For kidney transplant recipients, this not only elevates the risk of cross-contamination but also entails considerable economic burdens. Therefore, developing BKV quantification technologies independent of batch-specific standard curves is of great clinical significance for this patient population. The combination of CRISPR-Cas12a with recombinase polymerase amplification (RPA), termed DETECTR, offers notable advantages for digital nucleic acid analysis. However, due to the high viscosity of RPA reagents, the generation of high-throughput, uniform RPA microdroplets remains a significant technical challenge. In the present study, we developed a centrifugal RPA microdroplet generation method based on commercial capillaries, facilitating the production of high-throughput, uniform RPA microdroplets (23.1 μm in diameter) via simple centrifugation. Furthermore, by integrating a light-controlled RPA-CRISPR-Cas12a system, we established photoactivated digital DETECTR (pd-DETECTR) for precise, point-of-care, and cost-effective BKV quantification. When combined with a smartphone-based reader, the pd-DETECTR assay can be completed within 42 min. Clinical validation demonstrated a strong correlation (R[2] = 0.9801) with qPCR results, exhibiting high sensitivity (100.0%), specificity (98.0%), and accuracy (99.0%). The pd-DETECTR provides a rapid, convenient, and cost-effective tool for BKV load analysis, which can significantly reduce the economic burden and risk of opportunistic infections in kidney transplant recipients, thus holding significant clinical value.},
}
@article {pmid42008698,
year = {2026},
author = {Wang, H and Li, F and He, Y and Liu, X and Yin, Y and Xu, S},
title = {CRISPR/dCas9-Assisted On-Bead Multiplex Detection (BeadPlex2) for Genetically Modified Crops.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12586-12595},
doi = {10.1021/acs.analchem.5c08192},
pmid = {42008698},
issn = {1520-6882},
mesh = {*Plants, Genetically Modified/genetics ; *CRISPR-Cas Systems/genetics ; Glycine max/genetics ; Zea mays/genetics ; Spectrum Analysis, Raman ; *Crops, Agricultural/genetics ; Benzothiazoles ; },
abstract = {This study leverages the precise recognition ability of CRISPR/dCas9 and the Raman coding feature of the gap-enhanced Raman tag-encoded magnetic beads (MagGERTs) to create a unique on-bead nucleic acid detection platform (BeadPlex2) for accurate and multiplex nucleic acid detection, which was proven to be applicable for the identification of diverse genetically modified (GM) events. Five distinct MagGERTs (MB@Au[Ra]) encoded with different Raman reporters (Ras) were constructed, followed by the conjugation of dCas9/single guide RNA (sgRNA) complexes in which the sgRNAs were explicitly designed for different target genes of GM events. These coding units could recognize and capture target double-stranded nucleic acid (dsDNA) sequences by the dCas9/sgRNA complexes. Then, SYBR Green I was applied to highlight positive beads by binding to target dsDNA due to its fluorescent emission under an imaging system. Decoding Raman signals from the Ras of the MagGERTs achieved the high-specific identification of GM events. Our BeadPlex2 platform has been demonstrated to be applicable for detecting GM maize and soybean seeds with high accuracy comparable to qPCR. This platform opens a new way to detect multiple target nucleic acids simultaneously and offers a powerful strategy for identifying genetically modified organisms.},
}
@article {pmid42011754,
year = {2026},
author = {Yang, G and Fang, Y and Liu, Y and Deng, Y and Nie, L and Li, Z and Li, S and Chen, Z and Su, E and Zai, Y and Umar Siddiqui, AM and He, N},
title = {Rapid and Specific Detection of Gastric Cancer EVs Using a Cas12a-Powered Aptasensor with a Novel Targeting Aptamer.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12529-12541},
doi = {10.1021/acs.analchem.5c08039},
pmid = {42011754},
issn = {1520-6882},
mesh = {*Stomach Neoplasms/diagnosis/pathology/metabolism ; *Aptamers, Nucleotide/chemistry/metabolism ; Humans ; *Extracellular Vesicles/metabolism/chemistry ; SELEX Aptamer Technique ; *CRISPR-Associated Proteins/metabolism/genetics ; *Biosensing Techniques/methods ; Cell Line, Tumor ; CRISPR-Cas Systems ; Biomarkers, Tumor ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Gastric cancer remains a predominant contributor to global cancer-related mortality, characterized by a pronounced disparity in five-year survival rates between early stage (>90%) and advanced-stage (<30%) disease. This disparity underscores the urgent necessity for accessible early detection methods. Present diagnostic approaches, such as serum biomarkers and endoscopy, either lack adequate sensitivity or are invasive. Extracellular vesicles (EVs) represent promising biomarkers for liquid biopsy; however, a major limitation is the lack of probes that can specifically identify EVs derived from gastric cancer, as most existing markers are broad-spectrum and exhibit low specificity. To address this limitation, we isolated high-purity EVs from gastric cancer cells and utilized a combined immunomagnetic bead-based SELEX strategy to identify a novel aptamer, H-EV-4-1, which demonstrates high affinity (Kd = 13.32 ± 2.69 nM) and specificity for gastric cancer EVs. Subsequently, this aptamer was incorporated into a CRISPR-Cas12a-based biosensor. The aptamer was hybridized with a biotinylated oligonucleotide (H1-biotin) and immobilized on magnetic beads. Upon binding of the target EVs, the aptamer was displaced, thereby exposing H1-biotin to activate the Cas12a/crRNA complex. This activation induced the trans-cleavage of a fluorescent reporter, producing a quantifiable signal. This aptasensor facilitates the rapid, highly sensitive, and specific detection of gastric cancer EVs, presenting a promising platform for the development of noninvasive, point-of-care early diagnostic tools.},
}
@article {pmid42024570,
year = {2026},
author = {Liu, X and Shi, F and Luo, G and Wang, Q and Deng, F and Luo, X and Huo, D and Hou, C},
title = {A Disposable CRISPR-Nanozyme Electrochemical Biosensor for Rapid and Sensitive Detection of Breast Cancer Circulating Tumor DNA.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {12802-12810},
doi = {10.1021/acs.analchem.6c00173},
pmid = {42024570},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Circulating Tumor DNA/analysis/blood/genetics ; *Breast Neoplasms/blood/genetics/diagnosis ; *Electrochemical Techniques/methods ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Female ; *CRISPR-Cas Systems ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) serves as a promising next-generation biomarker for noninvasive cancer screening and monitoring. In this work, we report an electrochemical biosensor for ctDNA detection. The sensor is constructed based on the synergistic integration of CRISPR/Cas12a and PB-Au NPs. CRISPR/Cas12a provides precise target recognition and triggers trans-cleavage, while the nanozyme enables strong signal amplification through its catalytic activity. Using a disposable carbon-fiber paper as the biosensing interface, we developed this sensitive detection strategy. On this interface, a PB-AuNP-labeled single-stranded DNA reporter is immobilized. In the presence of target ctDNA, activated Cas12a cleaves the reporter, releasing the nanozyme and resulting in a quantifiable decrease in the TMB oxidation current. This dual-amplification strategy achieves a detection limit of 860 aM (S/N = 3) with a linear range from 1 fM to 1 nM. Overall, this approach provides a satisfactory demonstration toward the realization of a low-cost and highly sensitive biosensor for ctDNA detection.},
}
@article {pmid42027134,
year = {2026},
author = {Xu, L and Zhao, X and Meng, X and Chen, J and Chen, P},
title = {Multiselective Recognition of Metal Ion-Nucleic Acid Complexes by CRISPR/Cas12a and Quantum Dots Enables the Profiling of Circulating Tumor DNA in Breast Cancer.},
journal = {Analytical chemistry},
volume = {98},
number = {17},
pages = {13120-13133},
doi = {10.1021/acs.analchem.6c01333},
pmid = {42027134},
issn = {1520-6882},
mesh = {*Quantum Dots/chemistry ; *Breast Neoplasms/genetics/blood/diagnosis ; Humans ; *CRISPR-Cas Systems ; Female ; *Circulating Tumor DNA/blood/genetics ; *Silver/chemistry ; Class I Phosphatidylinositol 3-Kinases/genetics ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; },
abstract = {The rapid, noninvasive detection of circulating tumor DNA (ctDNA) is vital for the diagnosis and staging of breast cancer (BC). In this study, we developed a homogeneous CRISPR/Cas12a fluorescent platform using a hierarchical grape-cluster rolling circle amplification (GCRCA) nanomaterial to detect the PIK3CA E542K mutation. A pivotal discovery of this study is that activated Cas12a efficiently cleaves metal ion-mediated cytosine-Ag[+]-cytosine base pairs, which enables direct coupling between enzymatic activity and signal transduction. GCRCA, assembled by precise hybridization of long-chain RCA concatemers with auxiliary circular DNA, features Ag[+]-bridged dual-ring units that sequester both target sequences and Ag[+] reporters within a self-shielding framework. Upon target recognition, activated Cas12a dismantles the GCRCA architecture, initiating an autocatalytic feedback loop that releases caged Ag[+] to quench the quantum dot fluorescence. This label-free assay achieved attomolar sensitivity within 30 min without enzymatic preamplification or complex nucleic acid extraction. Importantly, the platform exhibits excellent sequence selectivity, enabling precise discrimination of single-base mutations against closely related sequences. Validation of 42 clinical plasma samples achieved 100% diagnostic specificity for BC. For staging, the platform yielded a sensitivity of 100%, a specificity of 92.3%, and an area under the curve of 0.978. With its exceptional sensitivity and operational simplicity, this platform offers a promising approach for precise ctDNA-based BC detection and staging, demonstrating significant translational clinical potential.},
}
@article {pmid42083602,
year = {2025},
author = {Taebi, S and Eskandari, F and Kohandani, M and Manoochehrabadi, T and Nasiri, H},
title = {Genetic Engineering in Hematopoietic Stem Cells for β-Hemoglobinopathies Treatment: Advances, Challenges, and Clinical Translation.},
journal = {International journal of hematology-oncology and stem cell research},
volume = {19},
number = {4},
pages = {399-423},
pmid = {42083602},
issn = {2008-3009},
abstract = {β-hemoglobinopathies rank among the most prevalent inherited blood disorders globally. Traditional management strategies are primarily palliative and often associated with significant challenges, including iron overload and limited long-term efficacy. Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative option for transfusion-dependent patients, but its broader applicability is constrained by factors that limit its use. Utilizing viral vectors and gene-editing tools, particularly CRISPR-Cas9 technology, researchers have developed therapies that target the root causes of these disorders. These innovative approaches have demonstrated substantial therapeutic potential, accompanied by favorable safety profiles, in clinical settings. Since the initial investigations, the genome editing tool has rapidly advanced for genetic abnormalities, particularly monogenic blood diseases, including β-hemoglobinopathies. This method suggests an approach with lower concerns in viral gene integration and insertional mutagenesis issues. This review comprehensively surveys the therapeutic strategies for β-thalassemia and sickle cell disease (SCD) currently in preclinical and clinical development, with a focus on the evolving treatment paradigm. Looking forward, critical research priorities include optimizing the efficiency and specificity of gene-editing platforms and pioneering novel delivery systems to guarantee both therapeutic efficacy and clinical safety.},
}
@article {pmid42084692,
year = {2026},
author = {Minami, A and Shimizu, M and Tamaki, S and Nishinarizki, V and Yosua, and Mochida, K},
title = {Affordable CRISPR RNP-Based Genome Editing in Euglena gracilis.},
journal = {Current protocols},
volume = {6},
number = {5},
pages = {e70357},
doi = {10.1002/cpz1.70357},
pmid = {42084692},
issn = {2691-1299},
support = {JPMJSA2204//This work was supported by the Japan Science and Technology Agency (JST) and the Japan International Cooperation Agency (JICA) through the Science and Technology Research Partnership for Sustainable Development (SATREPS) Program/ ; },
mesh = {*Gene Editing/methods/economics ; *Euglena gracilis/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Genome editing can enhance basic research and enable industrial applications of green algae. Here, we present an affordable, broadly applicable workflow for genome editing in the unicellular green alga Euglena gracilis using Cas9 nucleases. This method retains high editing efficiency while significantly lowering technical barriers. Unlike previous approaches that required specialized equipment, this protocol can be performed using a general-purpose laboratory electroporator and a simplified clonal isolation procedure without the need for specialized micromanipulation devices. This protocol is compatible with a range of editing outcomes, such as targeted deletions and precise base substitutions, enabling more widespread genome editing in Euglena. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culture of Euglena gracilis Basic Protocol 2: sgRNA synthesis Basic protocol 3: Transformation Basic protocol 4: Genotyping.},
}
@article {pmid42084860,
year = {2026},
author = {Tian, Y and Li, C and Zhao, E and Chen, Y and Shen, X and Xu, S and Yu, Y and Sun, L},
title = {Recent Advances in the Detection of Plant Diseases Based on the CRISPR-Cas System.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c07871},
pmid = {42084860},
issn = {1520-6882},
}
@article {pmid41188232,
year = {2025},
author = {Fu, BXH and Xu, A and Li, H and Johnson, DE and Grandis, JR and Gilbert, LA},
title = {Loss of Fanconi anemia proteins causes a reliance on lysosomal exocytosis.},
journal = {Cell death &amp; disease},
volume = {16},
number = {1},
pages = {791},
pmid = {41188232},
issn = {2041-4889},
mesh = {Humans ; Cell Line ; *Exocytosis ; *Lysosomes/metabolism ; *Fanconi Anemia/genetics/metabolism/pathology ; Autophagy ; CRISPR-Cas Systems ; Genome, Human ; DNA Repair ; Neoplasms/genetics ; },
abstract = {Mutations in the Fanconi Anemia (FA) pathway lead to a rare genetic disease that increases risk of bone marrow failure, acute myeloid leukemia, and solid tumors. FA patients have a 500 to 800-fold increase in head and neck squamous cell carcinoma compared to the general population and the treatments for these malignancies are ineffective and limited due to the deficiency in DNA damage repair. Using unbiased CRISPR-interference screening, we found the loss of FA pathway function renders cells dependent on key exocytosis genes such as SNAP23. Further investigation revealed that loss of FA pathway function induced deficiencies in lysosomal health, dysregulation of autophagy and increased lysosomal exocytosis. The compromised cellular state caused by the loss of FA genes is accompanied by decreased lysosome abundance and increased lysosomal membrane permeabilization in cells. We found these signatures in vitro across multiple cell types and cell lines and in clinically relevant FA patient cancers. Our findings are the first to connect the FA pathway to lysosomal exocytosis and thus expands our understanding of FA as a disease and of induced dependencies in FA mutant cancers.},
}
@article {pmid41400455,
year = {2026},
author = {Mukherjee, S and Kumar, M},
title = {CRISPR: a precise genome editing strategy for the treatment of hepatocellular carcinoma.},
journal = {Expert review of anticancer therapy},
volume = {26},
number = {5},
pages = {599-614},
doi = {10.1080/14737140.2025.2606090},
pmid = {41400455},
issn = {1744-8328},
mesh = {Humans ; *Carcinoma, Hepatocellular/therapy/genetics/pathology ; *Liver Neoplasms/therapy/genetics/pathology ; *Gene Editing/methods ; CRISPR-Cas Systems ; Animals ; Prognosis ; Clustered Regularly Interspaced Short Palindromic Repeats ; Immunotherapy, Adoptive/methods ; T-Lymphocytes/immunology ; Genetic Therapy/methods ; Receptors, Chimeric Antigen ; },
abstract = {INTRODUCTION: The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) gene-editing tool provides novel therapeutic alternatives by promoting the gene alteration in adaptive T cells or malignant cells to combat Hepatocellular Carcinoma (HCC). More successful cancer treatments are now possible due to the capacity of precisely locating and modifying particular genetic abnormalities that promote malignancy growth and metastasis.<br><br>AREAS COVERED: In this review, we address ongoing clinical trials, the possible similarities between CRISPR-based cancer treatments and current therapeutic choices, and how CRISPR technology can improve treatment outcomes for HCC while using the latest safety measures. Additionally, this analysis sheds light on the existing obstacles and potential future possibilities of applying CRISPR technology to the management of HCC, with a final objective of enhancing patient results and completely changing the field of HCC therapies.<br><br>EXPERT OPINION: The urgent need for innovative therapies is underscored by the poor prognosis associated with severe hepatocellular carcinoma, despite recent advancements in clinical therapies. Through a special emphasis on invivo cancer cell targeting along with the generation of chimeric antigen receptor (CAR) T cells, including T cell receptor (TCR) T cells, this review analyses the uses of CRISPR methods in the therapy of HCC.},
}
@article {pmid41690142,
year = {2026},
author = {Huang, Z and Ding, Y and Xu, H and Zhang, Q and Wang, X and Ding, H and Zhang, J and Wang, Y and Wang, Y and Jiao, P},
title = {Development of an RT-ERA-CRISPR/Cas12a assay for duck Tembusu virus.},
journal = {Poultry science},
volume = {105},
number = {5},
pages = {106612},
pmid = {41690142},
issn = {1525-3171},
mesh = {Animals ; *Ducks ; *Flavivirus/isolation &amp; purification/genetics ; *Poultry Diseases/virology/diagnosis ; *Flavivirus Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; China ; },
abstract = {Duck Tembusu virus (DTMUV) has continued to threaten the duck industry in China since 2010. Therefore, the establishment of a rapid, specific, and sensitive method for the field detection of DTMUV is urgently needed. Herein, a reverse transcription enzymatic recombinase amplification (RT-ERA) assay was combined with the CRISPR/Cas12a system to target the DTMUV C gene. This assay exhibited high specificity, effectively distinguishing DTMUV from other common avian viruses. Its limit of detection reached 1 copy/μL DTMUV RNA. Moreover, this assay can be completed at 42 °C within 15 min using a thermostatic water bath. Additionally, we tested 30 clinical samples from infected ducks using this assay, and the results showed 100% concordance with SYBR green quantitative PCR results. In summary, this rapid, specific, and sensitive assay shows promising potential for DTMUV detection in the field.},
}
@article {pmid41762975,
year = {2026},
author = {Guo, L and Cui, K and Yang, Y and Dong, S and Chen, Y and Liu, K and Lei, X and Duan, B and Zhao, Y and Lv, X and Bai, R and Zheng, M},
title = {Field-deployable multiplex RAA-CRISPR/Cas12a platform rapidly and simultaneously detects seven Eimeria species in chickens.},
journal = {Poultry science},
volume = {105},
number = {5},
pages = {106681},
pmid = {41762975},
issn = {1525-3171},
mesh = {Animals ; *Eimeria/isolation &amp; purification/classification/genetics ; *Chickens ; *Coccidiosis/veterinary/diagnosis/parasitology ; *Poultry Diseases/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Feces/parasitology ; Reproducibility of Results ; },
abstract = {Chicken coccidiosis, caused by infection with one or more of the seven Eimeria spp., is a major challenge in global poultry production. Rapid and accurate identification at the species level is critical for guiding targeted treatment strategies, minimizing antibiotic misuse, and mitigating disease transmission. In this study, we developed a point-of-care testing (POCT) platform, E-MRC12a (Eimeria-Multiplex RAA-CRISPR/Cas12a), which integrates multiplex recombinase-aided amplification (RAA) with CRISPR/Cas12a technology for the simultaneous detection of all seven Eimeria species in chicken fecal samples. Key assay parameters were optimized to balance detection performance and operational cost. The system was comprehensively evaluated for its sensitivity, specificity, reproducibility, and field applicability. E-MRC12a enables visual, one-pot detection of as few as 1 oocyst/μL. The process from sample loading to result interpretation required 1 h, while the total time from initial sample processing to final result readout was approximately 2 h. The assay exhibited high specificity with no cross-reactivity among Eimeria species, and demonstrated 100% concordance with conventional diagnostic methods in clinical validation. This rapid, field-deployable platform provides a species-specific coccidiosis diagnostic solution, supporting epidemiological surveillance and multivalent anticoccidial vaccine development.},
}
@article {pmid41813589,
year = {2026},
author = {Yu, T and Gao, P and Wang, K and Chen, B and Chu, Z and Liu, S and Hong, J and Zhang, X and Zhang, Z and Lv, R and Liu, B and Xun, H},
title = {Loss-of-Function Mutation in TaZIP4-B2 and TaMSH7-3D Fuels Karyotypic Variation, Phenotypic Diversity, and Enables Rapid Evolution of Tolerance to Salinity Stress.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {6},
pages = {3455-3469},
doi = {10.1111/pce.70487},
pmid = {41813589},
issn = {1365-3040},
support = {2022YFF1003303//National Key Research and Development Programme of China/ ; 32572354//Natural Science Foundation of China/ ; 32061143001//Israel Science Foundation (ISF)-China National Natural Science Foundation (NSFC)/ ; },
mesh = {*Triticum/genetics/physiology ; Phenotype ; *Loss of Function Mutation/genetics ; Karyotype ; *Plant Proteins/genetics/metabolism ; *Salt Stress/genetics ; *Salt Tolerance/genetics ; Meiosis/genetics ; CRISPR-Cas Systems ; },
abstract = {Allopolyploid species often contain specific genes dedicated to suppressing meiotic homoeologous pairing. In common wheat, TaZIP4-B2 and TaMSH7-3D fulfil this role. Nevertheless, to what extent the loss-of-function of these genes may lead to meiotic breakdown in wheat itself and hence generate karyotypic heterogeneity remains incompletely understood. Here, we show that CRISPR/Cas9-generated loss-of-function mutation of either or both TaZIP4-B2 and TaMSH7-3D leads to disrupted meiosis, triggering widespread karyotypic instability including both numerical and structural chromosomal variations (NCVs and SCVs). NCVs predominantly occurred in the D subgenome, involving preferential gains of 2A/4B/5D and losses of 6A/5B/2D, while frequencies of SCVs among subgenomes followed the order of subgenomes D > A > B, with 6A/5B/2D showing the most rearrangements. Notably, karyotypic variation in Tazip4-B2/Tamsh7-3D double mutants showed initial rapid accumulation followed by gradual stabilization across generations. Karyotypic heterogeneity caused extensive phenotypic diversity, including several key agronomic traits. Notably, Tazip4-B2/Tamsh7-3D double mutant showed more intercalary insertional translocations than the classical ph1b deletion mutant, suggesting its advantage in alien genetic introgression. Moreover, tolerance to strong salinity emerged in progenies of the mutants due to karyotypic variation. Our findings demonstrate that the loss-of-function mutation of TaZIP4-B2/TaMSH7-3D promotes rapid karyotype variability, phenotypic diversity, and environmental adaptability in wheat itself, suggesting a novel possibility for wheat improvement by karyotypic renovation.},
}
@article {pmid41874550,
year = {2026},
author = {Hsu, JY and Lu, WH and Chang, YY and Chiang, PC and King, PC and Hsu, YH and Huang, YS},
title = {Generation of Ucp1-ires-Cre knock-in mice to enhance specificity and efficiency of gene targeting in brown adipose tissue.},
journal = {American journal of physiology. Endocrinology and metabolism},
volume = {330},
number = {5},
pages = {E675-E683},
doi = {10.1152/ajpendo.00533.2025},
pmid = {41874550},
issn = {1522-1555},
support = {NHRI-EX109-10719SI//National Health Research Institutes (NHRI)/ ; NSTC111-2311-B-001-020-MY3//National Science and Technology Council (NSTC)/ ; AS-GC-111-L04//Academia Sinica (AS)/ ; },
mesh = {Animals ; *Uncoupling Protein 1/genetics/metabolism ; *Adipose Tissue, Brown/metabolism ; Mice ; *Integrases/genetics/metabolism ; Mice, Transgenic ; *Gene Knock-In Techniques/methods ; Thermogenesis/genetics ; Male ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; *Gene Targeting/methods ; Female ; },
abstract = {Brown adipose tissue (BAT) secretes cytokines that influence the function of other tissues. Given the widespread distribution of brown fat depots, we generated BAT-lacking (ΔBAT) mouse models by specifically eliminating brown adipocytes using the Cre-loxP system combined with a floxed-stop diphtheria toxin A (DTA) cassette. Uncoupling protein 1 (UCP1) is essential for BAT thermogenesis and exhibits a highly restricted expression pattern, so it was chosen to direct BAT-specific Cre recombinase expression. We used CRISPR-Cas9 to insert an ires-Cre sequence downstream of the UCP1 stop codon, developing the novel knock-in line, Ucp1-Cre[YH]. Ucp1-Cre[YH] and transgenic line TgUcp1-Cre[Evdr] mouse (Ucp1-Cre[Evdr]) were crossed with Ai14-tdTomato and floxed-CPEB2 mice to assess Cre specificity and efficiency. ΔBAT mice were then generated by crossing each Cre line with floxed-stop DTA mice, followed by assessments of locomotor activity, body weight, and glucose tolerance. Although both Cre lines showed cold-enhanced expression, Ucp1-Cre[Evdr] exhibited considerably lower Cre levels in BAT compared with Ucp1-Cre[YH] mice, leading to inefficient ablation of some floxed alleles, such as Cpeb2. Moreover, Ucp1-Cre[Evdr] mice displayed nonspecific Cre expression, whereas neither line showed evidence of substantial autonomous Cre activity in BAT-resident macrophages. Consequently, ΔBAT[Evdr] mice experienced off-target neuronal ablation, resulting in hyperactive locomotion and reduced body weight. Although ΔBAT[YH] mice showed normal locomotor activity and body weight, they had a modest weight gain and altered glucose homeostasis only after high-fat-diet feeding. In conclusion, novel Ucp1-Cre knock-in mouse showed specificity and efficiency for gene manipulation in brown adipocytes, highlighting its application in generating BAT-specific knockout and BAT-depleted mouse models.NEW &amp; NOTEWORTHY A novel Ucp1-CreYH knock-in mouse exhibits improved efficiency and specificity of Cre recombinase activity in brown adipose tissue (BAT) compared with the widely used Ucp1-CreEvdr line, which showed nonspecific Cre activity in many organs. A BAT-deprived mouse model, generated with this Cre line, showed normal locomotion and body weight, but altered body weight and glucose homeostasis after high-fat feeding, validating its use for BAT-specific gene manipulation.},
}
@article {pmid41914367,
year = {2026},
author = {Lu, Q and Ye, C and Chen, R and Xing, Z and Liu, Z and Zeng, F and Gong, J and Gao, Y and Sun, X and Tang, S and Song, Y},
title = {High-Entropy Alloy Synergized with Gene Editing for Cocktail-Sensitized Radioimmunotherapy of Lung Metastases.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {25},
pages = {e22618},
doi = {10.1002/adma.202522618},
pmid = {41914367},
issn = {1521-4095},
support = {22477056//National Natural Science Foundation of China/ ; 82272138//National Natural Science Foundation of China/ ; 22375089//National Natural Science Foundation of China/ ; 82572370//National Natural Science Foundation of China/ ; 2024300315//Fundamental Research Funds for the Central Universities/ ; 2019YFA0709200//National Key R&amp;D Program/ ; BE2021373//Jiangsu Province Key R&amp;D Program/ ; 5431ZZXM2304//State Key Laboratory of Analytical Chemistry for Life Science/ ; },
mesh = {*Lung Neoplasms/secondary/therapy/radiotherapy/pathology ; *Gene Editing ; Animals ; Humans ; *Alloys/chemistry/pharmacology ; Cell Line, Tumor ; Mice ; CRISPR-Cas Systems ; Palladium/chemistry ; Platinum/chemistry ; Gold/chemistry ; },
abstract = {Radiotherapy (RT) eliminates cancer cells either through direct DNA damage induced by ionizing radiation or indirectly by generating cytotoxic reactive oxygen species (ROS) via radiolysis. However, high-dose radiation often triggers DNA repair mechanisms, undermining therapeutic efficacy and causing damage to surrounding healthy tissues. Thus, enhancing anti-tumor effects at lower doses while minimizing normal tissue damage and improving safety remains a key challenge in advancing RT technologies. To tackle these issues, we developed an RT-sensitizing platform, referred to as HAEPRC, which integrates a novel high-entropy alloy (HEA) composed of gold (Au), bismuth (Bi), platinum (Pt), silver (Ag), and palladium (Pd), a CRISPR/Cas9 gene-editing system, and tumor cell membranes (CM) for enhanced home-targeting and biocompatibility. We demonstrated that HAEPRC exhibits exceptional dose enhancement factors (DEFs), significantly boosting RT sensitization and improving RT-induced immunotherapeutic outcomes. Furthermore, the gene-editing system modulates the cell cycle, transforming RT-resistant cancer cells into RT-sensitive ones and further amplifying RT efficacy. Additionally, Pd-mediated bioorthogonal catalysis activates immune adjuvant production, enhancing immune responses and reinforcing anti-tumor immunity. Collectively, these features synergistically promote an enhanced abscopal immune effect, inhibiting lung cancer growth and metastasis, and providing a promising strategy to improve the efficacy and safety of RT.},
}
@article {pmid41916134,
year = {2026},
author = {Buakaew, T and Thaiwong, R and Inthanachai, T and Palaga, T and Weissman, D and Suppipat, K and Ausavarungnirun, C and Tawinwung, S},
title = {Combining CRISPR/Cas9-mediated TRAC knockout with mRNA-based CAR expression enables flexible generation of allogeneic CAR T cells.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {198},
number = {},
pages = {119300},
doi = {10.1016/j.biopha.2026.119300},
pmid = {41916134},
issn = {1950-6007},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Messenger/genetics ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/metabolism ; Cryopreservation ; Gene Knockout Techniques ; *Receptors, Antigen, T-Cell/genetics ; Antigens, CD19/genetics ; Cell Survival ; Transfection ; Gene Editing ; },
abstract = {BACKGROUND: Autologous chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable efficacy in hematologic malignancies but remain limited by complex manufacturing processes. Allogeneic, off-the-shelf CAR T cells derived from healthy donors represent a promising alternative; however, safe implementation requires elimination of endogenous T-cell receptor (TCR) expression and flexible CAR expression strategies.<br><br>OBJECTIVE: This study aimed to develop an optimized manufacturing workflow for allogeneic CAR T cells by combining CRISPR/Cas9-mediated TCR knockout with mRNA-based CAR expression, and to evaluate cryopreservation strategies enabling on-demand CAR T-cell generation.<br><br>METHODS: Healthy donor T cells were edited at the TRAC locus using CRISPR/Cas9 to generate TCR-deficient T cells. These cells were cryopreserved and subsequently transfected with mRNA encoding CD117, BCMA, or CD19 CARs. CAR expression, cell viability, immunophenotype, cytokine secretion, and antigen-specific cytotoxicity were assessed under different cryopreservation-transfection conditions.<br><br>RESULTS: TCR knockout T cells exhibited efficient TCR disruption with reduced alloreactive proliferation. CD117 mRNA CAR T cells derived from TCR-deficient T cells demonstrated CAR expression kinetics, immunophenotypic profiles, and antigen-specific cytotoxicity comparable to wild-type CAR T cells. Evaluation of two cryopreservation strategies revealed that cryopreservation prior to mRNA electroporation preserved cell viability, phenotype, and cytotoxic function, whereas cryopreservation after mRNA transfection was associated with reduced functional activity. The optimized protocol was successfully extended to CD19- and BCMA-targeting CAR mRNAs.<br><br>CONCLUSION: Collectively, these findings establish a modular platform for producing allogeneic CAR T cells using mRNA technology, offering a practical approach for rapid, on-demand CAR T-cell therapy.},
}
@article {pmid41933706,
year = {2026},
author = {Wang, Q and Tang, J and Jiang, Y and Jiang, X and Wang, Y and Liu, W and Qin, L},
title = {Temperature-induced pupal pigmentation in Antheraea pernyi is affected by ApADC through the regulation of NBAD biosynthesis.},
journal = {Insect biochemistry and molecular biology},
volume = {191},
number = {},
pages = {104550},
doi = {10.1016/j.ibmb.2026.104550},
pmid = {41933706},
issn = {1879-0240},
mesh = {Animals ; Pupa/metabolism/growth &amp; development/genetics/physiology ; *Pigmentation/genetics ; *Moths/genetics/metabolism/growth &amp; development/physiology ; Temperature ; *Insect Proteins/metabolism/genetics ; *Dopamine/analogs &amp; derivatives/biosynthesis/metabolism ; CRISPR-Cas Systems ; },
abstract = {The Chinese oak silkworm, Antheraea pernyi, exhibits temperature-dependent pupal pigmentation, forming black pupae at 23 °C and yellow pupae at 29 °C. To address this, we elucidated the molecular mechanism of this phenotypic plasticity by integrating multi-omics analyses, RNA interference, and CRISPR/Cas9. We identified N-β-alanyl dopamine (NBAD) as essential for yellow pigmentation. Transcriptomic and metabolomic analyses revealed temperature-dependent regulation of A. pernyi aspartate 1-decarboxylase (ApADC) and NBAD synthase (Apebony). This regulation correlated with elevated β-alanine/aspartic acid in yellow pupae and enriched dopamine/N-acetyldopamine (NADA) in black pupae. Beta-alanine injection at 18 °C induced a dose-dependent transition to yellow, while RNAi of ApADC or Apebony at 29 °C triggered melanization. By establishing the first CRISPR/Cas9 platform for A. pernyi, we generated ApADC mutants with disrupted GadA domains, which developed black pupae even at 29 °C; this phenotype was rescued by β-alanine supplementation. Our results show that ApADC acts as a central temperature-responsive regulator of pupal pigmentation by modulating NBAD biosynthesis. These findings provide mechanistic insights into melanization and environmental adaptation in an important lepidopteran and are expected to guide the targeted breeding of stable genetic lines for the sustainable development of the A. pernyi industry.},
}
@article {pmid42072072,
year = {2026},
author = {Bartusik-Aebisher, D and Justin Raj, DR and Aebisher, D},
title = {Nanomaterial-Based Therapeutic Delivery: Integrating Redox Biology, Genetic Engineering, and Imaging-Guided Treatment.},
journal = {Antioxidants (Basel, Switzerland)},
volume = {15},
number = {4},
pages = {},
doi = {10.3390/antiox15040430},
pmid = {42072072},
issn = {2076-3921},
abstract = {Nanomaterials are emerging versatile platforms for therapeutic delivery, as they offer precise control over drug, antioxidant, and genetic payload transport across biological barriers. Inorganic, organic, hybrid, and biomimetic systems are the major classes of nanomaterials, which all have different physicochemical properties such as size, surface charge, and surface functionalization. These properties collectively influence stability, biodistribution, cellular uptake, and release kinetics. Engineering strategies are increasingly using stimuli-responsive designs that are triggered by pH, reactive oxygen species (ROS), and intracellular redox gradients to perform spatially and temporally controlled delivery. Antioxidant and redox-modulating nanocarriers are of great importance as they overcome the limited bioavailability and nonspecific activity of conventional antioxidants by improving stability, targeting oxidative microenvironments, and allowing for regulated release. Improvements in lipid, polymeric, and inorganic nanoplatforms have also developed gene delivery applications, including siRNA, mRNA, and CRISPR/Cas systems, to provide better cytosolic release and precise therapeutics. When diagnostic imaging is integrated with therapy through theranostic nanoparticles, real-time monitoring and personalized intervention are possible. Safety, scalable manufacturing, and regulatory alignment are some challenges that show the need for standardization and translational procedures to utilize the potential of theranostic nanomedicine.},
}
@article {pmid42074014,
year = {2026},
author = {Li, Y and Ma, S and Fei, T},
title = {CRISPR Applications in Alzheimer's Disease: From High-Throughput Genetic Screening to Precision Editing and CNS Delivery.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083371},
pmid = {42074014},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/therapy ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy/methods ; tau Proteins/genetics/metabolism ; *Genetic Testing/methods ; High-Throughput Screening Assays ; Induced Pluripotent Stem Cells/metabolism ; },
abstract = {Alzheimer's disease is a devastating progressive neurodegenerative disorder characterized by extracellular amyloid-beta plaques and intracellular tau tangles. Despite recent advancements in amyloid-beta-targeting immunotherapies, achieving safe and definitive disease control remains a profound clinical challenge. The CRISPR/Cas9 system has emerged as a powerful technology for precision neurogenetics, offering significant potential to address the fundamental questions behind Alzheimer's disease. This comprehensive review delineates the trajectory of CRISPR applications in Alzheimer's disease research and therapeutics. First, we explore the integration of CRISPR in engineering high-fidelity in vitro models, such as isogenic induced pluripotent stem cells and three-dimensional cerebral organoids, alongside advanced in vivo mammalian models. Second, we examine how these platforms facilitate unbiased high-throughput genetic screening to uncover molecular underpinnings regulating tau, lipid metabolism, and neuroinflammation. Third, we critically evaluate precision editing strategies targeting core risk genes (APP, MAPT, APOE, and TREM2), explicitly highlighting the severe physiopathological trade-offs between therapeutic efficacy and loss-of-function toxicity. Finally, we address the ultimate translational bottlenecks impeding clinical application. By dissecting the packaging limits of adeno-associated viral vectors and the physical barricade of the blood-brain barrier, we underscore the necessity of transitioning toward next-generation base editors and non-viral lipid nanoparticles to realize safe and efficacious in vivo clinical gene therapies against Alzheimer's disease.},
}
@article {pmid42074029,
year = {2026},
author = {O'Hanlon Cohrt, K and O'Dea, S},
title = {Clinical Trial Landscape of Gene-Edited Autologous Hematopoietic Stem Cells for Hemoglobinopathies and Immunodeficiencies.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083384},
pmid = {42074029},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; *Hemoglobinopathies/therapy/genetics ; *Hematopoietic Stem Cell Transplantation/methods ; *Hematopoietic Stem Cells/metabolism ; Transplantation, Autologous ; Clinical Trials as Topic ; *Immunologic Deficiency Syndromes/therapy/genetics ; CRISPR-Cas Systems ; Genetic Therapy/methods ; },
abstract = {Allogeneic hematopoietic cell transplantation (HCT) has been used for decades to treat certain malignant and non-malignant hematological conditions, but challenges remain. Increased understanding of disease mechanisms and recent developments in genome editing have enabled alternative strategies utilizing gene-edited autologous HCT and many of these have progressed to the clinic. We present here a comprehensive review of clinical trials of gene-edited autologous hematopoietic stem cells for the treatment of hemoglobinopathies and immunodeficiencies. Searches of major international clinical trial registries were carried out using specific key words. In total, 44 interventional clinical trials investigating gene-edited autologous stem cell therapies were identified, with CASGEVY (exagamglogene autotemcel) being the only product approved to date. Hemoglobinopathies were the most common indication (n = 37) followed by immunodeficiencies (n = 4), with single trials in HIV-1 infection, pyruvate kinase deficiency and limb-girdle muscular dystrophy. Gene-editing strategies fall into three categories: disruption of the BCL11A erythroid enhancer, editing of the γ-globin promoter and direct correction or disruption of disease-relevant genes. CD34[+] hematopoietic stem and progenitor cells are the most common cell types edited, and CRISPR-Cas9 is the most widely used gene-editing modality. While results are encouraging, efficient intracellular delivery of gene-editing tools, editing efficiencies and off-target editing remain challenges for the field.},
}
@article {pmid42074108,
year = {2026},
author = {An, L and Xu, Z and Zhang, X},
title = {Self-Assembling Short Peptide Carriers for Gene Delivery.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083464},
pmid = {42074108},
issn = {1422-0067},
support = {12574240//National Natural Science Foundation of China/ ; 12174462//National Natural Science Foundation of China/ ; IS24073//Beijing Natural Science Foundation/ ; 2025XYCM83//Organized Research Program in Minzu University of China/ ; },
mesh = {Humans ; *Gene Transfer Techniques ; *Peptides/chemistry ; Animals ; Genetic Therapy/methods ; Gene Editing ; *Drug Carriers/chemistry ; CRISPR-Cas Systems ; },
abstract = {Gene therapy relies on safe and efficient delivery systems, yet traditional viral vectors and synthetic polymers often fail to meet these requirements due to immunogenicity and biocompatibility concerns. This review highlights self-assembling short peptides as a highly programmable and biocompatible non-viral platform uniquely positioned to overcome these translational bottlenecks. To provide a comprehensive overview of next-generation gene delivery, we systematically trace the trajectory from fundamental chemistry to clinical applications. First, we elucidate the supramolecular interactions and mechanisms driving peptide-nucleic acid co-assembly. Second, we outline concrete design strategies, detailing how sequence engineering and environmental responsiveness dictate the formation of optimized nanomorphologies. Third, we critically analyze how these nanocarriers navigate critical physiological and intracellular barriers, with a specific focus on cellular uptake, endosomal escape, and cargo release. Finally, we demonstrate the platform's versatility in emerging frontiers, particularly mRNA vaccines and CRISPR/Cas9 gene editing. We conclude by identifying current obstacles to clinical translation and proposing future directions centered on multifunctional integration and stimuli-responsive design.},
}
@article {pmid42074129,
year = {2026},
author = {Lan, Z and Tian, M and Liu, J and Shi, W and Chen, T and Ma, Q and Jin, B and Zhao, Y and Zhang, H and Lai, CJ and Cui, G},
title = {Divergent Roles of SmHMGR2 and a Novel SmHMGR5 in Tanshinone Biosynthesis Revealed by CRISPR/Cas9-Mediated Knockout in Salvia miltiorrhiza.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083485},
pmid = {42074129},
issn = {1422-0067},
support = {CI2021A05051//China Academy of Chinese Medical Sciences/ ; },
mesh = {*Salvia miltiorrhiza/genetics/metabolism ; *CRISPR-Cas Systems ; *Abietanes/biosynthesis ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Phylogeny ; *Hydroxymethylglutaryl CoA Reductases/genetics/metabolism ; },
abstract = {3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) serves as a key rate-limiting enzyme in the mevalonate pathway and plays a central regulatory role in the biosynthesis of tanshinones. To date, four HMGR family members (SmHMGR1-4) have been identified in Salvia miltiorrhiza. Here, we cloned and identified a novel member, SmHMGR5, by integrating multiple genomic datasets. Genomically, SmHMGR5 formed an inverted repeat with SmHMGR3 (98.04% homology) and phylogenetically clustered with SmHMGR2. Based on the expression patterns of the five HMGR genes, we further generated SmHMGR2 and SmHMGR5 knockout mutants using CRISPR/Cas9 technology and compared their effects on the accumulation of 12 tanshinones and 4 phenolic acids via UPLC-MS-based metabolomic analysis. Knockout of SmHMGR2 significantly suppressed the accumulation of seven tanshinones, whereas SmHMGR5 knockout downregulated only three tanshinones, and neither mutation affected phenolic acids. Notably, the major compound tanshinone IIA remained stable across different mutants, but tanshinone IIB was markedly reduced upon SmHMGR2 knockout, suggesting complex regulatory mechanisms in tanshinone biosynthesis. These findings provide new insights into the biosynthetic network of tanshinones and establish a theoretical foundation for metabolic engineering strategies aimed at enhancing the production of bioactive constituents in S. miltiorrhiza.},
}
@article {pmid42074203,
year = {2026},
author = {Guo, B},
title = {CRISPR Interference to Inhibit Oncogenes for Cancer Therapy.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083564},
pmid = {42074203},
issn = {1422-0067},
support = {1R01CA293945-02/NH/NIH HHS/United States ; },
mesh = {Humans ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; *Oncogenes/genetics ; Animals ; Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {CRISPR interference (CRISPRi), a programmable transcriptional repression technology derived from nuclease-deficient CRISPR-Cas systems, has emerged as a powerful method for selectively inhibiting oncogene expression without altering the genomic DNA. This feature offers a major advantage over other oncogene targeting technologies such as CRISPR-mediated gene knockout, mRNA inhibition by siRNA or miRNA, or small-molecule inhibitors of the proteins encoded by the oncogenes, especially in cancers driven by transcriptional dysregulation or otherwise undruggable oncogenes. Here, I present a comprehensive review of CRISPRi mechanisms, delivery strategies, and preclinical applications in oncology (including advances in targeting core oncogenic drivers like MYC and KRAS). The advantages of CRISPRi as well as in vivo validation of CRISPRi-mediated tumor suppression are discussed. Finally, I outline translational challenges and future directions for incorporating CRISPRi into precision cancer therapies. The accumulated evidence suggests that CRISPRi could become a cornerstone for next-generation gene-regulatory therapeutics.},
}
@article {pmid42074279,
year = {2026},
author = {Sterckel, S and Chávez Martínez, IL and Schwach, V},
title = {CRISPR and the Future of Cardiac Disease Therapy: A New Genetic Frontier.},
journal = {International journal of molecular sciences},
volume = {27},
number = {8},
pages = {},
doi = {10.3390/ijms27083641},
pmid = {42074279},
issn = {1422-0067},
support = {10250042110011//ZonMw, The Dutch Organisation for knowledge and innovation in health, healthcare and well-being/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Heart Diseases/therapy/genetics ; *Genetic Therapy/methods ; Animals ; },
abstract = {CRISPR technologies are transforming cardiovascular therapy development by creating an increasingly seamless pipeline from potential target discovery to clinical translation. What began as a genome-editing tool has evolved into a versatile platform that enables researchers to precisely interrogate and modulate cardiac biology with tools such as base- and prime-editors, and CRISPR inhibition and activation. In this review, we follow the use of CRISPR across the stages of biomedical research through to bench-to-bedside application. This review begins by addressing how genome-wide and focused CRISPR screens discover developmental regulators, disease drivers, and drug-response pathways, making the first steps in identifying therapeutic targets. We then explore how CRISPR engineering creates progressively more relevant disease model systems to validate mechanisms of disease and test interventions, helping bridge the translational gaps between the lab and the clinic. Finally, we consider how CRISPR technologies are beginning to enter cardiovascular clinical trials, while highlighting the key challenges that still limit this translation. By linking the latest advances of modern CRISPR platforms to the stages of therapeutic development, this review highlights how CRISPR technology is reshaping the pipeline from molecular insight to clinical innovation in cardiac disease.},
}
@article {pmid42074580,
year = {2026},
author = {Gonzalez-Chavez, Z and Siddiqui, MA and Ganesan, S and Desai, SA},
title = {A CRISPR-Based Mutagenesis Strategy for Examining CLAG3 Helix 44 Contribution to Malaria Parasite Nutrient Uptake Channels.},
journal = {Genes},
volume = {17},
number = {4},
pages = {},
doi = {10.3390/genes17040462},
pmid = {42074580},
issn = {2073-4425},
support = {ZIA AI000882-25//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Protozoan Proteins/genetics/metabolism/chemistry ; *Plasmodium falciparum/genetics/metabolism ; Mutagenesis ; CRISPR-Cas Systems ; Erythrocytes/parasitology/metabolism ; Humans ; *Ion Channels/genetics/metabolism ; Nutrients/metabolism ; },
abstract = {Background: Malaria parasites import essential nutrients from plasma into their host erythrocytes through the plasmodial surface anion channel (PSAC), a conserved ion and nutrient channel on the infected cell surface. A parasite-encoded ternary complex consisting of CLAG3, RhopH2, and RhopH3 determines PSAC activity, but the precise contributions of each member to formation of the nutrient uptake pore remains uncertain. Methods: Here, we devised a two-step CRIPSR transfection strategy to examine an amphipathic CLAG3 helix, termed α-helix 44 (α-H44), as a candidate pore-lining domain. Results: A CLAG3 truncation protein without α-H44 phenocopies a CLAG3 knockout line, suggesting a critical role of α-H44 in formation of the nutrient channel; CLAG3 restoration using a recodonized α-H44 restores PSAC activity fully. A saturation mutagenesis library that splits the helix into four sequential segments was devised and implemented. Two engineered mutants exhibit distinct PSAC phenotypes; their cultures failed to expand in a modified medium that approximates in vivo nutrient availability. Conclusions: These studies support a α-H44 role in channel permeation and block by a strain-specific inhibitor. Our strategy will enable saturation mutagenesis to determine how PSAC achieves its unique ion and nutrient selectivity and should help guide drug discovery against this antimalarial target.},
}
@article {pmid42075136,
year = {2026},
author = {Zhang, E and Yan, J and Du, J and Chu, X and Chen, D},
title = {Application and Research Prospects of CRISPR/Cas Gene Editing Technology in Lactic Acid Bacteria.},
journal = {Microorganisms},
volume = {14},
number = {4},
pages = {},
doi = {10.3390/microorganisms14040739},
pmid = {42075136},
issn = {2076-2607},
abstract = {Lactic acid bacteria (LAB) are pivotal microorganisms in the food industry. Current approaches for functional gene validation and trait improvement in LAB primarily rely on traditional gene editing and homologous recombination techniques. These methods are often cumbersome, inefficient, and time-consuming, hindering the rapid and precise customization of strains. This limitation has, to some extent, constrained the rapid selection and industrial application of functional LAB strains. The engineering of LAB through gene editing technologies has significantly advanced both fundamental and applied research. Among these, CRISPR/Cas gene editing has successfully achieved precise modification of multiple genes in various LAB species. Compared to conventional methods, it offers superior editing efficiency and lower operational costs, opening new avenues for functional gene identification and genetic improvement in LAB. However, the application of exogenous CRISPR/Cas systems in LAB faces technical challenges such as high off-target rates, chromosomal abnormalities, and cytotoxicity. The development of endogenous CRISPR/Cas-based editing tools for LAB provides novel pathways for precise regulation, rational design, and flexible application. This paper first outlines the structural components and mechanistic principles of CRISPR/Cas gene editing tools. It then explores the research progress and applications of both endogenous and exogenous CRISPR/Cas systems in LAB. Finally, it provides an outlook on the future application of CRISPR/Cas gene editing technology in LAB, offering a reference for its implementation in this field. The advent of gene editing technologies has significantly propelled functional gene validation and trait improvement in lactic acid bacteria (LAB), thereby advancing both fundamental research and industrial applications. Notably, the CRISPR/Cas system has emerged as a transformative tool enabling precise genetic modification in diverse LAB species, offering marked improvements in editing efficiency and cost reduction relative to conventional approaches. CRISPR/Cas-based editing strategies in LAB are broadly classified into exogenous and endogenous systems. Exogenous systems operate independently of the host's native immune repertoire, conferring the advantages of broad strain applicability and high editing efficiency. These systems have been successfully deployed for functional gene characterization, metabolic pathway engineering, such as augmenting antimicrobial production, and probiotic safety enhancement via virulence gene deletion. Conversely, endogenous systems leverage the intrinsic CRISPR/Cas machinery of LAB, offering superior biocompatibility and minimized off-target risks. Notable applications include precise gene knockout and integration using the native Type I-E system in Lacticaseibacillus paracasei. This review provides a concise overview of CRISPR/Cas system architecture and mechanisms, followed by a systematic synthesis of research progress and applications for both exogenous and endogenous systems in LAB. Finally, future directions are outlined to guide the continued development and application of CRISPR/Cas technologies in this field.},
}
@article {pmid42075335,
year = {2026},
author = {Liu, Q and Qiu, Z and Yao, M and Jiao, B and Zhou, Y and Li, C and Liu, H and Xin, L},
title = {Progress of Rapid Detection Technology for Aquatic Microorganisms: A Comprehensive Review.},
journal = {Microorganisms},
volume = {14},
number = {4},
pages = {},
doi = {10.3390/microorganisms14040939},
pmid = {42075335},
issn = {2076-2607},
support = {No. 2023YFD2403000//National Key Research and Development Program of China/ ; 2024KJN029//the Ministry of Science and Technology of the People's Republic of China, Shandong Provincial University Youth Innovation Team Project/ ; },
abstract = {Microbial contamination in aquatic environments poses severe threats to aquaculture sustainability, ecological balance and public health. Traditional culture-based detection methods, while standardized, are time-consuming and labor-intensive, often failing to meet the urgent need for rapid on-site monitoring required to prevent disease outbreaks and manage water quality effectively. By integrating latest research advances (2020-2025), this study reviews advances in rapid detection technologies for aquatic microorganisms, including the evolution of nucleic acid amplification strategies, with a focused comparison of the analytical sensitivity and field deployability of quantitative polymerase chain reaction (qPCR) and mainstream isothermal amplification techniques (loop-mediated isothermal amplification, LAMP; recombinase polymerase amplification, RPA). Furthermore, this study reports on the emergence of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) systems as next-generation diagnostic tools, highlighting their integration with microfluidic Lab-on-a-Chip (LOC) platforms to achieve attomolar sensitivity. We also consider the application of portable nanopore sequencing for real-time pathogen identification and the growing role of Artificial Intelligence (AI) in analyzing complex diagnostic datasets. Advanced molecular methods have achieved significant reductions in time consumption-from days to less than one hour-while challenges regarding sample preparation and environmental matrix inhibition remain. The future of aquatic monitoring lies in integrated, automated systems that combine the specificity of CRISPR-Cas diagnostics with the connectivity of IoT-enabled biosensors. Comparative analysis indicates that isothermal amplification methods (LAMP, RPA) coupled with CRISPR-Cas systems offer the optimal balance of sensitivity, speed, and field deployability for point-of-care aquaculture diagnostics, while qPCR/dPCR remain indispensable for quantitative regulatory applications. We propose a structured technology selection framework to guide researchers and practitioners in choosing appropriate detection modalities based on specific sensitivity, cost, throughput, and deployment requirements.},
}
@article {pmid42075363,
year = {2026},
author = {Di Pinto, A and Forte, V and D'Attilia, C and Possenti, M and Felici, B and Augelletti, F and Sessa, G and Carabelli, M and Morelli, G and Frugis, G and D'Orso, F},
title = {A Rapid Hairy Root-Based Platform for CRISPR/Cas Optimization and Guide RNA Validation in Lettuce.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {8},
pages = {},
doi = {10.3390/plants15081161},
pmid = {42075363},
issn = {2223-7747},
support = {CN00000022//European Union Next-GenerationEU/ ; A0375E0166//Lazio Innova/ ; },
abstract = {Cultivated lettuce (Lactuca sativa L.) is a major leafy crop and an emerging model for functional genomics within the Asteraceae family, supported by high-quality reference genomes and efficient transformation systems. Although CRISPR/Cas technology offers powerful opportunities for crop improvement, editing efficiency depends on optimized construct architecture and reliable guide RNA (gRNA) validation. However, a rapid platform for evaluating CRISPR reagents in lettuce is still lacking. Here, we developed an efficient hairyroot-based system to accelerate CRISPR/Cas genome editing optimization in L. sativa. Four Agrobacterium rhizogenes strains were compared for hairy root induction in two cultivars, 'Saladin' and 'Osiride', identifying strain ATCC15834 as the most effective based on transformation frequency and root production. Using this platform, we evaluated multiple CRISPR construct configurations, including alternative promoters for nuclease and gRNA expression. A plant-derived promoter combined with At-pU6-26 variant significantly improved editing efficiency. As a proof of concept, we targeted LsHB2, the putative ortholog of Arabidopsis thaliana ATHB2, a key regulator of the shade avoidance response using SpCas9, SaCas9, and LbCas12a nucleases. The system enabled rapid genotyping and quantitative indel profiling. Overall, this workflow provides a robust framework for efficient guide selection and construct optimization in lettuce genome editing.},
}
@article {pmid42079440,
year = {2026},
author = {Yang, L and Luo, R and Zhou, W and Yin, P and Feng, Y and Zhang, Y},
title = {Recent advances in noncanonical inhibition mechanisms of anti-CRISPR proteins.},
journal = {mLife},
volume = {5},
number = {2},
pages = {133-147},
pmid = {42079440},
issn = {2770-100X},
abstract = {The CRISPR-Cas system constitutes an adaptive immune mechanism in prokaryotes that defends against mobile genetic elements. Within the perpetual co-evolutionary arms race between bacteria and their viral predators, bacteriophages encode anti-CRISPR (Acr) proteins that use sophisticated molecular strategies to sabotage CRISPR-Cas function. While canonical Acr proteins rely on steric blockade of Cas effectors, recent discoveries reveal unprecedented noncanonical mechanisms spanning CRISPR immunity stages. This review synthesizes recent mechanistic advances in this field since 2023, highlighting the expansion of noncanonical inhibition mechanisms beyond type I to include types II, V, and VI, as well as novel Acr interventions targeting multiple functional stages, such as spacer acquisition, translation-coupled inhibition, complex assembly/disassembly, and R-loop DNA binding. Structural insights demonstrate how Acr proteins achieve substoichiometric inhibition via conformational hijacking, catalytic repurposing, and molecular mimicry. Forged by the intense selective pressure of the phage-host conflict, these molecular innovations represent both remarkable evolutionary adaptations and versatile precision tools. They enable spatiotemporal control of CRISPR technologies, from engineered off-switches to diagnostic reset mechanisms, while posing critical challenges for therapeutic safety and microbiome management.},
}
@article {pmid42079647,
year = {2026},
author = {Requejo Cier, CJ and Valentini, N and Boudreau, G and Delisle, JS and Lamarche, C},
title = {Engineering human Tregs to resist tacrolimus via FKBP12 gene editing.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1756624},
pmid = {42079647},
issn = {1664-3224},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology/drug effects/metabolism ; *Tacrolimus/pharmacology ; *Tacrolimus Binding Protein 1A/genetics/metabolism ; *Gene Editing ; Cell Proliferation/drug effects ; CRISPR-Cas Systems ; *Immunosuppressive Agents/pharmacology ; Interleukin-2 ; Lymphocyte Activation/drug effects ; Cells, Cultured ; },
abstract = {Regulatory T cells (Tregs) are essential for immune tolerance and are under active development as cell therapy in transplantation. However, the widespread use of the calcineurin inhibitor tacrolimus may inadvertently suppress Treg proliferation and activation, undermining their therapeutic potential. Tacrolimus binds to the FKBP12 protein in T cells, forming a complex that blocks calcineurin-NFAT signaling and suppresses IL-2 gene transcription, thereby inhibiting T cell activation. In this study, we investigated whether deleting FKBP12 in human Tregs could prevent tacrolimus-mediated suppression. Using CRISPR-Cas9 gene editing, FKBP12 was knocked out in ex vivo expanded human Tregs, which were then cultured for seven days with tacrolimus (10 ng/mL) or control, under varying IL-2 concentrations (100-500 IU/mL). We observed that tacrolimus significantly reduced the proliferation of control Tregs, even in conditions with 500 IU/mL IL-2, whereas FKBP12-knockout Tregs maintained robust proliferation comparable to untreated cells. We found no discernible changes in Treg phenotype or stability following FKBP12 deletion or tacrolimus exposure: edited Tregs retained normal expression of the lineage-defining marker FOXP3, displayed a global transcriptomic profile nearly indistinguishable from controls, and were similarly suppressive, indicating that they remained bona fide Tregs. These findings demonstrate that the antiproliferative effect of tacrolimus on Tregs is critically dependent on FKBP12, mirroring its mechanism in conventional T cells. By genetically uncoupling tacrolimus from its target in Tregs, this approach suggests a strategy to preserve Treg numbers during tacrolimus-based immunosuppression in transplant recipients, potentially enhancing Treg-based therapies for transplantation tolerance.},
}
@article {pmid42080108,
year = {2026},
author = {Yousefian, M and Baharmast, M},
title = {Artificial Intelligence-Assisted CRISPR Gene Editing: Current Advances, Clinical Challenges, and Future Directions in Precision Medicine.},
journal = {Avicenna journal of medical biotechnology},
volume = {18},
number = {1},
pages = {3-15},
pmid = {42080108},
issn = {2008-2835},
abstract = {Recent advances in Artificial Intelligence (AI) have profoundly transformed the field of genome editing, particularly through integration with the Clustered Regularly Inter-spaced Short Palindromic Repeats (CRISPR) technology. This review highlights how AI-driven computational models are reshaping guide RNA (gRNA) design, off-target prediction, and editing precision in CRISPR-Cas systems. A PRISMA-informed literature survey was conducted using PubMed, Scopus, EMBASE, and Google Scholar databases to identify studies exploring AI-assisted CRISPR applications in gene therapy and biomedical research. The results demonstrate that deep learning, machine learning, and reinforcement learning approaches significantly enhance prediction accuracy, algorithmic efficiency, and translational potential across genetic diseases such as β-thalassemia, muscular dystrophy, and cancer. Moreover, ethical challenges, algorithmic bias, and data security concerns remain critical barriers to clinical adoption. This review also discusses the emerging landscape of AI-assisted CRISPR research in Iran, emphasizing national progress, infrastructural constraints, and future opportunities. Overall, the convergence of AI and CRISPR technologies promises to advance precision medicine by accelerating the development of personalized, efficient, and ethically responsible genome-editing solutions.},
}
@article {pmid42080254,
year = {2026},
author = {Cho, SW and Kim, T and Yang, J and Byun, G and Seo, SW},
title = {Multiplexed CRISPR base editing enables pulse-activated irreversible biocontainment of engineered bacteria.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag422},
pmid = {42080254},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2024-0035256//Ministry of Science and ICT/ ; RS-2025-02214910//Ministry of Science and ICT/ ; RS-2025-02309093//Ministry of Science and ICT/ ; RS-2024-00345885//Korean government/ ; SRFC-MA1901-11//Samsung Research Funding &amp; Incubation Center of Samsung Electronics/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *CRISPR-Cas Systems ; Genes, Essential ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Escherichia coli Proteins/genetics ; },
abstract = {The environmental and therapeutic application of genetically engineered microorganisms necessitates the development of robust, irreversible biocontainment systems. In this study, we present an eEGM (editing-driven essential gene multiplex inactivation) module that utilizes CRISPR-mediated cytidine base editing to induce permanent self-killing via a single transient induction. By targeting the start codons of essential genes, we achieved an irreversible translational blockade that avoids the fitness costs associated with basal toxicity in nuclease-based systems. Multiplexed targeting of non-redundant essential loci (holA, ftsB, and dfp) yielded escape frequencies at or below the NIH guideline criterion (10-8) within 1 h of pulse induction. Furthermore, the eEGM system exhibited robust functional orthogonality and portability across laboratory, industrial, and therapeutic Escherichia coli strains, including MG1655, W3110, and Nissle 1917, without detectable interference with heterologous protein expression. This work establishes base editing as a cleavage-free CRISPR effector for pulse-activated, irreversible biocontainment and provides a practical framework for safer deployment of engineered microbes.},
}
@article {pmid42080258,
year = {2026},
author = {Yang, H and Shen, B and Wang, Y and Liu, J and Zhou, F and Liu, M and Li, J and Fan, J and Ding, S and Guo, J and Zhang, J and Li, X},
title = {Flexible regulation of CRISPR/Cas12a activity by spatial confinement effect.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag414},
pmid = {42080258},
issn = {1362-4962},
support = {82202649//National Natural Science Foundation of China/ ; 82202649//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0007//Chongqing Natural Science Foundation Joint Fund for Innovation and Development/ ; CSTB2023NSCQ-MSX0897//Natural Science Foundation of Chongqing/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Single-Stranded/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics ; HIV-1/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Precise regulation of the trans-cleavage activity of CRISPR/Cas12a has substantially expanded its utility in molecular diagnostics. However, existing strategies rely predominantly on systems with freely diffusing components, necessitating intricate CRISPR RNA (crRNA) designs or specialized chemical modifications, which hinder their simplicity and broader applicability. Here, we demonstrate that the activity of spatially confined Cas12a on fluid membranes (CAS-FLIER) can be facilely modulated by simply adjusting the length of crRNA and the duplex-strand reporters. We reveal that fine-tuning the movement range of membrane-Cas12a and the accessibility of the reporter to Cas12a enables precise, scalable control over trans-cleavage activity. As a proof of concept, we show that the activity of confined Cas12a can be co-activated by single-stranded DNA (ssDNA) and RNA inputs, a capability that remains unattainable in conventional freely diffusing systems. Furthermore, by incorporating a DNA reverse-transcriptor into the CAS-FLIER system, we achieve one-pot, highly sensitive detection of HIV RNA, supporting accurate diagnosis of HIV infection. Notably, this assay is compatible with a lateral-flow format for direct visual readout, highlighting its potential as a point-of-care diagnostic tool for HIV. Collectively, our findings shed new light on modulating Cas12a activity, advancing its applications in molecular diagnostics.},
}
@article {pmid42080260,
year = {2026},
author = {Luo, W and Wu, Y and Ni, D and Zhang, L and Zhang, Y and Han, X and Zhang, Y and Pu, J and He, Y and Yin, N and Wang, W and Huang, R and Guo, Y and Sun, Y and Xie, G},
title = {A universal and orthogonal safety valve for CRISPR/Cas12a without chemical modification or external stimulation.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag436},
pmid = {42080260},
issn = {1362-4962},
support = {82372351//National Natural Science Foundation of China/ ; 82572673//National Natural Science Foundation of China/ ; 82501041//National Natural Science Foundation of China/ ; BJRC202410//Chongqing Medical University/ ; GZC20251421//National Postdoctoral Researcher Support Program/ ; CSTB2025NSCQ-GPX1184//Chongqing Natural Science Foundation General Project/ ; HBRC202404//Chongqing National Reserve Talent Program in Health and Wellness/ ; CSTB2025NSCQ-JQX0016//Chongqing Outstanding Youth Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *CRISPR-Associated Proteins/metabolism/genetics/antagonists &amp; inhibitors/chemistry ; Humans ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; *Bacterial Proteins/genetics/metabolism ; RNA/genetics/antagonists &amp; inhibitors/chemistry ; Proprotein Convertase 9/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {CRISPR/Cas-based gene editing technologies have achieved remarkable progress over the past decade, yet their broad practical applications remain limited by safety concerns. Although regulatory strategies applied before or during CRISPR/Cas activation have substantially improved sequence, temporal, and spatial specificity, persistent activity of already activated Cas nucleases may still increase the risk of uncontrolled editing. Therefore, an effective post-activation control strategy is urgently needed. Here, we report a modification- and stimulation-free RNA inhibitor (iRNA) that functions as a post-activation safety valve for CRISPR/Cas12a. By exploiting Cas12a's allosteric sensitivity and the thermodynamic and kinetic programmability of nucleic acid strand displacement, iRNA drives already activated Cas12a ribonucleoproteins back to an inactive state, enabling universal, sequence-programmable, and orthogonal post-activation inhibition within the validated Cas12a framework. Experiments and simulations elucidate the mechanistic basis of iRNA-mediated strand displacement and demonstrate its high inhibitory efficiency, reversible cyclic control, compatibility, expandability, orthogonality, and universality. Importantly, iRNA also acts as a programmable, autonomously operating safety valve in cells, suppressing uncontrolled editing while preserving PCSK9 gene knockout. With its simple design, excellent biocompatibility, and autonomous intracellular expression, iRNA provides a foundation for next-generation controllable CRISPR systems and holds broad potential for precision therapeutics, cell therapy, and molecular diagnostics.},
}
@article {pmid42080263,
year = {2026},
author = {Feng, W and Hu, J and Zhang, H and Le, XC},
title = {A kinetic approach for mapping seed regions of CRISPR ribonucleoprotein and improving specificity.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag413},
pmid = {42080263},
issn = {1362-4962},
support = {TIPS CBRF2-2023-001//Canadian Biomedical Research Fund/ ; RGPIN-2024-05974//Natural Sciences and Engineering Research Council of Canada/ ; 22506171//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Kinetics ; *Ribonucleoproteins/metabolism/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Binding ; Binding Sites ; },
abstract = {The binding of an activator (target nucleic acid) to a crRNA-Cas ribonucleoprotein (RNP) in CRISPR systems is critical to the activation, kinetics, and specificity of the CRISPR technology. Key to this activation process is the interaction between the protospacer region of the activator and the spacer region of the crRNA in the RNP complex. However, how the nucleotides in the spacer region of the crRNA contribute to the kinetics of RNP binding is not well characterized. We report here profiling of the kinetically critical regions in the process of RNP binding to activators (RNA targets). We introduced the concept and strategy of kinetic manipulators, which enabled mapping of the seed regions (6-9 nucleotides within the spacer that is sensitive to mismatches) of the CRISPR-Cas13a system, including the LbuCas13a and LwaCas13a homologs. The characterization of the binding kinetics and the introduction of kinetic manipulators provided the foundation for a new kinetic approach to improve the specificity of CRISPR techniques without sacrificing the activity. Profiling the kinetically critical regions in the CRISPR system and designing corresponding manipulators maximized the kinetic differences, between the on-target and off-target, and increased discrimination of single-nucleotide mismatches.},
}
@article {pmid42080266,
year = {2026},
author = {Purcell, J and Liu, L and Calvert, RW and Hayes, BK and Huang, C and Davidovich, C and Knott, GJ and Rosenbluh, J},
title = {DUSP11 is an RNA triphosphatase that limits PspCas13b activity by destabilizing gRNA abundance in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag412},
pmid = {42080266},
issn = {1362-4962},
support = {APP2011329//National Health and Medical Research Council/ ; //Australian Government Research Training Program Scholarship/ ; APP1175568//National Health and Medical Research Council/ ; SMRF2021-276//Snow Medical Research Foundation/ ; },
mesh = {Humans ; CRISPR-Cas Systems ; *Dual-Specificity Phosphatases/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; HEK293 Cells ; *Mitogen-Activated Protein Kinase Phosphatases/genetics/metabolism ; RNA Stability ; Animals ; Gene Knockout Techniques ; RNA Polymerase III/metabolism/genetics ; },
abstract = {The CRISPR-Cas13 system enables programmable RNA targeting with potential applications in therapeutics and research. However, while PspCas13b mediates efficient RNA knockdown following transient transfection, stable lentiviral delivery results in minimal activity, limiting its utility. Here, we performed a genome-wide CRISPR-Cas9 knockout screen to identify mammalian factors that restrict PspCas13b activity. We discovered that DUSP11, an RNA triphosphatase, suppresses PspCas13b function by dephosphorylating the 5'-triphosphate of Pol III-transcribed guide RNAs (gRNAs), triggering their degradation. DUSP11 knockout increased gRNA levels 2.5-4-fold and enhanced PspCas13b-mediated knockdown across multiple cell lines. This enhancement was sustained for at least 27 days and enabled targeting of endogenous transcripts previously refractory to PspCas13b. Our findings reveal an unexpected host restriction of bacterial CRISPR systems and demonstrate that gRNA levels are a limiting factor. We provide a simple strategy to improve PspCas13b activity in mammalian cells. These results have implications for developing PspCas13b-based therapeutics and suggest that systematic identification of host factors regulating CRISPR components could enhance genome editing technologies.},
}
@article {pmid42080267,
year = {2026},
author = {Pan, L and Sang, R and Xue, R and Ma, Y and Goldys, E and Deng, F},
title = {AlphaFold3-guided tracrRNA redesign yields small monomeric Cas12f RNPs.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag430},
pmid = {42080267},
issn = {1362-4962},
support = {//Henan Province High-level Talent International Exchange Project/ ; 252300423880//Henan Provincial Natural Science Foundation/ ; //UNSW SHARP/ ; 2024/ECF1573//Cancer Institute NSW/ ; DP240103024//ARC/ ; 2030464//NHMRC/ ; 2024/ECF1573//Cancer Institute NSW/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; *Ribonucleoproteins/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *CRISPR-Cas Systems ; Gene Editing ; DNA, Single-Stranded/metabolism ; Models, Molecular ; Protein Multimerization ; },
abstract = {Although Cas12f (Cas14) is among the smallest Class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effectors, it assembles into dimeric ribonucleoprotein (RNP) complexes with guide RNA, substantially increasing its functional size and limiting its suitability for gene editing and biosensing applications. To overcome this limitation, we systematically investigate the structural and functional roles of Cas12f dimerization using a combination of computational modeling and experimental validation. Structural analysis using Protein Data Bank data and AlphaFold-3 predictions revealed that the 5'-end sequence of tracrRNA is essential for dimer formation but dispensable for substrate cleavage. Based on this, we designed a truncated tracrRNA by removing 70 nucleotides from its 5'-end. This shortened tracrRNA successfully loaded into Cas12f to form a one guide RNA-one Cas12f monomer RNP. This functionally monomeric RNP demonstrated substantially enhanced trans-cleavage activity: 4.5-fold for ssDNA, 3.5-fold for dsDNA, and 2.5-fold for RNA, resulting in markedly improved detection sensitivity: 10-fold for ssDNA and dsDNA, and 4-fold for RNA. In addition, the functionally monomeric RNP exhibits cis-cleavage activity and gene editing efficiency comparable to that of the dimeric RNP, thereby restoring the advantage of Cas12f as a compact enzyme for in vivo gene editing. These results highlight that the functionally monomeric Cas12f RNP combines enhanced biosensing performance with retention of its uniquely compact size, benefiting gene editing applications.},
}
@article {pmid42080294,
year = {2026},
author = {Liu, Q and Guan, J and He, X and Xie, P and Zhao, Z and Liu, X and Lee, TY and Chiang, YC and Yao, L},
title = {EnAcrPred: A robust ensemble machine learning framework for identifying anti-CRISPR proteins.},
journal = {Protein science : a publication of the Protein Society},
volume = {35},
number = {6},
pages = {e70559},
doi = {10.1002/pro.70559},
pmid = {42080294},
issn = {1469-896X},
support = {JCYJ20230807114206014//Science, Technology and Innovation Commission of Shenzhen Municipality/ ; 2025A1515011753//Guangdong Province Basic and Applied Basic Research Fund/ ; 2025XAKJ0102017//Scientific Research Foundation of State Key Laboratory of Vaccines for Infectious Diseases, Xiang An Biomedicine Laboratory/ ; 20720250172//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Machine Learning ; *CRISPR-Cas Systems ; Gene Editing ; *Software ; },
abstract = {The identification of anti-CRISPR proteins (Acrs) is crucial for understanding the regulation of CRISPR-Cas systems and their application in gene editing. However, current experimental methods face challenges, particularly in detecting Acrs with low similarity to known protein sequences. To address these challenges, we propose EnAcrPred, an advanced prediction framework based on ensemble learning. The model combines features such as sequence composition, order correlation, and inferred structure and utilizes a stacking ensemble architecture to integrate multiple base models, which enhances both the accuracy and generalization ability of the predictions. Experimental results demonstrate that EnAcrPred achieves superior performance over existing methods across multiple evaluation metrics, further confirming its robustness. Additionally, SHapley Additive exPlanations (SHAP) value analysis identifies the key features influencing Acrs recognition. To facilitate broad adoption in practice, we developed an online platform where users can quickly obtain Acrs predictions by entering a protein sequence. EnAcrPred offers an effective solution for Acrs identification, contributing to the advancement of gene editing research and safety. The platform is accessible via the link at https://ycclab.cuhk.edu.cn/EnAcrPred/.},
}
@article {pmid42080605,
year = {2026},
author = {Merwaiss, F and Aragonés, V and García, A and Daròs, JA},
title = {A Modified Cas9 Scaffold Allows Extension of the Virus-Induced Gene Editing Technology to the Large Potyvirus Genus.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70675},
pmid = {42080605},
issn = {1467-7652},
support = {PID2023-146418OB-I00//Spanish Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; FPU20/05477//Spanish Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; CIPROM/2022/21//Generalitat Valenciana/ ; },
abstract = {Plant viruses are recognized as rapid and effective vectors to deliver CRISPR-Cas reaction components into plants, a strategy termed virus-induced gene editing (VIGE). However, VIGE is limited by the host range of the viral vectors. Development of new viral vectors to target a broad range of plant species will potentially enable the delivery of the editing components to new cultivars. Potyviruses (genus Potyvirus) comprise the largest group of plant RNA viruses. The main limitation of potyviral vectors to express a non-coding RNA consists of potential insertion of stop codons that interrupt the large open reading frame that encompasses most potyviral genome. This is the case with the Streptococcus pyogenes Cas9 sgRNA scaffold, which contains stop codons in all three possible frames. In this work, we first built on a visual reporter system targeting the two homeologs of Nicotiana benthamiana Magnesium chelatase subunit I (CHLI). Second, we developed a tobacco etch virus (Potyvirus nicotianainsculpentis)-derived vector for VIGE by engineering a modified Cas9 scaffold, free of stop codons, to maintain the potyviral polyprotein reading frame while ensuring effective editing. This vector self-replicates and moves systemically, delivering sgRNAs efficiently throughout the plant. This allowed us to obtain plants exhibiting a white phenotype with their four alleles edited through in vitro regeneration from infected leaves, and also to produce edited progeny. We further demonstrated the vector utility in tomato. Given the conserved biological properties within the genus Potyvirus, these findings may be broadly applicable to other potyviruses, expanding the reach of the VIGE technology.},
}
@article {pmid42081667,
year = {2026},
author = {Xie, H and Geng, L and Hu, Z and Chen, F and Tan, M and Wang, D and Huang, Z and Ye, W and Chen, P and Zhu, J},
title = {Genome editing generates high oleic soybean and eliminates beany flavors.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70282},
pmid = {42081667},
issn = {1744-7909},
abstract = {Soybeans serve as excellent sources of vegetable oil, protein, and other valuable nutrients for human consumption, materials for diverse industries, including the cosmetics and medical industries, and feedstocks for animals. Nevertheless, some people do not favor soy oil or other various food products derived from soybeans, due to inadequate levels of oleic acid in the oil and the presence of undesirable grassy and beany flavors associated with oxidation products of polyunsaturated fatty acids in the seeds. In this study, we developed soybean cultivars with very high levels of oleic acid in the seeds, and without grassy and beany flavors. We achieved this by using CRISPR-Cas-SF01 to edit genes in the elite cultivar Xudou 18 (XD18), targeting two microsomal Δ-12 fatty acid desaturase 2 (GmFAD2-1A and GmFAD2-1B) and three lipoxygenase (GmLOX1, GmLOX2, and GmLOX3) genes. Our findings showed that fad2-1a/b and fad2-1a/b/lox1/2/3 plants performed similarly to XD18 plants in the field, indicating no obvious growth penalties. Overall, this research has demonstrated that the development of soybean germplasms with high levels of oleic acid and without undesirable beany flavors through gene-editing of multiple relevant genes is effective, and this endeavor can contribute to the health of a broader global consumer population.},
}
@article {pmid42082082,
year = {2026},
author = {Saliani, N and Hejazi, MS and Vahed, SZ and Aghdam, EM and Mori, Z and Hasheminejad, N and Parvar, MD and Montazersaheb, S and Ebrahimi, V},
title = {Synthetic Biology-Driven Innovations in Triple-Negative Breast Cancer: Integrating Engineering Design with Targeted Therapeutics.},
journal = {Journal of biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jbiotec.2026.04.017},
pmid = {42082082},
issn = {1873-4863},
abstract = {Triple-negative breast cancer (TNBC) is a highly aggressive malignancy with limited therapeutic options and poor clinical outcomes due to the absence of hormone-responsive receptors. The advent of synthetic biology, which integrates molecular biology with engineering design principles, has introduced new opportunities to develop precise and programmable therapeutic and diagnostic strategies for TNBC. Engineered immune cells, such as chimeric antigen receptor (CAR)-T constructs, can selectively recognize tumor-associated antigens and overcome immunosuppressive barriers. Synthetic gene circuits and engineered bacteria enable tumor-specific delivery of cytotoxic or immunomodulatory agents, while induced pluripotent stem cells (iPSCs) provide patient-specific platforms for disease modeling and drug screening. In parallel, CRISPR/Cas-based genome editing facilitates targeted modulation of oncogenic and tumor-suppressor networks, offering both mechanistic insights and therapeutic innovation. This review highlights current advances in synthetic biology-driven approaches for TNBC, encompassing cell-based, microbial, and nucleic acid-engineered systems. It also discusses their synergistic potential to mitigate tumor heterogeneity, enhance therapeutic specificity, and overcome drug resistance. Collectively, the intersection of synthetic biology, immuno-oncology, and precision medicine holds significant promise for next-generation, adaptive, and patient-tailored treatments for TNBC.},
}
@article {pmid41634255,
year = {2026},
author = {Liu, G and Tian, X and Ye, L and Han, P and Wang, Q and Cao, Y and Wu, Y and Lu, Y},
title = {CRISPR-Cas9-mediated uATG introduction in the 5'UTR of the Uox gene for hyperuricemia mouse models: implications for gout and metabolic disorders.},
journal = {Science China. Life sciences},
volume = {69},
number = {5},
pages = {1620-1633},
pmid = {41634255},
issn = {1869-1889},
mesh = {Animals ; *Hyperuricemia/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Mice ; Humans ; *5' Untranslated Regions/genetics ; Gene Editing/methods ; HEK293 Cells ; *Gout/genetics/metabolism ; *Metabolic Diseases/genetics ; Gene Knockdown Techniques ; Uric Acid/blood ; Male ; Mice, Inbred C57BL ; },
abstract = {Sequence-specific gene knockdown technologies are crucial for fundamental research and therapeutic applications. RNA interference and CRISPR interference, while extensively utilized for gene expression manipulation, face limitations due to their ectopic or transient expression. In this study, we developed a generalizable and efficient method to downregulate gene expression in human 293T cells by introducing de novo upstream ATGs (uATGs) of genes using CRISPR-Cas9-mediated genome editing. Through CRISPR library screening, in-depth sequencing, and flow cytometry analysis, we validated that the introduction of uATGs served as an effective method to suppress protein expression. Our findings further revealed that this strategy can be tailored to diminish endogenous gene expression in tumor cells without affecting the mRNA transcription levels. Importantly, by introducing a uATG into the 5' untranslated region (UTR) of the Uox gene, we successfully established a Uox-knockdown (KD) mouse model of hyperuricemia associated with metabolic disorders. This model demonstrated hyperuricemia, with serum uric acid levels that exceeded 400 µmol L[-1], along with renal dysfunction, as indicated by elevated serum creatinine and blood urea nitrogen levels. Examination of the kidneys from 8-week-old Uox-KD mice revealed abnormal histopathological characteristics, including partial dilation of Bowman's capsules and renal tubules, focal nephron collapse and necrosis, and lymphocytic infiltration. In addition, the mice exhibited lipid and glucose metabolism disorders, all while maintaining a normal lifespan. This spontaneous hyperuricemia model has potential as a valuable tool for long-term studies on hyperuricemia and gout. Taken together, we present an efficient approach for the constant suppression of specific gene expression in mammalian cells and the development of a Uox-KD mouse model of hyperuricemia via CRISPR-Cas9-mediated uATG introduction. This offers broad implications for fundamental research and therapeutic applications.},
}
@article {pmid41866453,
year = {2026},
author = {Wang, X and Xie, Y and Lin, Q and Xiong, Y and Liu, Y and Ge, S and Tan, Q and He, Z and Jiang, Y and Han, Q and Jin, S and Huang, P and Wang, Y and Guo, W and Ren, F and Gui, JF and Mei, J},
title = {Genetic deletion of miR-200a/200b increases growth and feed conversion efficiency in yellow catfish.},
journal = {Science China. Life sciences},
volume = {69},
number = {5},
pages = {1674-1687},
pmid = {41866453},
issn = {1869-1889},
mesh = {Animals ; *MicroRNAs/genetics ; *Catfishes/genetics/growth &amp; development/metabolism ; Animal Feed ; *Gene Deletion ; Polymorphism, Single Nucleotide ; Energy Metabolism/genetics ; Genome-Wide Association Study ; Animals, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {The most effective approach for minimizing feed cost and maximizing animal production is the creation of breeding materials with simultaneous increases in growth and feed conversion efficiency (FCE). However, the key genes that regulate FCE are unknown. Here, we artificially selected specific strains of yellow catfish with simultaneous improvements in growth and FCE traits and then conducted a genome-wide association study to screen candidate SNPs and genes associated with these traits. A particular locus in the miR-200 cluster on chromosome 23 was identified, and the causal relationships between miR-200a/200b expression and growth/FCE were further validated. Genetic deletion of miR-200a/200b by CRISPR/Cas9 in yellow catfish significantly underpins phenotypic gains in growth and FCE by regulating genes involved in energy intake and energy metabolism without significantly affecting average feed intake or the expression of appetite-regulating genes. Several critical target genes of miR-200a/200b, such as stat5b and fasn, were identified via RNA-RNA pulldown and RNA-seq analyses, and stat5b-transgenic yellow catfish exhibited significantly increased growth and FCE. These findings highlight the pivotal role of the miR-200a/200b-stat5b signaling axis in controlling growth, metabolism, and FCE in yellow catfish, thus providing a strategy toward achieving more effective and sustainable animal agriculture by gene editing.},
}
@article {pmid42068455,
year = {2026},
author = {Chakraborty, A and Yu, ASL},
title = {Miniaturization of CRISPRa plasmids for efficient delivery into renal epithelial cells and Pkd1 transactivation.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42068455},
issn = {1573-4978},
support = {24PRE1194472//American Heart Association/ ; U54 DK126126/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *TRPP Cation Channels/genetics/metabolism ; *Plasmids/genetics ; Mice ; Epithelial Cells/metabolism ; Kidney/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Transcriptional Activation/genetics ; Polycystic Kidney, Autosomal Dominant/genetics/therapy ; Genetic Vectors/genetics ; Gene Editing/methods ; },
abstract = {BACKGROUND: Autosomal Dominant Polycystic Kidney Disease is caused by loss-of-function mutations in PKD1 or PKD2 genes, leading to reduced polycystin protein levels. Increasing PKD1 expression via CRISPR activation (CRISPRa) represents a promising therapeutic strategy; however, delivery of large CRISPRa plasmids into renal epithelial cells, and particularly primary cells, remains inefficient due to size-related barriers. We aimed to enable Pkd1 transactivation by miniaturizing CRISPRa plasmids into ~ 6 kb vectors using a one-pot method to enhance cellular uptake in mouse kidney epithelial cells.<br><br>METHODS AND RESULTS: Using type IIS restriction enzymes, we excised the mammalian expression cassette from full-length large 9-11 kB plasmids. The excised cassette was engineered to have complimentary overhangs. Thermocycling with T4 DNA ligase promoted circularization of the excised cassette (forming&#x2009;~&#x2009;6kB mini-CRISPRa vectors), and T5 exonuclease digestion removed residual backbone fragments. These mini vectors substantially enhanced nucleofection efficiency from 16.10% &#xb1; 0.53 to 54.17% &#xb1; 2.10 in Pkd1[RC/-] cells, and from 10.14% &#xb1; 1.40 to 31.27% &#xb1; 0.12 in primary Pkd1[RC/Cond]; Pkhd1[Cre+] cells. Functionally, the mini-CRISPRa plasmid (mdCas9-VPR) with Pkd1-targeting sgRNAs induced robust endogenous Pkd1 upregulation compared with non-targeting controls: a 4.1-fold increase in Pkd1[RC/-] cells (p&#x2009;<&#x2009;0.001) and a 2.9-fold increase in primary cells (p&#x2009;<&#x2009;0.001). Full-length plasmids produced no significant activation in either cell type.<br><br>CONCLUSIONS: Miniaturization of CRISPRa vectors with this one-pot approach overcomes delivery limitations in hard-to-transfect renal epithelial cells and enables efficient, functional Pkd1 activation, in vitro.},
}
@article {pmid42069941,
year = {2026},
author = {Singh, A and Bhattacharjee, S and Singh, Y and Kostova, I},
title = {Parabiotics as Next-Generation Microbiome Therapeutics: Insights into Mechanisms, Evidence, and Therapeutic Potential.},
journal = {Current microbiology},
volume = {83},
number = {6},
pages = {},
pmid = {42069941},
issn = {1432-0991},
mesh = {Humans ; *Prebiotics/administration &amp; dosage ; Animals ; *Gastrointestinal Microbiome/drug effects ; Probiotics ; *Microbiota ; },
abstract = {Parabiotics (also termed paraprobiotics) are defined as non-viable microbial cells or their components, including peptidoglycans, teichoic acids, surface proteins, that confer health benefits without requiring viability which distinguishes them from traditional probiotics. Their non-viable nature eliminates risks such as microbial translocation, bacteremia, and sepsis, making them suitable for vulnerable populations including immunocompromised, critically ill, paediatric and elderly individuals. In addition, parabiotic exhibit improved thermal stability, extended shelf life, and easier incorporation into functional foods, nutraceuticals, and pharmaceutical formulations without cold-chain requirements. Mechanistically, parabiotics retain immunomodulatory, anti-inflammatory and have barrier-enhancing activities through interactions with host pattern recognition receptors, including Toll-like receptors, modulation of cytokine responses, and reinforcement of gut epithelial integrity. Preclinical and clinical studies support their therapeutic potential such as in case of heat-killed Lactobacillus acidophilus LB (L. acidophilus) has shown efficiency in managing acute paediatric diarrhoea, while heat-inactivated Lacticaseibacillus paracasei PS23 (Lcb. paracasei) has demonstrated improvements in muscle strength and inflammatory markers, including reduced C-reactive protein and interleukin-6 and increased interlukin-10 in elderly individuals. Similarly, inactivated Lactiplantibacillus plantarum (Lpb. plantarum) and Bifidobacterium strains have been associated with benefits in irritable bowel syndrome, atopic dermatitis, respiratory infections, visceral fat reduction, and antibiotic-associated dysbiosis. Synergistic combinations with prebiotics, postbiotics and related bioactives further enhance therapeutic outcomes in inflammatory, metabolic and infectious conditions. Advances in metagenomics, next-generation sequencing, proteomics, metabolomics, CRISPR-Cas systems, and synthetic biology are accelerating strain characterization, functional evaluation, and scalable production. Despite ongoing challenges in standardization and regulated harmonization, parabiotics represent a safe and effective approach for microbiome-targeted interventions. This review synthesizes current evidence on their therapeutic applications, technological advancements, and translational potential, highlighting their role in precision health and next-generation functional nutrition.},
}
@article {pmid42070239,
year = {2026},
author = {Geerthana, S and Yogi, D and Kumar, A and Asokan, R and Suresh, K and Prabhakar, N and Rani, BU and Thangaraj, K and Paramasivam, M and Kaninika, V and Chiranth, RK and Shankarnarayan, AM and Pradeep, C and Manamohan, M},
title = {Towards CRISPR/Cas9 Genome Editing in Spodoptera frugiperda: A Proof-of-Concept Targeting Wing and Eye Color Genes.},
journal = {Archives of insect biochemistry and physiology},
volume = {122},
number = {1},
pages = {e70161},
doi = {10.1002/arch.70161},
pmid = {42070239},
issn = {1520-6327},
support = {//ICAR-IASRI for funding under the CABin programme/ ; },
mesh = {Animals ; *Gene Editing ; *CRISPR-Cas Systems ; *Spodoptera/genetics/growth &amp; development ; Wings, Animal ; *Eye Color/genetics ; Pigmentation/genetics ; Tryptophan Oxygenase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; },
abstract = {Fall armyworm (Spodoptera frugiperda), a globally significant destructive lepidopteran invasive pest, has recently invaded Africa and Asia, threatening food security. Conventional method of management, including chemical insecticides, are often ineffective due to various reasons compelling the need to explore alternative strategies. In this regard, CRISPR/Cas9 based genome editing has emerged as a powerful tool for functional genomics in insects, enabling to introduce site-specific mutations for various purposes. In this study, we applied multiplex CRISPR/Cas9 ribonucleoprotein (RNP) injections to disrupt two key genes in S. frugiperda: the spalt (Sfspalt), which regulates wing patterning, and tryptophan 2,3-dioxygenase (Sfto/vermillion) gene, involved in eye pigmentation. Microinjection of sgRNA/Cas9 ribonucleoprotein complex into freshly laid eggs resulted in distinct phenotypic alterations, including altered wing pigmentation and modified eyespot patterns, as well as golden-yellow eye color mutants. Genotyping and ICE analysis confirmed the presence of frameshift mutations in the target loci, supporting the phenotypic changes. Notably, while mutations were detected, only a single individual was confirmed to carry mutations in both genes simultaneously. These results demonstrate the technical feasibility of multiplex CRISPR/Cas9 editing in S. frugiperda, but also reveal a low frequency of confirmed events under the present experimental conditions. Therefore, this study is considered a proof of concept establishing a preliminary workflow in multiplex platform. The findings provide foundational insights for further optimization of genome editing strategies targeting sex related genes in this agriculturally important pest.},
}
@article {pmid41870705,
year = {2026},
author = {Khoshraftar, SH and Alirezae, P and Kiani Darabi, AH and Hadi, S and Gholami, A and Amirfiroozi, A and Pourseif, MM and Mansoori-Derakhshan, S},
title = {The role of circular RNAs as miRNA sponges in the mechanisms and therapeutic potential of triple negative breast cancer.},
journal = {Discover oncology},
volume = {17},
number = {1},
pages = {},
pmid = {41870705},
issn = {2730-6011},
support = {73169//Tabriz University of Medical Sciences/ ; },
abstract = {Triple-negative breast cancer (TNBC) is an aggressive subtype of breast carcinoma which lacks estrogen receptors, progesterone receptors and HER2 along with limited therapeutic options mainly based on chemotherapy. In this review, we outline the emerging function of circRNAs as key regulators in TNBC pathogenesis. CircRNAs are endogenous non-coding RNAs with a closed-loop structure, in contrast to the linear form. In TNBC cells, the underlying molecular mechanism mainly relies on their functions as a competitive sponge of miRNAs, which can absorb or bind to microRNAs (miRNAs) and hence regulate the expression of target genes. Such sponging can result in the activation of oncogenes or repression of tumor suppressor genes, which eventually affect cellular proliferation, apoptosis, and drug sensitivity. Crucial mechanisms include certain circRNAs, such as circEPSTI1, circRAD18, and hsacirc0000199 that enhance tumorigenesis and resistance to chemotherapy by targeting tumor-suppressor miRNAs and activation of oncogenic pathways (e.g., PI3K/Akt/mTOR pathway or Wnt/β-catenin). The potential clinical implications and dysregulation of circRNA-miRNA axes are highlighted, indicating that these may constitute promising diagnostic or prognostic markers by their stability in biofluids. Additionally, this review outlines the innovative treatment approaches regarding these interactions which have recently been addressed and described, novel methods include ASOs therapy, CRISPR/Cas system and nanoplatforms that may help to get over current therapeutic drawbacks in treating TNBC patients.},
}
@article {pmid40356240,
year = {2025},
author = {Coveney, CR and Maridas, D and Chen, H and Muthuirulan, P and Liu, Z and Jagoda, E and Yarlagadda, S and Movahhedi, M and Proffen, B and Dashtdar, B and Aghaalikhani, M and Richard, D and Rosen, V and Kiapour, AM and Capellini, TD},
title = {Complex Regulatory Interactions at GDF5 Shape Joint Morphology and Osteoarthritis Disease Risk.},
journal = {Arthritis &amp; rheumatology (Hoboken, N.J.)},
volume = {77},
number = {11},
pages = {1488-1502},
pmid = {40356240},
issn = {2326-5205},
support = {//Institutional Centers for Clinical and Translational Research at Boston Children's Hospital/ ; R01 AR070139/AR/NIAMS NIH HHS/United States ; P30 AR075042/AR/NIAMS NIH HHS/United States ; //Harvard University Dean's Competitive Fund/ ; 1R01AR070139/NH/NIH HHS/United States ; 1UM1TR004408-01/NH/NIH HHS/United States ; //Harvard Clinical and Translational Science Center/ ; //The Children's Orthopaedic Surgery Foundation/ ; UM1 TR004408/TR/NCATS NIH HHS/United States ; //Harvard University Milton Fund/ ; P30 AR075042/NH/NIH HHS/United States ; 1R01AR070139/NH/NIH HHS/United States ; 1UM1TR004408-01/NH/NIH HHS/United States ; P30 AR075042/NH/NIH HHS/United States ; },
mesh = {*Osteoarthritis/genetics/metabolism/pathology ; *Growth Differentiation Factor 5/genetics/metabolism ; Disease Models, Animal ; Gene Expression Regulation ; Humans ; Female ; Animals ; Mice/embryology/metabolism ; Male ; Cartilage, Articular/metabolism/pathology ; Joints/metabolism ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {OBJECTIVE: The objective of this study was to reveal causal-level osteoarthritis (OA) disease biology by targeting regulatory interactions at GDF5.<br><br>METHODS: By investigating different GDF5 regulatory regions (R2, R3-R5, R7-R9, R18-R20, GROW1), we explored their functional impacts on gene expression and joint morphology in vivo and in vitro. We additionally modeled OA variants in said enhancers in in vitro and in vivo mouse models for expression and disease effects.<br><br>RESULTS: For all regulatory regions, we found evidence of activation and repression between or within said regions that impacted patterns of joint-specific expression. Examples are as follows: (1) the R4 enhancer, although considered to be activating, has dual roles repressing expression in adjacent tissues and sites, and (2) growth plate-specific expression patterns by the GROW1 regulatory region are confined by adjacent sequences to restrict its expression to the perichondrium. We next targeted different regions and variants in vivo. Testing the R2de region resulted in ~40% reduction in Gdf5 expression and joint morphology changes but no increase in OA risk; likewise, modeling the most cited OA risk variant (rs143384) in mice had no impact on expression, joint morphology, or disease. However, we identified epistatic interactions between this rs143384 risk variant and downstream disease risk variants lying within regulatory regions subject to repression, which compound to impact expression.<br><br>CONCLUSION: These findings, at the best studied OA locus to date, serve as lessons on the nature of how gene regulatory interactions and local epistasis work in the etiology of OA disease risk, and that assessment of individual variants of high genome-wide association study significance need not alone be considered causal.},
}
@article {pmid41394860,
year = {2025},
author = {Andersch, L and Grunewald, L and Stecklum, M and Klironomos, F and Haase, K and Hollek, V and Lam, T and Jung, BA and Winkler, A and Schwiebert, S and Astrahantseff, K and Launspach, M and Jens, M and Henssen, A and Kloke, L and Blüthgen, N and Eggert, A and Schulte, JH and Anders, K and Künkele, A},
title = {Investigating genetic modifications to enhance L1CAM-CAR T cell migration in solid tumors in a 3D bioprinted neuroblastoma model.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1677361},
pmid = {41394860},
issn = {1664-3224},
mesh = {*Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; *Neuroblastoma/immunology/pathology/therapy ; Bioprinting ; *Immunotherapy, Adoptive/methods ; *Cell Movement/genetics/immunology ; *T-Lymphocytes/immunology/metabolism ; Receptors, Chimeric Antigen/immunology/metabolism ; Neural Cell Adhesion Molecule L1/immunology/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Membrane Glycoproteins/genetics/metabolism ; Adaptor Proteins, Signal Transducing/deficiency/genetics/metabolism ; Xenograft Model Antitumor Assays ; Single-Cell Gene Expression Analysis ; Humans ; Female ; Animals ; Mice ; Cell Line, Tumor ; Coculture Techniques ; Gene Expression Regulation/immunology ; },
abstract = {INTRODUCTION: Effective CAR T cell infiltration into solid tumors remains a major barrier to therapy success. Despite their clinical potential, few studies have evaluated phenotypes of CAR T cells successfully invading the tumor mass following infusion. Phenotypic information would enrich our understanding of the mechanisms governing CAR T cell migration into solid tumors. Here we implemented an in vitro strategy to identify genes driving L1CAM-CAR T cell migration into a 3D tumor mass.<br><br>METHODS: L1CAM-CAR T cells were separated into 2 groups by their capability to infiltrate (or not) a 3D bioprinted neuroblastoma model. Single-cell and bulk RNA sequencing was performed, and infiltrating CAR T cells were compared to noninfiltrating cells to seek genetic drivers of CAR T cell migration. CRISPR/Cas9 technology was used to generate modified L1CAM-CAR T cells.<br><br>RESULTS: Tumor-infiltrating L1CAM-CAR T cells expressed lower levels of the selectin P ligand (SELPLG) glycoprotein and higher levels of the T cell-specific adaptor protein, SH2D2A. Functional characterization of L1CAM-CAR T cells genetically modified to enforce these characteristics demonstrated that neither trait negatively impacted L1CAM-CAR T cell cytotoxicity, activation and cytokine release upon coculture with neuroblastoma target cells. Transgenic SH2D2A expression did not improve CAR T cell migration in an endothelial transmembrane assay. SELPLG knockout benefited CAR T cell in vitro trans-endothelial migration, but did not enhance anti-tumor efficacy in an immunodeficient mouse model.<br><br>DISCUSSION: Our findings reveal a key limitation of murine xenograft models, which are widely used as the gold standard for preclinical CAR T cell testing. The lack of conservation between the human and murine SELPLG proteins likely accounts for the discrepancy between enhanced in vitro migration of SELPLG-deficient L1CAM-CAR T cells and their lack of improved efficacy in the mouse model. This underscores the need for more predictive human-relevant models to better preclinically evaluate CAR T cell function.},
}
@article {pmid41877120,
year = {2026},
author = {Wang, Y and Cao, M and Hu, M and Xu, H and Du, Y and Sun, C and Kong, L and Luo, Y and Liu, X and Yang, J and Liang, B},
title = {Systematic engineering of Escherichia coli for biosynthesis of 3-hydroxypropionic acid from glucose and malonate.},
journal = {BMC biotechnology},
volume = {26},
number = {1},
pages = {},
pmid = {41877120},
issn = {1472-6750},
support = {22278233//National Natural Science Foundation of China/ ; 22378222//National Natural Science Foundation of China/ ; ZR2025MS127//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Malonates/metabolism ; *Metabolic Engineering/methods ; *Glucose/metabolism ; *Lactic Acid/analogs &amp; derivatives/biosynthesis/metabolism ; CRISPR-Cas Systems ; Fermentation ; Malonyl Coenzyme A/metabolism ; },
abstract = {BACKGROUND: 3-Hydroxypropionic acid (3-HP) is a promising C3 platform chemical with wide industrial applications. However, its microbial production remains limited by insufficient intracellular malonyl-CoA availability and metabolic imbalance.<br><br>RESULT: In this study, we systematically engineered Escherichia coli for enhanced 3-HP biosynthesis. The malonate assimilation genes (matB, smatPQM) and 3-HP biosynthesis gene (mcr) were chromosomally integrated using CRISPR/Cas9, resulting in a plasmid-free, antibiotic-free strain (WYY04) that produced 21.97 mM 3-HP, 0.51-fold higher than the plasmid-based system. Further improvement was achieved by CRISPRi-mediated repression of fatty acid biosynthesis genes (fabD, fabF), increasing 3-HP titer by 66%. Introduction of a malonyl-CoA-responsive FapR/fapO biosensor enabled dynamic regulation of mcr expression, enhancing 3-HP production by 59%. Through all these above engineering, the 3-HP production of the strain WYY19 increased by 2.29 times compared to that of the plasmid-expressing system. Under optimized fermentation conditions, the final engineered strain WYY19 produced 42.22&#xa0;g/L 3-HP with the specific productivity of 0.69&#xa0;g/g and 0.46&#xa0;g/L/h from glucose and malonate in fed-batch bioreactor.<br><br>CONCLUSIONS: This study demonstrates a robust, genetically stable, and scalable microbial platform for 3-HP biosynthesis.},
}
@article {pmid41945404,
year = {2026},
author = {Yu, F and Zhang, D and Peng, C and Qin, H and Xu, F and Zhang, Y and Pan, Z and Xiao, P and Li, N},
title = {Development of a rapid and sensitive single-tube RAA-CRISPR/Cas12a assay for monkeypox virus detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {16},
pages = {3312-3320},
doi = {10.1039/d6ay00065g},
pmid = {41945404},
issn = {1759-9679},
mesh = {*Monkeypox virus/isolation &amp; purification/genetics ; *Mpox, Monkeypox/diagnosis/virology ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; Sensitivity and Specificity ; Limit of Detection ; Recombinases/metabolism ; },
abstract = {Monkeypox (mpox) is a zoonotic disease caused by the monkeypox virus (MPXV), with outbreaks historically concentrated in West and Central Africa. The current global outbreak of MPXV highlights the urgent need for an efficient detection system. This study presents a novel single-tube recombinase-aided amplification (RAA)-CRISPR/Cas12a assay for the rapid, sensitive, and specific detection of MPXV. Optimized to achieve a fluorescence-based sensitivity as low as 0.5 copies per µL within 35 min at 37 °C, the system demonstrates excellent specificity, accurately distinguishing MPXV from other viruses, including vaccinia virus, with no cross-reactivity observed. Additionally, a lateral flow assay (LFA) format was developed, enabling visual detection of concentrations as low as 5 copies per µL within 40 min. Validated with simulated clinical samples, the assay achieved 100% accuracy in distinguishing positives from negatives. This integrated single-tube approach eliminates the need for costly thermal cyclers, simplifying the detection process, reducing contamination risks, and delivering reliable results in a short time, making it ideal for point-of-care testing (POCT) in resource-limited settings. Furthermore, the RAA-CRISPR/Cas12a platform offers significant cost savings by requiring fewer reagents for virus detection. The extremely low template volume requirement of just 1 µL maximizes detection efficiency. This configuration allows for repeated testing without compromising result integrity, further enhancing the utility of this approach for surveillance and outbreak control, particularly in low-resource environments.},
}
@article {pmid41960606,
year = {2026},
author = {Zhang, X and Nie, X and Yu, W and Du, G and Liu, S and Song, Y},
title = {Catalytic hairpin assembly-assisted split-T7 promoter-regulated CRISPR/Cas12a system for the sensitive analysis of microRNAs associated with coronary heart disease.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {16},
pages = {3303-3311},
doi = {10.1039/d6ay00524a},
pmid = {41960606},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *Coronary Disease/genetics/diagnosis ; *Promoter Regions, Genetic/genetics ; *Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) have emerged as promising non-invasive biomarkers for coronary heart disease (CHD); however, their accurate quantification remains challenging due to their short length, low abundance, and high sequence homology. Herein, we report a highly sensitive and specific biosensing platform by integrating catalytic hairpin assembly (CHA) with a split-T7 promoter-regulated CRISPR/Cas12a system. The key innovation lies in the split-T7 promoter architecture, which enables target-responsive reconstitution of a functional T7 promoter, driving robust in vitro transcription to generate abundant crRNA transcripts eliminating the need for pre-synthesized exogenous crRNA. This strategy achieves triple cascade amplification through three synergistic modules: CHA-mediated target recycling ensures efficient signal initiation; split-T7 promoter-driven transcription provides substantial signal enrichment; and CRISPR/Cas12a-based trans-cleavage delivers highly specific fluorescence readout. By eliminating pre-synthesized crRNA, the platform significantly reduces assay cost and complexity while maintaining excellent amplification efficiency. The method achieves a limit of detection as low as 38.9 aM for miRNA-155 within 90 min, exhibits single-base mismatch discrimination capability, and performs reliably in clinical specimens from CHD patients. With its modular design and robust performance, this cascade amplification platform offers a versatile and cost-effective tool for miRNA analysis, holding great promise for cardiovascular disease diagnosis and broader molecular diagnostics.},
}
@article {pmid42064640,
year = {2026},
author = {Pandey, H and Sharma, A and Misra, V and Mall, AK and Ceasar, SA},
title = {Pre-validation strategies for CRISPR/Cas-based genome editing in plants: a critical analysis of in vitro RNP cleavage assays.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {32},
number = {4},
pages = {677-691},
pmid = {42064640},
issn = {0971-5894},
abstract = {The advent of CRISPR/Cas-based genome editing has revolutionized crop improvement. However, the genome editing success rate remains a major challenge in many crops, especially those with challenging transformation protocols. We critically evaluate the integration of in vitro cleavage assays using naked target DNA and guide RNA-Cas9 nuclease (gRNA-Cas9) ribonucleoprotein (RNP) complexes as a pre-transformation validation step in genome editing workflows. We also compare other pre-validation methods with in vitro cleavage assays and present their advantages and limitations. In vitro assays can help directly confirm target cleavage and biochemically validate gRNA specificity. This strategy may facilitate the functional screening of gRNAs for plants with challenging and low transformation efficiency. In vitro assays can also reduce the unnecessary waste of resources and time associated with intensive transformation processes using non-specific gRNAs. Researchers can prioritize effective constructs based on the cleavage efficiency and specificity of the gRNAs. However, this assay may not guarantee simulation of the natural cellular environment for in vivo editing. We also present this pre-validation approach, which is particularly helpful for polyploid crops like wheat and cotton. In vitro cleavage assays offer a reliable pre-transformation screening step to identify highly active and specific gRNAs, thereby reducing resource-intensive transformation attempts. Future studies should integrate in vitro assays with advanced computational and in vivo validation tools to create a more predictive and efficient gRNA selection pipeline.},
}
@article {pmid42065027,
year = {2026},
author = {Ezeobiora, CE and Igbokwe, NH and Amin, DH and Mendie, UE},
title = {Rare endophytic actinobacteria from nigeria harbor unique biosynthetic gene clusters with novel antibiotic potential.},
journal = {3 Biotech},
volume = {16},
number = {5},
pages = {174},
pmid = {42065027},
issn = {2190-572X},
abstract = {UNLABELLED: Actinobacteria are prolific producers of specialized metabolites, including antibiotics; however, much of their biosynthetic potential remains unexplored, particularly within rare genera. This study presents the first genomic insights into the biosynthetic capacity of two rare endophytic actinobacteria, Saccharomonospora xinjiangensis strain PNSac2 and Saccharopolyspora cebuensis strain PGLac3, isolated from medicinal plants in Nigeria. The strains were characterized using morphological analysis, molecular sequencing, phylogenetic inference, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH). Whole-genome sequencing revealed that PNSac2 possesses a 4.7 Mb genome with 45 tRNA genes, 3 rRNA operons, and 4,541 coding sequences (CDSs), while PGLac3 harbors a 6.4 Mb genome comprising 48 tRNA genes, 4 rRNA operons, and 6,372 CDSs. Genome mining using antiSMASH identified 24 biosynthetic gene clusters (BGCs) in PNSac2 and 28 in PGLac3, including clusters encoding polyketides, nonribosomal peptides, siderophores, terpenes, and ribosomally synthesized and post-translationally modified peptides (RiPPs). Many BGCs showed low similarity to known clusters, indicating a strong potential for novel metabolite discovery. Notably, PNSac2 encoded BGCs related to bleomycin, oxalomycin, desertomycin, and ossamycin, while PGLac3 harbored predicted arylpolyene, lanthipeptide, and a unique lassopeptide cluster. Comparative genomics revealed conserved synteny with related species alongside strain-specific BGCs, and phylogenomic analysis confirmed their taxonomic placement. Overall, these findings highlight the untapped biosynthetic diversity of rare Nigerian endophytic actinobacteria and underscore their promise as sources of novel antimicrobial compounds. Targeted genome engineering approaches, including CRISPR-Cas-based strategies, may further enable the activation and exploitation of cryptic biosynthetic pathways in these strains.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-026-04781-4.},
}
@article {pmid42067284,
year = {2026},
author = {Zhang, X and Tian, C and Wang, M and Jia, H and Li, X and Tian, G},
title = {Performance enhancement of CRISPR-Cas system based on improved guide RNA: a review.},
journal = {Analytica chimica acta},
volume = {1406},
number = {},
pages = {345472},
doi = {10.1016/j.aca.2026.345472},
pmid = {42067284},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Humans ; Biosensing Techniques ; },
abstract = {BACKGROUND: CRISPR-Cas technology has emerged as a transformative tool with widespread applications in gene editing and biosensing research; nevertheless, it is plagued by a suite of performance-related bottlenecks, including suboptimal targeting efficiency, undesirable off-target effects, insufficient sensitivity and recognition specificity, restricted target scope, limited multiplexing capacity, incompatible reaction systems, and compromised stability. As gRNA optimization has emerged as a core strategy to address these bottlenecks, there is an urgent need to consolidate recent breakthroughs in this rapidly advancing field. Existing literature lacks a comprehensive, focused synthesis of how gRNA optimization mitigates these key limitations, alongside an analysis of current challenges and future directions.<br><br>RESULTS: Herein, this review comprehensively summarizes recent breakthroughs in augmenting CRISPR-Cas system performance through guide RNA (gRNA) optimization, and further dissects the current challenges, future prospects, and promising research directions in this rapidly advancing field.<br><br>SIGNIFICANCE: It is timely to guide researchers in overcoming CRISPR-Cas performance barriers and accelerating its applications in gene editing and biosensing.},
}
@article {pmid42067668,
year = {2026},
author = {Wu, X and Lam, WH and Zhao, Z and Cao, Y and Lin, H and Feng, X and Zhai, Y and Hsing, IM},
title = {DNA-guided CRISPR-Cas12a effectors for programmable RNA recognition and cleavage.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {42067668},
issn = {1546-1696},
support = {16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 6107-20G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 6107-20G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16303522//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 16304225//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C6053-25G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C7035-23GF//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C6053-25G//Research Grants Council, University Grants Committee (RGC, UGC)/ ; C7035-23GF//Research Grants Council, University Grants Committee (RGC, UGC)/ ; 325014//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {CRISPR-Cas effectors typically rely on RNA guides to recognize target sequences. In Cas12a, the protospacer adjacent motif on DNA engages conserved protein residues, triggering target binding and nuclease activation. Here we reprogram Cas12a into a DNA-guided, RNA-targeting effector. Exploiting protospacer-adjacent motif-dependent interaction, we engineer synthetic CRISPR DNA that engages Cas12a to form a functional deoxyribonucleoprotein complex, while repurposing solely RNA as the programmable target. Structural, biophysical and biochemical analyses reveal the molecular basis of this DNA-guided, RNA-targeting configuration and support an activation pathway distinct from that of canonical RNA-guided systems. DNA-guided Cas12a enables direct RNA detection and efficient intracellular RNA knockdown, establishing a modular activation architecture for CRISPR-Cas12a and expanding the design space for programmable RNA manipulation.},
}
@article {pmid41520142,
year = {2026},
author = {Brown, A and Strickland, LM and Erman, EN and Pirozzi, CJ and Low, JT and Diplas, BH and Gibson, E and Shobande, M and Khambati, T and Krylova, M and Liu, H and McLendon, RE and Reitman, ZJ and Keir, ST and Zou, L and Ashley, DM and Waitkus, MS},
title = {SMARCAL1 is a targetable synthetic lethal therapeutic vulnerability in ATRX-deficient gliomas that use alternative lengthening of telomeres.},
journal = {Neuro-oncology},
volume = {28},
number = {4},
pages = {895-910},
pmid = {41520142},
issn = {1523-5866},
support = {P50 CA108786/CA/NCI NIH HHS/United States ; F32 CA298007/CA/NCI NIH HHS/United States ; P50 CA190991/CA/NCI NIH HHS/United States ; K22 CA258965/CA/NCI NIH HHS/United States ; R01 CA091438/CA/NCI NIH HHS/United States ; P30 CA014236/CA/NCI NIH HHS/United States ; T32 GM145449/GM/NIGMS NIH HHS/United States ; //The Preston Robert Tisch Brain Tumor Center/ ; //Uncle Kory Foundation Seed/ ; SPORE 5P50CA190991//Duke Brain Tumor/ ; 1K22CA258965-01A1/GF/NIH HHS/United States ; 1F32CA298007-01/GF/NIH HHS/United States ; },
mesh = {Humans ; *X-linked Nuclear Protein/deficiency/genetics ; Animals ; *Glioma/genetics/pathology/metabolism ; *Telomere Homeostasis ; Mice ; *Brain Neoplasms/genetics/pathology/metabolism ; Xenograft Model Antitumor Assays ; *Telomere/genetics ; *DNA Helicases/genetics ; Tumor Cells, Cultured ; *Synthetic Lethal Mutations ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {BACKGROUND: Approximately 10% of cancers achieve replicative immortality through a telomerase-independent mechanism of telomere maintenance, termed Alternative Lengthening of Telomeres (ALT). ALT is particularly prevalent in certain subtypes of malignant gliomas, such as IDH-mutant astrocytoma and pediatric glioblastoma, and frequently co-occurs with ATRX (ATRX chromatin remodeler) inactivating mutations. Although ALT is an adaptive mechanism through which cancer cells achieve proliferative immortality, the elevated levels of replication stress observed in ALT tumors constitute a potential therapeutic vulnerability.<br><br>METHODS: Leveraging CRISPR/Cas9 screening data from the Cancer Dependency Mapping Project, coupled with patient-derived cell lines and xenografts, we identified SMARCAL1 as a novel synthetic lethal vulnerability in ATRX-deficient glioma models that engage ALT. Using complementary molecular assays for DNA damage, telomere maintenance, and telomeric replication stress, we define the mechanisms underlying cytotoxicity induced by SMARCAL1 depletion in ALT-positive glioma cells.<br><br>RESULTS: Our data demonstrate the annealing helicase SMARCAL1 is a highly specific synthetical lethal vulnerability in cancers that use ALT. SMARCAL1 localizes to ALT-associated PML (Promyelocytic leukemia protein) bodies in ALT-positive glioma cell lines, including IDH-mutant astrocytomas. SMARCAL1 depletion, via doxycycline-induced RNAi, led to a hyperactivation of the ALT phenotype, high levels of DNA double-strand breaks in G2 phase, and cell death via mitotic catastrophe. In mice bearing intracranial xenografts derived from high-grade IDH-mutant astrocytoma, inducible SMARCAL1 depletion prolonged animal survival.<br><br>CONCLUSIONS: Our findings demonstrate that the molecular processes orchestrating ALT-mediated telomere maintenance constitute a targetable synthetic lethal vulnerability that can be exploited by SMARCAL1 inhibition, thus supporting the future development of small molecule inhibitors of SMARCAL1 as anti-cancer therapeutics.},
}
@article {pmid41851324,
year = {2026},
author = {Zaboroski-Silva, I and da Silva Brandão, E and de Freitas Brenha, B and Landry, C and Carrara, J and Ferreira, RS and Vaz, IM and Jamur, VR and Schaffer, AE and Miranda, HC and Shigunov, P},
title = {Impact of the CYFIP2 R87C variant in a human neuronal model in vitro.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41851324},
issn = {2045-2322},
support = {PDPG-FAP 88887.629870/2021-0//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; R01 NS121374/NS/NINDS NIH HHS/United States ; 15/2019-PROEP/ICC-442324/2019-7//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; PEP ICC-007-FEX-23-2-1-36//Financiadora de Estudos e Projetos/ ; R01 NS121374/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *Neurons/metabolism/cytology ; *Neural Stem Cells/metabolism/cytology ; Organoids/metabolism/cytology ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Cell Differentiation/genetics ; Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Mutation ; Cell Line ; Neurogenesis/genetics ; },
abstract = {Mutations in the CYFIP2 gene, particularly the R87C variant, are associated with severe epileptic encephalopathy, and present challenges for therapeutic development. This study utilized CRISPR/Cas9-edited human pluripotent stem cell (hPSC) lines to investigate the impact of R87C variant on neuronal morphology and function. hPSCs were differentiated into neural progenitor cells (NPCs), cortical neurons (CNs), and cortical organoids. Phenotypic characterization included immunofluorescence, scanning electron microscopy (SEM), high-throughput scanning (HTS), multi-electrode array (MEA) recordings, and Western blotting. Edited hPSC lines maintained pluripotency, and neurogenic differentiation yielded NPCs and CNs without significant differences in neural progenitor marker expression. However, mutated NPCs exhibited reduced motility in cell tracking assays, and SEM revealed altered cell morphology, suggesting an impact on lamellipodia formation. While both mutant and wild-type CNs expressed appropriate neuronal and glial markers and showed similar electrophysiological properties, R87C/R87C cortical organoids displayed decreased CYFIP2 protein levels and, by day 30, showed increased size alongside an absence of SOX2 + cells, suggesting premature depletion of the progenitor pool. These findings highlight a marked divergence between 2D and 3D models, with organoids revealing neurodevelopmental abnormalities not evident in monolayer cultures. Together, our results suggest that the CYFIP2 R87C variant impacts NPC cytoskeletal dynamics and early cortical development, warranting further investigation into its role in epileptic encephalopathy.},
}
@article {pmid42044121,
year = {2026},
author = {Varderesian, HV and Utaegbulam, JN and Brown, HE and Ramirez, B and Velcani, M and Ryder, SP},
title = {The pos-1 3' untranslated region governs germline specification and proliferation to ensure reproductive robustness.},
journal = {PLoS genetics},
volume = {22},
number = {4},
pages = {e1012129},
pmid = {42044121},
issn = {1553-7404},
mesh = {Animals ; *3' Untranslated Regions/genetics ; Caenorhabditis elegans/genetics ; *Caenorhabditis elegans Proteins/genetics/metabolism ; *RNA-Binding Proteins/genetics/metabolism ; Female ; Male ; Germ Cells/metabolism ; Gene Expression Regulation, Developmental ; Cell Proliferation/genetics ; Zygote/metabolism/growth &amp; development ; Reproduction/genetics ; Fertilization/genetics ; Oocytes/metabolism ; Fertility/genetics ; CRISPR-Cas Systems ; },
abstract = {During fertilization, haploid gametes combine to form a zygote. The male (sperm) and female (oocyte) gametes contribute a similar amount of DNA, but the oocyte contributes nearly all the cytoplasm. Oocytes are loaded with maternal mRNAs thought to be essential for embryonic patterning after fertilization. A conserved suite of RNA-binding proteins (RBPs) regulates the spatiotemporal translation and stability of maternal mRNAs. POS-1 is a CCCH-type tandem zinc finger RBP expressed in fertilized Caenorhabditis elegans zygotes from maternally supplied mRNA. POS-1 accumulates in the posterior of the embryo where it promotes posterior cell fate. Here, we show that the pos-1 3' untranslated region (UTR) is essential for POS-1 patterning and contributes to maximal reproductive fecundity. We engineered a pos-1 mutant where most of the endogenous pos-1 3'UTR was removed using CRISPR genome editing. Our results show that the 3'UTR represses POS-1 expression in the maternal germline but increases POS-1 protein levels in embryos after fertilization. In a wild-type background, POS-1 repression via the 3'UTR has little impact on fertility. In a sensitized background, the deletion mutant has a complex pleiotropic phenotype where most adult homozygous progeny lack either one or both gonad arms. Most phenotypes become more penetrant at elevated temperature. Together, our results support an emerging model where the 3'UTRs of maternal transcripts, rather than being essential, contribute to reproductive robustness during stress.},
}
@article {pmid42057199,
year = {2026},
author = {Hatem, H and Mysara, M and Ramadan, R},
title = {Trends of nucleic acid - based point-of-care diagnostics for infectious diseases.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {},
pmid = {42057199},
issn = {1754-1611},
abstract = {UNLABELLED: The global emergence and spread of infectious diseases highlights the importance of having an easily accessible, decentralized testing modality that is adept at quickly identifying the nucleic acids present in a Point-of-Care (PoC) setting. PoC-based nucleic acid diagnostics encompass a broad range of platforms, such as lateral flow, biochips, and biosensors, whose primary goal has always been to offer sensitive, selective, and economically viable testing beyond conventional laboratory facilities. The current review illustrates a comprehensive overview of PoC-based nucleic acid diagnostics for infectious diseases, as it is divided into three major operational steps: nucleic acid extraction, amplification, and detection. Firstly, it describes strategies that could effectively work as a PoC, such as magnetic bead-based, paper-based, and integrated microfluidic approaches, while further focusing on their ability to remain simplified, rugged, and equipment-free. Secondly, it summarizes key amplification methods, such as Polymerase Chain Reaction (PCR), Nucleic Acid Sequence-Based Amplification (NASBA), Recombinase Polymerase Amplification (RPA), and Loop-Mediated Isothermal Amplification (LAMP), as they have all been modified to effectively work under more rapid, low-power, and portable conditions. Lastly, it introduces all presently known detection platforms, starting from simpler colorimetric and fluorescent assays present in lateral flow or device platforms, to innovative and advanced biosensors like those exploiting CRISPR/Cas systems and toehold switch principles, as these detectively offer highly sensitive and highly programmable nucleic acid recognition.<br><br>GRAPHICAL ABSTRACT: [Image: see text]},
}
@article {pmid42058176,
year = {2026},
author = {McNiven, C and Carnielli Trindade, JB and Geoghegan, V and Faria, JRC and Mottram, JC},
title = {CRISPR-Cas9 precision editing of kinetochore protein phosphosite codons in Leishmania mexicana.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1788564},
pmid = {42058176},
issn = {2235-2988},
mesh = {*Leishmania mexicana/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Kinetochores/metabolism ; *Protozoan Proteins/genetics/metabolism ; Phosphorylation ; *Codon/genetics ; },
abstract = {Leishmania mexicana, like other trypanosomatids, possess a unique kinetochore-the protein complex crucial for chromosome segregation during mitosis. To investigate the functional significance of specific phosphorylation sites on essential kinetochore proteins, we adapted a selection-free precision editing strategy using CRISPR-Cas9 in Leishmania mexicana promastigotes. Our method targeted genomic DNA with 160-bp double-stranded DNA repair templates and guide RNAs to introduce targeted modifications. We focused on six phosphosites within the kinetochore proteins KKT2, KKT4, and KKT7, generating phosphodeficient, phosphomimetic, and synonymous mutants for each site. Across 18 independent transfections, we achieved a successful editing rate of 27.5% as determined by PCR screening, with 30.4% of clones confirmed as edited by Sanger sequencing. A significant portion of these edited clones (22.1%) were homozygous. Despite these precise genomic modifications, none of the phosphosite mutant clones exhibited any apparent growth defects or cell cycle dysregulation, suggesting these phosphorylation sites individually may not be critical for these processes under standard culture conditions. To facilitate higher-throughput precision editing, we developed a Python script that automates the design of the 160 bp repair templates. This script uses a FASTA file, a codon usage table, and a simple configuration file to design templates with a single nonsynonymous mutation and additional synonymous mutations for screening purposes. It also generates a corresponding synonymous-only repair template and primers for both screening and repair template generation, offering a "ready-to-go" approach. While designed for Leishmania, this powerful tool is adaptable for use with other kinetoplastids.},
}
@article {pmid42059198,
year = {2026},
author = {Hellmer, H and Mayer, T and Bauersachs, L and Simmel, FC},
title = {Operating CRISPR/Cas12a in a complex nucleic acid sequence background.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag390},
pmid = {42059198},
issn = {1362-4962},
support = {SI761/5-1//Deutsche Forschungsgemeinschaft/ ; 453249455//Deutsche Forschungsgemeinschaft/ ; CRC392 TP A5//Deutsche Forschungsgemeinschaft/ ; 521256690-TPA5//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; DNA/metabolism/genetics/chemistry ; Kinetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Gene Editing/methods ; Base Composition ; Base Sequence ; Humans ; },
abstract = {Since their discovery, CRISPR-Cas systems have been widely applied in areas ranging from genome editing to biosensing, owing to their specific, RNA-guided target recognition. Their performance in complex biological environments has been extensively studied, particularly to optimize guide RNA (gRNA) design and minimize off-target cleavage. Here, we focus on the kinetic inhibition of the interaction between Cas12a-a Class 2, Type V effector-and its target, caused by interference from non-cognate background nucleic acids. This effect is particularly relevant for sensing applications in complex mixtures or cellular contexts, where genome- and transcriptome-derived sequences may impede CRISPR-Cas activity. Using in vitro assays under defined conditions, we systematically examine the influence of background single-stranded RNA and double-stranded DNA (dsDNA) on reaction kinetics. We find that both the purine-to-pyrimidine ratio and the GC content of the gRNA seed region significantly affect kinetic inhibition by background polynucleotides. gRNAs with low GC content and a high purine fraction in the seed region were least affected by background sequences. A gRNA with high uracil content in the seed region exhibited particularly strong inhibition in the presence of a dsDNA background. Experiments with dCas12a-based gene activation in living cells indicate that our in vitro findings may also be relevant for in vivo applications.},
}
@article {pmid42060714,
year = {2026},
author = {Jung, M and Wen, Z and Humbert, S and Lu, F and DeLeon, A and Marshall, L and Hastings, C and Cartwright, H and Thilges, K and Wang, N and Breckenridge, K and Wu, E and Ryan, L and Fengler, K and Simcox, K and Thatcher, S and Llaca, V and Woollums, G and Sander, J and Xu, D and Beatty, M and Brink, K and Fedorova, M and Jones, M and Ohlson, E and Suresh, LM and Beyene, Y and Olsen, M and Ogugo, V and Alakonya, A and Murithi, A and Mugo, S and Karanja, J and Boddupalli, P and Pixley, K and Albertsen, M and Jones, T and Meeley, R and Gutterson, N and Mazur, B and Dhugga, KS},
title = {Targeted knockout of a host peroxisomal peptidase confers field resistance to maize lethal necrosis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {18},
pages = {e2535202123},
doi = {10.1073/pnas.2535202123},
pmid = {42060714},
issn = {1091-6490},
support = {OPP1193833//Bill and Melinda Gates Foundation (GF)/ ; INV-007833/GATES/Gates Foundation/United States ; },
mesh = {*Plant Diseases/virology/genetics ; *Zea mays/virology/genetics ; *Disease Resistance/genetics ; *Peroxisomes/enzymology/genetics ; Potyvirus/pathogenicity ; Gene Knockout Techniques ; *Peptide Hydrolases/genetics/metabolism ; Quantitative Trait Loci/genetics ; Tombusviridae/pathogenicity ; CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; },
abstract = {Maize lethal necrosis (MLN) is a severe disease caused by the combined infection of maize chlorotic mottle virus (MCMV) and a potyvirus, most often sugarcane mosaic virus (SCMV). This disease seriously threatens food security across sub-Saharan Africa (SSA). We investigated a major-effect quantitative trait locus for resistance on chromosome 6, named the maize lethal necrosis susceptibility locus 1 (qMLNS1), derived from the Thai line KS23-6. Fine mapping and CRISPR-Cas9 editing of the candidate genes within the narrowed 105 kb interval revealed a peroxisomal peptidase as the underlying cause of susceptibility. Confocal microscopy confirmed the localization of the MLNS1 protein within peroxisomes. Targeted knockout of the Mlns1 gene in the susceptible elite line CML536 from SSA conferred resistance comparable to KS23-6 in field trials conducted in Naivasha, Kenya. This knockout specifically blocked MCMV accumulation without affecting SCMV. The edited lines showed no yield penalty or agronomic defects under disease-free conditions. Our findings uncover a mechanistic link between a peroxisomal enzyme and viral susceptibility. They also establish a rapid, scalable gene editing strategy for incorporating MLN resistance into elite germplasm, offering a model for combating similar viral diseases in staple crops globally.},
}
@article {pmid41927370,
year = {2026},
author = {Zheng, J and Wang, X and Wu, M and Liu, J and Feng, H and Yang, H and Li, D and Wang, H and Hu, J and Zuo, E},
title = {Large-scale parallel characterization of RNA-guided nuclease activity and specificity.},
journal = {Science bulletin},
volume = {71},
number = {8},
pages = {2044-2054},
doi = {10.1016/j.scib.2026.03.047},
pmid = {41927370},
issn = {2095-9281},
mesh = {*Gene Editing/methods ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems ; *Endonucleases/metabolism/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {As systematic comparisons of editing efficiency and specificity seldom keep pace with rapid developments in RNA-guided nucleases (RGNs), the current study examined 50 such editing systems and characterized the off-target effects and genomic structural impacts of a subset of high-efficiency RGNs. Among them, AsCas12a-Ultra, LbCpf1, and AsCas12a-Plus demonstrated similar or higher efficiency compared to SpCas9, while the relatively high efficiency and small size of enOsCas12f1 together support its suitability for in vivo delivery. AsCpf1-YH and FnCpf1 exhibited the lowest single-guide RNA-dependent (sgRNA-dependent) off-target risks, whereas DpFNuc showed the highest. Genomic structural analysis revealed that enCas12f-HKRA frequently introduces chromosomal translocations, while Cas12j-SF05 poses a lower risk of such mutations. Notably, the high-efficiency RGNs were associated with translocation hotspots. Additionally, enRhCas12f1 and SpaCas12f1 had the lowest cytotoxicity, while enAsCpf1-HF strongly inhibited cell proliferation. This study establishes the first multidimensional performance evaluation framework for RGNs, providing a data-driven tool to support precise genome editing.},
}
@article {pmid42051075,
year = {2026},
author = {Vats, S and Jadhav, H and Mahakalkar, B and Patil, G and Sonah, H and Sharma, TR and Deshmukh, R},
title = {Genome Editing of a Carotenogenic Gene for Lycopene Enhancement Increases Heavy Metal Stress Susceptibility in Tomato (Solanum lycopersicum L.).},
journal = {Physiologia plantarum},
volume = {178},
number = {3},
pages = {e70884},
doi = {10.1111/ppl.70884},
pmid = {42051075},
issn = {1399-3054},
support = {BT/PR38279/GET/119/351/2020//Department of Biotechnology, Ministry of Science and Technology, India/ ; //Anusandhan National Research Foundation, JC Bose Fellowship, Government of India./ ; HSCSIT/R&amp;D/2024/511//Haryana State Council for Science Innovation and Technology (HSCSIT)/ ; },
mesh = {*Solanum lycopersicum/genetics/drug effects/physiology/metabolism ; *Gene Editing/methods ; *Lycopene/metabolism ; Stress, Physiological/genetics/drug effects ; *Metals, Heavy/toxicity ; Fruit/genetics ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Intramolecular Lyases/genetics/metabolism ; Gene Expression Regulation, Plant ; Carotenoids/metabolism ; Plants, Genetically Modified ; },
abstract = {Improving the nutritional quality and abiotic stress tolerance of crop plants is essential for sustainable agriculture and global food security. Recent advances in genome editing, particularly the CRISPR/Cas9 system, have enabled precise modification of metabolic pathways to enhance valuable traits such as carotenoid accumulation. The present study aimed to enhance fruit lycopene content and assess associated stress responses in tomato (Solanum lycopersicum L.) through targeted genome editing of the lycopene beta cyclase (β-LCY, EC 5.5.1.19) gene, encoding for a key enzyme in the carotenoid biosynthetic pathway. A Csy4-based multiplex CRISPR/Cas9 approach was applied to edit β-LCY in three tomato genotypes, including two cultivated varieties and the wild species S. peruvianum L. Genotypic analysis revealed significant genotype-dependent differences in editing efficiency. The β-LCY knockout lines exhibited markedly increased lycopene accumulation in fruits, resulting in enhanced pigmentation. However, when subjected to cadmium stress, these lines showed greater susceptibility than wild-type plants, with pronounced wilting and stress symptoms. Physiological, biochemical, and metabolomic analyses confirmed disruption of stress-response mechanisms associated with carotenoid pathway modification. These findings demonstrate that while genome editing can successfully enhance desirable metabolic traits, it may also impair abiotic stress tolerance. This study provides new insight into the complex interplay between the carotenoid biosynthetic pathway and stress adaptation in tomato.},
}
@article {pmid42053263,
year = {2026},
author = {Turowski, P and Gatermann, SG and Pfennigwerth, N},
title = {Cefiderocol resistance mediated by mutation of the miniconductance mechanosensitive channel MscM in Klebsiella oxytoca.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {81},
number = {5},
pages = {},
doi = {10.1093/jac/dkag147},
pmid = {42053263},
issn = {1460-2091},
support = {//Robert Koch Institute/ ; 1369-402//German Ministry of Health/ ; },
mesh = {*Klebsiella oxytoca/drug effects/genetics/growth &amp; development/isolation &amp; purification ; *Anti-Bacterial Agents/pharmacology ; *Cephalosporins/pharmacology ; Microbial Sensitivity Tests ; Humans ; Klebsiella Infections/microbiology ; Cefiderocol/pharmacology ; *Mutation ; *Drug Resistance, Bacterial/genetics ; *Ion Channels/genetics ; *Bacterial Proteins/genetics ; Whole Genome Sequencing ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {OBJECTIVE: To investigate the emergence of cefiderocol resistance in a clinical Klebsiella oxytoca isolate and to identify the underlying mechanism.<br><br>METHODS: A clinical isolate of K. oxytoca susceptible to cefiderocol was exposed to stepwise increasing cefiderocol concentrations via broth microdilution in iron-depleted CAMHB (ID-CAMHB) to select spontaneous mutants. WGS identified potential resistance-associated mutations. CRISPR-Cas9 genome editing was used to confirm causality. Growth curves in CAMHB and ID-CAMHB were performed to assess potential growth alterations.<br><br>RESULTS: A spontaneous mutant with elevated cefiderocol MIC (16&#x2005;mg/L) carried a Q1008L substitution in the miniconductance mechanosensitive channel MscM. CRISPR-edited strains reproduced this phenotype. Growth kinetics did not reveal an obvious growth defect under the tested in vitro conditions.<br><br>CONCLUSION: This is the first report linking cefiderocol resistance to a mutation in MscM in K. oxytoca. Although observed in a single isolate, the lack of an apparent growth defect under the tested conditions suggests that this resistance mechanism may persist in the absence of antibiotic pressure.},
}
@article {pmid42053665,
year = {2026},
author = {Lei, M and Bakhsh, MZM and Zhang, X and He, D and Dai, C and Ma, C and Tu, J and Shen, J and Wen, J and Fu, T and Yi, B},
title = {From haploid inducer to CMS donor: repurposing of CENH3 to create a CMS line in a single step in Brassica napus.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {42053665},
issn = {1432-203X},
support = {2024ZD04077//Biological Breeding-National Science and Technology/ ; },
mesh = {*Haploidy ; *Brassica napus/genetics ; *Plant Infertility/genetics ; *Plant Proteins/genetics/metabolism ; Plant Breeding/methods ; Cytoplasm/genetics ; CRISPR-Cas Systems/genetics ; *Histones/genetics/metabolism ; Mutation ; Centromere/genetics/metabolism ; },
abstract = {Mutation in centromere histone H3 (CENH3) protein could induce a paternal haploid with maternal cytoplasm in rapeseed. By paternal haploid induction, a cytoplasmic male sterile line can be created in any genetic background within one breeding cycle. Hybrid development in rapeseed relies primarily on the three-line system, which includes a cytoplasmic male sterile (CMS) line. Conventionally, these CMS lines are developed through backcrossing, a process that requires several breeding cycles to complete. More recently, the doubled haploid technique has been employed in various crops to generate homozygous lines within a single breeding cycle. In the present study, we utilized a haploid induction (HI) strategy to produce fertile homozygous lines and CMS lines via paternal haploid induction. We have created single homozygous and double heterozygous mutants of the BnaCENH3 gene in the rapeseed cultivar ganA (hau-CMS) using CRISPR/Cas9 technique. Upon hybridization of CMS-HI line with wild type can successfully induced paternal haploids with maternal sterile cytoplasm. This system offers the ability to introduce sterile cytoplasm into any genetic background within a single generation.},
}
@article {pmid42053928,
year = {2026},
author = {Deres, D and Terefe, M},
title = {Beyond CRISPR/Cas9: emerging genome editing technologies for next-generation crop improvement.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {42053928},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Genome, Plant/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Genome editing has changed plant biology and accelerated crop improvement. CRISPR/Cas9 allows precise and efficient genetic changes in many species. Still, Cas9 has limits like PAM restrictions, off-target effects, and varying editing success. This led to new systems. Editors like Cas12a, CasΦ, CasMINI, and CasX offer more targeting options, can edit RNA, and work better with hard to edit plant genomes. Precision tools such as base editors and prime editors make precise changes by swapping nucleotides or adding small pieces without cutting both DNA strands. This improves accuracy. Beyond single tools, combined and step by step editing methods can be used for handling complex traits controlled by many genes. Using several methods like CRISPR knockouts, base and prime editing, epigenome editing and recombinase systems-breeders can improve traits while reducing unwanted side effects. Stepwise editing helps to test changes, confirm their effects, and improve entire biological pathways. Combining these approaches with AI-driven analysis, target prediction, and design optimization makes it easier to pick the best genes and edits for desired traits. These advanced editing methods are used to boost stress tolerance, fight diseases, improve nutrition, increase yields, and enhance quality after harvest. Despite progress, problems remain with how efficient edits can be made, delivering tools into plants, reliance on specific genotypes, unclear regulations, and acceptance by society. Looking ahead, joying genome editing with AI, fast breeding techniques help develop stronger, high yielding crops and support global food security.},
}
@article {pmid42056528,
year = {2026},
author = {Marino, ND and Talaie, A and Gerovac, M and Rodriguez, JL and Schmidt, AD and Astmann, TJ and Carion, H and Taylor, AF and Liliedahl, J and Haniyur, S and Zoga, K and Johnson, MC and Buhlmann, L and Chen, KH and Silas, S and Yuping, L and Zhang, Y and Swaney, DL and Vogel, J and Bondy-Denomy, J},
title = {Translation-dependent degradation of cas12 mRNA triggered by an anti-CRISPR.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {42056528},
issn = {1476-4687},
abstract = {Bacteria encode diverse defence systems, including CRISPR-Cas, to recognize and cleave the DNA of bacteriophages (phages) and other mobile genetic elements[1]. In response, phages encode anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas activity by blocking DNA binding or cleavage[2]. Here we report an unexpected mechanism by which the anti-CRISPR AcrVA2 inhibits Cas12a biogenesis. AcrVA2 binds conserved and functionally important amino acid residues near the Cas12a N-terminus and triggers selective degradation of cas12a mRNA as it is translated. Additionally, conserved residues in the AcrVA2 C-terminal domain enable co-sedimentation with ribosomes and polysomes, which is required to achieve targeted co-translational mRNA degradation. The AcrVA2 C-terminal domain is broadly conserved in homologs encoded by diverse mobile genetic elements, typically in hosts that lack cas12a, suggesting that these homologues may recognize and downregulate alternative substrates in other bacteria. These findings reveal a novel mechanism for molecular conflict and gene regulation in bacteria.},
}
@article {pmid41839994,
year = {2026},
author = {Fu, X and Zhao, F and Ge, J and Guan, H and Lv, P and Guo, F and Ma, H and Ainiwaer, M and Hu, S},
title = {Establishment of a rapid Brucella detection method based on MCDA-CRISPR dual signal amplification system for reducing transfusion-transmitted diseases.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41839994},
issn = {2045-2322},
support = {CXCY2025024//the Xinjiang Medical University Graduate Innovation and Entrepreneurship Project/ ; KYQDJJ2024006//the Doctoral Scientific Research Startup Fund of Zhengzhou Central Hospital/ ; 2022-2-6042//the Capital's Funds for Health Improvement and Research/ ; 2025YLZDJH193//The Guidance Plan Project for Scientific and Technological Innovation in the Medical and Health Field of Zhengzhou City/ ; },
mesh = {*Brucella/genetics/isolation &amp; purification ; Humans ; *Brucellosis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Blood Donors ; Sensitivity and Specificity ; DNA, Bacterial/genetics ; China ; Blood Transfusion ; },
abstract = {Brucellosis is a common zoonotic disease caused by Brucella and remains a globally concerning public health issue. Timely and effective detection methods are crucial for clinical diagnosis. We developed a novel Brucella detection platform (MCDA-CRISPR) by integrating multiple cross displacement amplification (MCDA) with a CRISPR-Cas12a-based biosensing system, and preliminarily applied it for the first time to screen for Brucella in voluntary blood donors from Xinjiang, China. This technology enables amplification under isothermal conditions at 64 °C using only a water bath, requires no specialized equipment, and completes detection within 60 min. Amplification products can be directly visualized under UV light without complex interpretation. Performance results demonstrated a minimum detection limit of 1 fg/μL for Brucella DNA, making the method 100 times more sensitive than conventional PCR. The assay showed 100% specificity for Brucella detection with no cross-reactivity to non-Brucella pathogens. The assay could also detect Brucella in blood donors samples and showed the same sensitivity and specificity as the culture method. The assay is a visual, sensitive, and highly specific detection technique. When applied to routine blood transfusion screening in areas with high prevalence of brucellosis, such as Xinjiang, can effectively reduce the risk of transfusion-transmitted brucellosis, and hold broad application prospects in resource-limited primary or field testing scenarios.},
}
@article {pmid41885428,
year = {2026},
author = {Carrington, E and Ballmer, D and Niederwieser, I and Thommen, BT and Brancucci, NMB and Voss, TS},
title = {A heterologous marker-free selection approach for CRISPR/Cas9-based gene editing in the malaria parasite Plasmodium falciparum.},
journal = {mSphere},
volume = {11},
number = {4},
pages = {e0088425},
doi = {10.1128/msphere.00884-25},
pmid = {41885428},
issn = {2379-5042},
support = {310030_184785,310030_220001//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; 310030_200683//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung/ ; ALTF 920-2024//European Molecular Biology Organization/ ; },
mesh = {*Plasmodium falciparum/genetics/drug effects ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Drug Resistance/genetics ; Pyrimethamine/pharmacology ; Tetrahydrofolate Dehydrogenase/genetics ; Protozoan Proteins/genetics ; Antimalarials/pharmacology ; Thymidylate Synthase/genetics ; Humans ; Malaria, Falciparum/parasitology ; Plasmids ; Multienzyme Complexes ; },
abstract = {CRISPR/Cas9-based gene editing of the malaria parasite Plasmodium falciparum has emerged as a transformative tool for advancing functional studies on parasite biology and identifying new therapeutic targets. Currently applied CRISPR/Cas9 methodologies depend on a limited set of heterologous drug resistance markers for the selection of transgenic parasites, which restricts the potential for iterative genetic modifications. Here, we developed a heterologous marker-free CRISPR/Cas9 gene editing strategy (CRISPR/Cas9[pyrR]) for P. falciparum based on the simultaneous editing of a gene of interest and introduction of pyrimethamine (PYR) resistance-conferring mutations into the dihydrofolate reductase-thymidylate synthase (pfdhfr-ts) gene. By providing a pfdhfr[pyrR] donor sequence and the Cas9 expression cassette on separate plasmids, CRISPR/Cas9[pyrR] ensures that only parasites acquiring both plasmids survive under PYR pressure. As a proof of principle, we applied CRISPR/Cas9[pyrR] to generate two transgenic parasite lines expressing GFP-tagged versions of the putative nuclear envelope protein PfGEX1 and nuclear pore protein PfNUP116, respectively. We show that PfGEX1-GFP marks the nuclear envelope specifically in gametocytes, but not in asexual blood stage parasites. Similarly, and against previous reports, we find PfNUP116-GFP expression is undetectable in asexual parasites but instead localizes to a distinct perinuclear region in early gametocytes. These results suggest dynamic compositional changes of the nuclear periphery during sexual differentiation. We further demonstrate sequential genetic engineering of the PfNUP116-GFP-expressing line using the human dhfr drug resistance marker combined with WR99210-based selection by additionally tagging PfAP2-G, the master transcriptional regulator of sexual commitment, and the nuclear pore protein PfNUP313. Hence, CRISPR/Cas9[pyrR] provides a versatile and effective new method that enhances and complements the current genetic toolkit for malaria research.IMPORTANCEMalaria tropica, which is caused by the unicellular parasite Plasmodium falciparum, is one of the most devastating infectious diseases worldwide. The development of urgently needed effective vaccines and new antimalarial drugs with novel modes of action requires a profound understanding of parasite biology. CRISPR/Cas9-based genome engineering is beyond doubt the most important experimental approach to study the function and essentiality of parasite proteins and to identify and validate new vaccine and drug targets. In this study, we developed and successfully applied a modified CRISPR/Cas9 strategy, termed CRISPR/Cas9[pyrR], that avoids the use of a heterologous drug resistance marker for the selection of genetically modified parasites. CRISPR/Cas9[pyrR] thus complements the CRISPR/Cas9 toolbox available for gene editing in P. falciparum and overcomes some of the limitations of currently employed protocols.},
}
@article {pmid41964558,
year = {2026},
author = {Guo, A and Guo, W and Guo, Y and Zhang, Y and Zhang, Z and Zou, X and Sun, Z},
title = {Clustered Regularly Interspaced Short Palindromic Repeat-Based Colorimetric Aptasensor Combined with Smartphone Imaging and Deep Learning Enables Selective Recycling and Visual Prediction of Microplastics in the Environment.},
journal = {Analytical chemistry},
volume = {98},
number = {16},
pages = {11886-11898},
doi = {10.1021/acs.analchem.5c08138},
pmid = {41964558},
issn = {1520-6882},
mesh = {*Colorimetry/methods ; *Smartphone ; *Aptamers, Nucleotide/chemistry/genetics ; *Microplastics/analysis ; *Deep Learning ; Polystyrenes/analysis ; *Biosensing Techniques/methods ; Polyvinyl Chloride/analysis ; *Water Pollutants, Chemical/analysis ; CRISPR-Cas Systems ; },
abstract = {Microplastics present significant risks to human health and ecosystem stability, creating an urgent need for analytical methods that are simple, rapid, sensitive, and field-deployable. Herein, we report a clustered regularly interspaced short palindromic repeat (CRISPR)-based colorimetric aptasensor for the detection of poly(vinyl chloride) (PVC) and polystyrene (PS) microplastics. This platform leverages the high specificity of PVC and PS aptamers integrated into a Fe3O4@Au-DNA magnetic complex, which facilitates capture, separation, and detection. Upon microplastic binding, a competitive reaction releases an activator DNA, initiating a dual CRISPR-Cas12a system for signal amplification. The activated Cas12a trans-cleavage activity is then linked to a hemin-aptamer DNAzyme colorimetric reaction, converting the signal into a visible color change. This colorimetric output is captured by smartphone imaging and processed in real time. Furthermore, a deep-learning-based regression model was developed to enable the quantitative prediction of PVC and PS micro/nanoplastics in diverse environmental matrices. The method exhibited high selectivity and a broad dynamic range from 10[-2] to 10[3] μg/mL. In smartphone detection mode, the limits of detection for PVC and PS reached 3.1 ng/mL and 3.7 ng/mL, respectively. This approach significantly enhances detection performance and stability, enabling visual monitoring of microplastics in complex real samples. Collectively, this work provides a rapid and effective strategy for the extraction and real-time quantification of small molecules.},
}
@article {pmid42046057,
year = {2026},
author = {Hsu, CY and Abdelgawwad El-Sehrawy, AAM and Alshkarchy, SS and Abdul, AS and Ganesan, S and Gupta, PK and Sharma, R and Nayak, PP and Ebrahimpour, A and Khazaei, Y},
title = {Innovative approaches in the treatment of hematologic malignancies: the role of CRISPR-engineered microbiomes along the gut-immune axis in immunotherapy development.},
journal = {Cancer cell international},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12935-026-04316-0},
pmid = {42046057},
issn = {1475-2867},
}
@article {pmid42048304,
year = {2026},
author = {Mallon, J and Lenihan, CJ and Shridhar, S and Bailey, S},
title = {Target discrimination and PAM profiling of the Thermotoga maritima type I-B CRISPR system.},
journal = {The Biochemical journal},
volume = {},
number = {},
pages = {},
doi = {10.1042/BCJ20260189},
pmid = {42048304},
issn = {1470-8728},
abstract = {Type I-B CRISPR-Cas systems represent the most abundant CRISPR subtype in nature and have emerged as powerful tools for endogenous genome editing in diverse prokaryotes. Here we reconstitute and characterize the type I-B1 system from the thermophile Thermotoga maritima (Tma) using purified components. We demonstrate that Tma Cascade requires standalone Cas11 expression, as the cryptic internal translation start site within cas8b1 is non-functional in E. coli. The reconstituted system exhibits canonical type I function including RNA-guided DNA binding, PAM-dependent target discrimination, Cas3-mediated degradation, and seed region interrogation spanning seven PAM-proximal nucleotides. Using next-generation sequencing-based PAM library screens, we define a YYD consensus PAM (Y = C/T; D = G/A/T) with strong discrimination against the array repeat-adjacent sequence (AAC). Comprehensive PAM profiling reveals context-dependent tolerance for non-consensus sequences and identifies numerous intermediate-activity PAMs that may function in priming. Comparison with other characterized type I-B systems reveals correlation between Cas8b variant and position -3 specificity, conserved pyrimidine preference at position -2, and variability at position -1. This work establishes a thermostable type I-B platform for biotechnological applications and provides insights into evolutionary mechanisms balancing PAM promiscuity with self-discrimination in the most abundant CRISPR-Cas subtype.},
}
@article {pmid42048640,
year = {2026},
author = {Dominy, C},
title = {CRISPR Diagnostics, in Your Pocket.},
journal = {Journal of medical Internet research},
volume = {28},
number = {},
pages = {e98572},
doi = {10.2196/98572},
pmid = {42048640},
issn = {1438-8871},
}
@article {pmid42048928,
year = {2026},
author = {Li, L and Ju, R and Yan, Y and Lou, Y and Chen, H and Wen, Q and Wang, M and Lei, R and Liu, J and Wang, X},
title = {Nanomaterial-enabled CRISPR-Cas biosensing for non-nucleic acid targets: Strategies, mechanisms, and applications.},
journal = {Talanta},
volume = {307},
number = {},
pages = {129831},
doi = {10.1016/j.talanta.2026.129831},
pmid = {42048928},
issn = {1873-3573},
abstract = {Nanomaterial-assisted CRISPR-Cas biosensing has transcended nucleic acid detection, emerging as a prominent paradigm for the ultrasensitive analysis of non-nucleic acid targets. This review systematically elucidates recent advances by proposing a novel three-dimensional framework encompassing transduction efficiency, signal output, and application adaptation. We first focus on molecular engineering approaches that convert non-nucleic acid recognition events into CRISPR activation signals, critically assessing the inherent efficiency limits and bottlenecks of key techniques such as aptamer switches, nuclease allosteric effects, substituent steric hindrance, Cas protein allosteric regulation, and Cas protein engineering. We then detail how various nanomaterials, including AuNPs, QDs, UCNPs, MOFs, nanozymes, synergistically enhance the efficiency of optical, electrochemical, and multimodal signal output. Utilizing this framework, the review analyzes representative applications in detecting proteins, small molecules, and heavy metal ions, clarifying the mechanisms behind performance enhancement and current limitations. Finally, we discuss persistent challenges and prospectively proposes disruptive future directions, such as constructing CRISPR sensing networks, developing intelligent nanocarriers, and creating closed-loop theranostic systems. This study provides a technical overview, a critical framework, and a design roadmap, facilitating the rational design of biosensors and enabling the realization of integrated bio-regulatory platforms.},
}
@article {pmid42049209,
year = {2026},
author = {Da Lage, JL and Bonneau, M and Moreno, C and Le Rouzic, A},
title = {Is the alpha-amylase paralogue Amyrel dispensable in Drosophila melanogaster?.},
journal = {Open biology},
volume = {16},
number = {4},
pages = {},
doi = {10.1098/rsob.250411},
pmid = {42049209},
issn = {2046-2441},
support = {//CNRS/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics/metabolism/enzymology ; *Drosophila Proteins/genetics/metabolism ; Female ; *alpha-Amylases/genetics/metabolism ; Male ; CRISPR-Cas Systems ; },
abstract = {Divergent duplicated gene copies are considered to get new or variant function or regulation through sub- or neofunctionalization. In Drosophila and other flies (Muscomorpha), the alpha-amylase paralogue Amyrel is known to have peculiar enzymological properties compared with the classical enzyme Amy. Yet, its real function in fly biology is unclear. Here, we show that Amyrel and Amy share similar regulation patterns such as sugar downregulation and midgut-specific expression in Drosophila melanogaster. Most regulatory information lies within 500 bp of the upstream sequence, as enhanced green fluorescent protein expression under the Amyrel promoter mimics Amyrel expression quite well. To get an insight into Amyrel function, we knocked out the gene using CRISPR-Cas9. Setting a competition experiment between wild-type (wt) and null alleles over 40 generations, we estimated the selective advantage of the wt to be 2%. However, Amyrel-null mutant lines exhibited no clear defect in several life history traits. Interestingly, while Amyrel had very low expression in young adults, it was significantly upregulated in females aged two months; however, lifespan was not affected. Overall, we were able to document substantial functional and regulatory differences between the Amyrel copy and the regular amylase, and we showed that carrying an Amyrel gene conferred a competitive fitness advantage.},
}
@article {pmid42049231,
year = {2026},
author = {Zhou, J and Wang, X and Zhou, L and Zhang, H and Ye, S and Yuan, YJ},
title = {Automated linear DNA assembly of A. thaliana's chloroplast and mitochondrial genome.},
journal = {Nucleic acids research},
volume = {54},
number = {8},
pages = {},
doi = {10.1093/nar/gkag380},
pmid = {42049231},
issn = {1362-4962},
support = {2021YFA0909300//National Key Research and Development Program of China/ ; //Ministry of Science and Technology of the People's Republic of China/ ; 22527901//National Natural Science Foundation of China/ ; //National Major Research Instrumentation Program/ ; JYB2025XDXM503//Fundamental and Interdisciplinary Disciplines Breakthrough Plan of the Ministry of Education of China/ ; 25ZXWCSY00330//Tianjin Municipal Science and Technology Major Program/ ; },
mesh = {*Arabidopsis/genetics ; *Genome, Mitochondrial ; *Genome, Chloroplast ; Escherichia coli/genetics ; Genome, Plant ; Synthetic Biology/methods ; CRISPR-Cas Systems ; },
abstract = {Synthetic genomics is advancing from microbial toward multicellular organisms. However, current manual methods for DNA and genome assembly remain inadequate for the efficient, large-scale production of long DNA constructs. Here, we present Programmed DNA Assembly via Cas9 and Conjugative Transfer (PACT), a method that integrates a linear vector system, bacterial conjugation, and programmable Cas9-mediated cleavage to achieve highly efficient, iterative assembly of large DNA fragments. PACT enhances assembly efficiency by ~30-fold compared to conventional circular vector strategies, enabling one-step assembly of DNA up to 80 kb. We engineered four single guide-RNA-Marker donor cassettes to support iterative assembly workflows. PACT can utilize low-recombination Escherichia coli strains as hosts to efficiently assemble Arabidopsis thaliana mitochondrial genome with high repeat units. Integrated with an automated robotic platform, we developed an unattended, high-throughput pipeline (aPACT) toward large-scale parallel DNA assembly. Using aPACT, we successfully assembled three large DNA constructs: a 210 kb digital DNA, the designed chloroplast (120 kb) and mitochondrial (350 kb) genome of A. thaliana. This automated system offers a powerful tool for scalable assembly of large DNA molecules, accelerating synthetic genomics research toward complex multicellular organisms.},
}
@article {pmid41820368,
year = {2026},
author = {Bertlin, JAC and Pauzaite, T and Liang, Q and Wit, N and Williamson, JC and Sia, JJ and Matheson, NJ and Ortmann, BM and Mitchell, TJ and Speak, AO and Zhang, Q and Nathan, JA},
title = {VHL synthetic lethality screens uncover CBF-β as a negative regulator of STING.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41820368},
issn = {2041-1723},
support = {R01 CA284591/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Membrane Proteins/metabolism/genetics ; Cell Line, Tumor ; Animals ; Signal Transduction ; Mice ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Interferon Type I/metabolism ; *Synthetic Lethal Mutations ; STING Protein ; },
abstract = {Clear cell renal cell carcinoma (ccRCC) represents the most common form of kidney cancer and is typified by biallelic inactivation of the von Hippel-Lindau (VHL) tumour suppressor gene. Here, we undertake genome-wide CRISPR/Cas9 screening to reveal synthetic lethal interactors of VHL, and uncover that loss of Core Binding Factor β (CBF-β) causes cell death in VHL-null ccRCC cell lines and impairs tumour establishment and growth in vivo. This synthetic relationship is independent of the elevated activity of hypoxia inducible factors (HIFs) in VHL-null cells, but does involve the RUNX transcription factors that are known binding partners of CBF-β. Mechanistically, CBF-β loss leads to upregulation of type I interferon signalling, and we uncover a direct inhibitory role for CBF-β at the STING locus controlling Interferon Stimulated Gene expression. Targeting CBF-β in kidney cancer both selectively induces tumour cell lethality and promotes activation of type I interferon signalling.},
}
@article {pmid41955813,
year = {2026},
author = {Ji, Z and Zhao, Y and Huang, J and Shan, Y and Li, J and Huang, Q and Wu, M and Liu, F},
title = {Portable amplification-free digital droplet CRISPR/Cas12a platform for one-pot multiplexed viruses detection with attomolar sensitivity.},
journal = {Biosensors &amp; bioelectronics},
volume = {305},
number = {},
pages = {118668},
doi = {10.1016/j.bios.2026.118668},
pmid = {41955813},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/instrumentation ; Animals ; Swine ; *Circovirus/isolation &amp; purification/genetics ; Limit of Detection ; *Porcine epidemic diarrhea virus/isolation &amp; purification/genetics ; *Porcine respiratory and reproductive syndrome virus/isolation &amp; purification/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {High-throughput, sensitive, and field-deployable nucleic acid detection is essential for timely pathogen surveillance. Here, we developed a novel amplification-free and portable platform based on an instrument-free polydisperse digital droplet CRISPR/Cas12a (DD-Cas12a) assay, enabling the simultaneous detection of three synthetic nucleic acid targets in a single reaction. The platform achieved attomolar-level sensitivity (0.15 fM) without pre-amplification, generated detection results within 40 min, and maintained high specificity even in the presence of a 10-fold excess of non-target nucleic acids. The DD-Cas12a was further applied to detect one DNA virus, porcine circovirus type 2 (PCV2), and two RNA viruses, porcine epidemic diarrhea virus (PEDV) and porcine reproductive and respiratory syndrome virus (PRRSV). The detection limits for PCV2, PEDV, and PRRSV were 126.0, 673.5, and 584.5 fg/μL, respectively, corresponding to sensitivity comparable to qPCR for PCV2 and 10-fold higher sensitivity for PEDV and PRRSV. To improve analytical throughput and objectivity, we established an Algorithm-Driven DD-Cas12a (ADC) platform for automated image analysis and result decoding. The custom-designed miniaturized portable instrument was developed for signal acquisition and field deployment. By combining the DD-Cas12a assay with the ADC platform and the portable instrument, we established a portable, digital, amplification-free, and ultrasensitive system for multiplex pathogen detection, providing a powerful tool for diagnostics, biosurveillance, and environmental monitoring.},
}
@article {pmid41962435,
year = {2026},
author = {Jia, M and Ru, X and Bing, X and Zhai, F and Huang, M and Du, X},
title = {CRISPR/Cas12a-mediated aggregation of unmodified AuNPs via microwave-assisted heating-dry for label-free detection of Escherichia coli O157:H7.},
journal = {Biosensors &amp; bioelectronics},
volume = {305},
number = {},
pages = {118676},
doi = {10.1016/j.bios.2026.118676},
pmid = {41962435},
issn = {1873-4235},
mesh = {*Escherichia coli O157/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Gold/chemistry ; Microwaves ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Colorimetry/methods ; Endodeoxyribonucleases/chemistry ; *Escherichia coli Infections/microbiology/diagnosis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as a powerful platform for developing next-generation biosensors, yet achieving rapid, instrument-free visual detection remains a challenging. Conventional CRISPR-AuNP assays rely on pre-functionalized, thiol-modified gold nanoparticles (AuNPs), which introduce steric hindrance that slows Cas12a kinetics and increases cost and complexity. Here, we developed a rapid, label-free colorimetric platform that integrates a mismatched catalytic hairpin assembly (MCHA) for low-background amplification with CRISPR/Cas12a trans-cleavage, coupled to a microwave-assisted dry-heating method for in-situ probe conjugation. Unlike conventional approaches where CRISPR acts on pre-conjugated probes, our method allows Cas12a to cleave free, non-thiolated ssDNA probes. Intact probes rapidly conjugate to unmodified AuNPs under microwave irradiation via their high-affinity domain, forming a protective corona that prevents salt-induced aggregation. Conversely, probes that have been cleaved by target-activated CRISPR/Cas12a lose this conjugation ability, rendering AuNPs susceptible to aggregation and producing a visible color change. To address the conflicting ionic requirements between the CRISPR reaction and AuNP stability, a Ba(OH)2-mediated ionic reset step is introduced to remove excess Mg[2+] prior to probe conjugation. This platform enables the sensitive detection of Escherichia coli O157:H7 within 2 h a limit of detection of 5 CFU/mL and robust performance in complex food and clinical matrices. Combined with smartphone-based RGB analysis, the instrument-minimized strategy eliminates the need for chemical modification of AuNPs and specialized operators, offering a versatile, low-cost, and user-friendly platform well-suited for pathogen detection in resource-limited settings.},
}
@article {pmid41990423,
year = {2026},
author = {Wang, Q and Zhu, Y and Chen, B and Zhai, C and Xu, J and Xia, J},
title = {Asymmetric RPA-primed hybridization chain reaction enabling one-input-multiple-Cas12a activation for ultrasensitive Salmonella detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {305},
number = {},
pages = {118689},
doi = {10.1016/j.bios.2026.118689},
pmid = {41990423},
issn = {1873-4235},
mesh = {*Salmonella/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; Nucleic Acid Hybridization ; Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Food Microbiology ; DNA, Single-Stranded/chemistry/genetics ; DNA, Bacterial/genetics ; Food Contamination/analysis ; Bacterial Proteins/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; Endodeoxyribonucleases ; },
abstract = {Ensuring food safety requires accurate, simple detection of pathogens such as Salmonella. However, conventional methods suffer from long processing times, complicated procedures, and inadequate accuracy. To address this, we developed an integrated platform combining asymmetric recombinase polymerase amplification (aRPA), programmable hybridization chain reaction (HCR), and CRISPR/Cas12a readout in a simplified single-tube integrated format. Unlike conventional CRISPR assays, in which a single target activates a single Cas12a complex in a PAM-dependent manner, our design uses aRPA-generated single-stranded DNA to trigger HCR, converting each target into a long dsDNA molecule with multiple PAM sites. This architecture enables one-input, multiple-Cas12a activation, representing a shift from linear to cooperative amplification. Under optimized conditions, the assay achieves a detection limit of 1.8 × 10[2] colony-forming units/mL (CFU/mL) in direct screening, and reaches as low as 5 CFU/mL after a 6 h enrichment step. The method shows high specificity toward common foodborne pathogens and reliable performance in spiked food samples. Furthermore, a DNA releaser simplifies sample preparation and enhances convenience. In summary, this integrated strategy offers a reliable and practical tool for food safety monitoring.},
}
@article {pmid41999797,
year = {2026},
author = {Wang, Z and Zhou, L and Ming, L},
title = {From "Simulation" to "Mirror": Gene editing and humanization redefines the next-generation precision oncology animal model.},
journal = {Gene},
volume = {998},
number = {},
pages = {150161},
doi = {10.1016/j.gene.2026.150161},
pmid = {41999797},
issn = {1879-0038},
mesh = {*Gene Editing/methods ; Animals ; Humans ; *Precision Medicine/methods ; Disease Models, Animal ; *Neoplasms/genetics/therapy ; CRISPR-Cas Systems ; Tumor Microenvironment/genetics ; Mice ; Xenograft Model Antitumor Assays/methods ; Mutation ; },
abstract = {Patient-derived xenograft (PDX) models, although conventionally used in oncology, exhibit critical limitations: they frequently lose patient-specific genetic mutations and lack the human leukocyte antigen (HLA) diversity essential for immune recognition, and fail to recapitulate the human tumor microenvironment (TME). These deficiencies contribute to immunotherapy prediction failure rates exceeding 80% in clinical translation. To address these gaps, we propose a Tumor Model 2.0 framework. This framework integrates multi-omics data (whole-genome, transcriptomic, and proteomic) with precision genome editing technologies (CRISPR-Cas9 and Prime Editing) to reconstruct patient-specific mutations across multiple biological layers. Employing an organoid-animal coupling platform with stepwise immune system construction and microenvironment remodeling-subsequently validated in large animals-the framework enables the creation of programmable, patient-specific digital twins. These high-fidelity models support personalized N-of-1 clinical trials, bridging the gap between preclinical research and clinical precision oncology.},
}
@article {pmid42037564,
year = {2026},
author = {de Barros Rodrigues, DK and Leeuwerik, M and Brankovics, B and Fava, WS and Venturini, J and Meyer, W and Arai, T and Majima, H and Watanabe, A and de Souza Carvalho Melhem, M},
title = {Environmental Circulation of Aspergillus fumigatus With Reduced Susceptibility to Agricultural Triazole in Brazil: Clonal Dissemination of Potentially Resistant Genotypes.},
journal = {Mycoses},
volume = {69},
number = {5},
pages = {e70179},
doi = {10.1111/myc.70179},
pmid = {42037564},
issn = {1439-0507},
support = {201422/2024-7//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 443813/2023-0//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; 88887.658397/2021-00//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; 88887.819536/2023-00//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; },
mesh = {*Aspergillus fumigatus/genetics/drug effects/isolation &amp; purification/classification ; *Triazoles/pharmacology ; *Drug Resistance, Fungal/genetics ; Genotype ; *Antifungal Agents/pharmacology ; Microbial Sensitivity Tests ; Humans ; Brazil/epidemiology ; Aspergillosis/microbiology ; Fungal Proteins/genetics ; Whole Genome Sequencing ; Microsatellite Repeats ; Agriculture ; Cytochrome P-450 Enzyme System/genetics ; Tubulin/genetics ; Polymorphism, Single Nucleotide ; Air Microbiology ; Mutation ; },
abstract = {BACKGROUND: Aspergillus fumigatus resistance to triazole antifungals poses an increasing global health concern. Moreover, the cross-resistance between azole antifungal agents used in clinical settings and those applied in agriculture has become an important emerging issue.<br><br>OBJECTIVES: In this study, we investigated the five environmental A. fumigatus strains showing reduced susceptibility to tebuconazole.<br><br>METHODS: Fungal strains were recovered from air samples collected around the homes of two patients with suspected aspergillosis caused by resistant isolates. Species identification was performed by sequencing the &#x3b2;-tubulin gene, and minimum inhibitory concentrations were determined by broth microdilution. The cyp51A gene was sequenced to detect mutations, and CRISPR-Cas9 genome editing was employed to investigate their influence on susceptibility patterns. Microsatellite genotyping was performed to assess genetic variability, followed by whole genome sequencing and single nucleotide polymorphism analysis.<br><br>RESULTS: The environmental strains presented the same cyp51A genotype characterised by the M172V substitution and silent mutations. Microsatellite genotyping and whole genome sequencing confirmed that the strains were clonal. Functional validation demonstrated that the M172V and silent mutations partially contribute to reduced susceptibility to tebuconazole but are not the main mechanism of resistance involved. Analysis of polymorphisms in genes other than cyp51A revealed no resistance-conferring mutations.<br><br>CONCLUSIONS: The findings described herein suggest the possibility of local clonal dissemination of environmental strains under selective pressure from agricultural azoles in a major agribusiness region of the Midwest of Brazil. This study highlights the silent spread of potentially resistant genotypes in urban areas and reinforces the need for environmental surveillance and expanded genomic monitoring in South America.},
}
@article {pmid42040301,
year = {2026},
author = {Iqbal, Z and Awan, AZ and Atta, S and Hussain, K and Khurshid, M and Ahmad, F and Munir, M and Ghafoor, A and Hashedi, SAA and Ramadan, KMA and El-Gananiny, SM and AlSaleh, MA},
title = {Unveiling the landscape of plant virology in Saudi Arabia: seven decades of progress and future directions toward Vision 2030.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1758142},
pmid = {42040301},
issn = {1664-462X},
abstract = {Plant viruses pose a persistent and escalating threat to global agriculture and food security, inflicting over $30 billion in annual losses - a challenge acutely felt in Saudi Arabia as it strives for agricultural self-sufficiency under Vision 2030. This is the first comprehensive review which presents seven decades of plant virology research in the Kingdom, from early symptom-based diagnosis to advanced molecular, genomic, and bioinformatics advances. A total of ~81 plant viral species infecting 46 plant host species have been documented across the major agroecological regions, dominated by positive-sense single-stranded RNA viruses (~70%). Among these viruses, some are economically most destructive-including alfalfa mosaic virus, cucumber mosaic virus, soilborne cereal mosaic virus, tomato yellow leaf curl virus, zucchini yellow mosaic virus, watermelon chlorotic stunt virus, and barley mild mosaic virus-posing recurrent challenges to key crops such as alfalfa, cucurbits, and tomatoes. Network analysis of virus distribution revealed strong epidemiological linkages among central and western agricultural regions, possibly driven by intensive cultivation and vector ecology. The review highlights emerging management strategies including CRISPR-Cas diagnostics, RNA interference, AI-based detection, nanotechnology, and plant growth promoting rhizobacteria. Gaps persist in genomic surveillance, vector ecology, and biosecurity enforcement. The review concludes with future research priorities emphasizing innovation, interdisciplinary collaboration, and the development of a national plant virus genomic and surveillance framework to secure sustainable agriculture in line with Vision 2030.},
}
@article {pmid42041587,
year = {2026},
author = {Elias, A and Stern, S},
title = {Gene Editing Strategies for Neurological and Mental Disorders: Advances in Delivery, Methodology, and Clinical Translation.},
journal = {Cells},
volume = {15},
number = {8},
pages = {},
doi = {10.3390/cells15080720},
pmid = {42041587},
issn = {2073-4409},
mesh = {Humans ; *Gene Editing/methods ; *Nervous System Diseases/therapy/genetics ; *Mental Disorders/therapy/genetics ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems/genetics ; *Gene Transfer Techniques ; *Translational Research, Biomedical ; },
abstract = {Neurological and mental disorders are among the main causes of disability worldwide, affecting over three billion people and increasing the socioeconomic burden. Advances in molecular genetics and genome engineering have led to gene-targeted therapies that address root causes rather than just symptoms. This review covers current genome-editing tools, including CRISPR/Cas, base editing, and prime editing. The focus is on the benefits of gene editing in the central nervous system, where post-mitotic neurons allow lasting effects after a single treatment. It also discusses emerging delivery platforms such as viral vectors, nanoparticles, and exosome systems, as well as methods to bypass the blood-brain barrier. Recent clinical progress in spinal muscular atrophy, Parkinson's disease, Huntington's disease, and Alzheimer's disease is highlighted, with promising preclinical results for autism, bipolar disorder, epilepsy, and other neurogenetic conditions. The review concludes with regulatory issues, market trends, and ongoing clinical trials, underscoring the potential of gene therapies to transform disease management and provide long-term solutions.},
}
@article {pmid42042483,
year = {2026},
author = {Zhang, C and Wang, YX and Liu, XD and Iqbal, A and Wang, Q and Wang, Y},
title = {Integrated Pest Management Strategies for Controlling Phthorimaea (Tuta) absoluta: Advances in Biological, Pheromone, and Cultural Control Methods.},
journal = {Insects},
volume = {17},
number = {4},
pages = {},
doi = {10.3390/insects17040441},
pmid = {42042483},
issn = {2075-4450},
support = {No. XJ202411439006 and No. XJ202411439010.//Jilin Agricultural Science and Technology College Student Innovation and Entrepreneurship Training Program Project and the Jilin Agricultural Science and Technology University College Student Innovation and Entrepreneurship Training Program Project/ ; },
abstract = {The tomato leaf miner, Phthorimaea (Tuta) absoluta, Meyrick 1917 is recognized as a highly destructive pest, causing significant economic losses to crops in both greenhouse and open field environments across four continents: Asia, Africa, Europe, and South America. High genetic homogeneity among populations from various regions and countries indicates significant gene flow between P. absoluta populations, suggesting a lack of geographical barriers to dispersion. Furthermore, P. absoluta has developed resistance to insecticides due to target-site mutations or metabolic resistance, which enable the insect to withstand lethal doses of insecticides. To control this insect pest, the plant-mediated RNA interference (RNAi) is most promising host-induced gene silencing technique, utilized the plant's machinery to express double-stranded (dsRNA), which triggers the RNAi pathway in P. absoluta. Due to thermal tolerance, the P. absoluta has increased its area of invasion by 600 km per year over 9 years. Female P. absoluta releases pheromones that are recognized by males with a sophisticated olfactory circuit on their antenna. Pheromone binding proteins (PBPs) play a crucial role in mate recognition and attraction, and their expression peaks during courtship, specifically around 6:00 a.m. Given its potential to significantly alter the insect genome, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) offer a revolutionary strategy to control P. absoluta. Furthermore, this pest has developed remarkable adaptations to survive on unfavorable hosts by secreting specific proteins from its salivary glands that detoxify plant defenses. Insecticide resistance is likely the cause of field control failures of P. absoluta. Biological control, sex pheromone traps, and cultural control are the most promising approaches to address insecticide resistance resulting from these failures. Therefore, the implementation of integrated control programs and appropriate resistance management strategies is necessary to keep P. absoluta infestations under economic damage thresholds.},
}
@article {pmid42043550,
year = {2026},
author = {Yao, X and Yao, X and Luo, M and Luo, L and Zhou, L and Li, X},
title = {Establishment of a CRISPR-Cas12a based electrochemical detection method for Burkholderia gladioli and its subspecies cocovenenans in fresh noodles and tremella.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42043550},
issn = {1432-072X},
support = {2024CZ19//Science and Technology Plan Project of Administration for Market Regulation of Guangdong Province/ ; },
mesh = {*Electrochemical Techniques/methods ; *Biosensing Techniques/methods ; *Burkholderia gladioli/isolation &amp; purification/genetics/classification ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; Sensitivity and Specificity ; Bacterial Proteins/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Bacterial/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Burkholderia gladioli pv. cocovenenans is a pathogenic bacterium of concern due to its potential threat to food safety. This study presents a rapid electrochemical detection method based on dual recombinase-aided amplification (RAA) coupled with CRISPR-Cas12a for the sensitive and specific identification of B. gladioli and its toxigenic subspecies B. gladioli pv. cocovenenans in food. The 16 S rDNA and bonA genes were selected as species- and subspecies-specific targets, respectively, and corresponding CRISPR-Cas12a reaction systems were established. An electrochemical biosensor incorporating a gold electrode functionalized with single-stranded DNA probes was constructed, and its specificity and sensitivity were evaluated using artificially contaminated fresh noodles and tremella samples. The method precisely distinguished B. gladioli and its toxigenic cocovenenans subspecies within 1 h, achieving a limit of detection limit of 10[2] CFU/g in both food matrices. This strategy provides a rapid and field-deployable approach for distinguishing non-toxigenic and toxigenic B. gladioli strains in food, supporting timely screening and food safety monitoring.},
}
@article {pmid42043584,
year = {2026},
author = {Ray, S and Vijayan, J and Vanchinathan, S and Dhakar, R and Nasrullah, N and Nagar, S and Dutta, TK and Chinnusamy, V},
title = {CRISP-PTG-Assembler Ver. 1.0: a primer design tool for polycistronic tRNA-gRNA (PTG) assembly for Cas9-based multiplex genome editing in plants.},
journal = {Planta},
volume = {263},
number = {6},
pages = {},
pmid = {42043584},
issn = {1432-2048},
support = {CRG/2021/002949//Science and Engineering Research Board (SERB), GoI/ ; Genome Editing Project//Indian Council of Agricultural Research/ ; Crop Science EFC-10//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *RNA, Transfer/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; DNA Primers/genetics ; },
abstract = {Multiplex genome editing using the CRISPR/Cas9 system allows simultaneous modifications at several genomic sites, offering great potential for crop improvement. Among various approaches, the polycistronic tRNA-gRNA (PTG) system is widely adopted due to its use of the host's native tRNA processing machinery, enabling the generation of multiple sgRNAs from a single transcript without the need for expressing any foreign RNA processing enzymes or ribozymes. However, designing the complete set of primers suitable for performing in vitro PTG assembly is complex and needs expertise, as a single mistake can lead to complete failure of the assembly process or subsequent editing. To overcome this challenge, we developed CRISP-PTG-Assembler Ver. 1.0, a user-friendly tool that takes only (i) 20-nucleotide sgRNA spacers and (ii) 4-nucleotide joiners as inputs; and produces colour-coded outputs in forms of (i) Primer-set required for complete PTG assembly, (ii) Primary PCR Amplicons, (iii) Overlap-Extension PCR Amplicons and (iv) Expected PTG assembly, for easy interpretation and construct making. Our novel assembly approach provides flexibility in sticky-end choice during golden gate ligation and ensures the fidelity of component sgRNAs in the PTG assembly by buffering against ligation errors (~ 1.5-40%) that may occur during the Golden Gate assembly process, thereby safeguarding the functionality of the in vivo-generated individual sgRNA molecules. We validated its effectiveness by editing two loci of the matrix metalloproteinase 1 gene in rice and demonstrated its applicability across various plant systems. With an intuitive interface and robust features, CRISP-PTG-Assembler empowers researchers of all levels to effectively implement PTG-based multiplex genome editing in plants.},
}
@article {pmid42045369,
year = {2026},
author = {Ragucci, AE and Antine, SP and Leviss, EM and Mooney, SE and Garcia, JM and Shyrokova, L and Hauryliuk, V and Lee, ASY and Kranzusch, PJ},
title = {Nuclease-NTPase antiphage defence systems use conserved molecular features to control bacterial immunity.},
journal = {Nature microbiology},
volume = {},
number = {},
pages = {},
pmid = {42045369},
issn = {2058-5276},
support = {1DP2GM146250-01//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
abstract = {Bacteria encode diverse defence systems, including restriction-modification and CRISPR-Cas, that cleave nucleic acid to protect against phage infection. Bioinformatic analyses demonstrate that many recently identified antiphage defence operons comprise a nuclease and NTPase protein, suggesting that additional nucleic acid-targeting systems remain to be understood. Here we develop large-scale comparative cell biology and biochemical approaches to analyse 16 nuclease-NTPase systems and define molecular features that control antiphage defence. Purification, biochemical characterization and in vitro reconstitution of nucleic acid degradation demonstrates that protein-protein complex formation is a shared feature of multigene nuclease-NTPase systems. We show that PaAbpAB, BtHachiman and EcPD-T4-8 system nucleases use highly degenerate recognition site preferences to enable broad nucleic acid degradation, and the Azaca system exhibits specific phage targeting through the recognition of modified phage genomic DNA. Our results uncover principles of antiphage defence system function and highlight the mechanistic diversity of nuclease-NTPase systems in bacterial immunity.},
}
@article {pmid41553078,
year = {2026},
author = {Xu, T and Wang, T and Zhang, C and Cao, Y and He, X},
title = {Bibliometric-Based Analysis of Global Trends and Collaborative Networks in Plant Genetic Engineering (1994-2024).},
journal = {Plant biotechnology journal},
volume = {24},
number = {5},
pages = {3065-3083},
doi = {10.1111/pbi.70550},
pmid = {41553078},
issn = {1467-7652},
support = {2023ZD0406310//Science and Technology Innovation 2030-Biological Breeding-Major Projects/ ; },
mesh = {*Bibliometrics ; Plants, Genetically Modified/genetics ; *Genetic Engineering/trends ; Gene Editing ; CRISPR-Cas Systems ; Crops, Agricultural/genetics ; },
abstract = {Agricultural sustainability faces serious challenges from population growth, climate change and ecological degradation. Genetic modification (GM) technology can be regarded as a precise extension of the Green Revolution, aiming to balance yield enhancement with ecological integrity through biotechnology. To systematically examine global trend, this study conducts a bibliometric analysis using worldwide literature data from 1994 to 2024. The findings reveal a dual-core structure of international collaboration, centered on China and the United States. The United States is closely connected with Korea, Japan and the United Kingdom, forming a high-density cluster, while China engages with emerging regions in Southeast Asia and Africa through the Belt and Road Initiative. This initiative is intended to strengthen China's influence and is accompanied by the proliferation of technology in countries less endowed with resources. The technology lifecycle has been evolved through three distinct phases. Initially, the process of Agrobacterium-mediated transformation in tobacco plants was carried out, marking the beginning of transgenic development. This was followed by the implementation of RNA interference (RNAi) technology to silence multiple genes. Finally, a breakthrough happened through the development of CRISPR-Cas9 genome editing technologies. The analyses conducted in this study demonstrate the preponderance of CRISPR in contemporary research, thus suggesting that the industry places a premium on technological refinement. Hence, the future technological trajectory is predicted to focus on germplasm digitization, multi-gene editing, intelligent breeding and synthetic biology. Transgenic technology will serve as a foundational support for achieving sustainable food security in the forthcoming second green revolution.},
}
@article {pmid41825104,
year = {2026},
author = {Li, P and Wang, L and Li, G and Li, R and Li, Y and Zhang, Z and Yang, S and Tang, H and Liu, Z},
title = {Transcriptomic and phenotypic analysis of maize with CRISPR/Cas9-mediated targeted mutagenesis of melatonin synthesis genes under drought stress.},
journal = {Plant physiology and biochemistry : PPB},
volume = {233},
number = {},
pages = {111189},
doi = {10.1016/j.plaphy.2026.111189},
pmid = {41825104},
issn = {1873-2690},
mesh = {*Melatonin/biosynthesis/genetics ; *Zea mays/genetics/metabolism/physiology ; *Droughts ; *CRISPR-Cas Systems/genetics ; *Transcriptome ; *Stress, Physiological/genetics ; Abscisic Acid/metabolism ; Reactive Oxygen Species/metabolism ; Gene Expression Regulation, Plant ; Mutagenesis ; Plant Proteins/genetics/metabolism ; Phenotype ; Genes, Plant ; Gene Expression Profiling ; },
abstract = {Melatonin, a pleiotropic regulatory factor, plays a key role in mediating crop drought resistance. Herein, we conducted an integrated physiological and transcriptomic approach to elucidate the mitigating effect of endogenous melatonin in mitigating drought stress in maize. We generated a comt snat asmt maize mutant via CRISPR-Cas9-mediated simultaneous editing of ZmCOMT, ZmSNAT, and ZmASMT1-genes encoding rate-limiting enzymes in the endogenous melatonin biosynthesis pathway. Sequencing of the mutant lines revealed key amino acid substitutions (Gly168→Ala in ZmSNAT, Asp175→Glu in ZmCOMT, and Asp150→Glu in ZmASMT1) within critical protein domains, resulting from CRISPR-induced small insertions or deletions (indels), which led to subtle alterations in the tertiary conformation of corresponding proteins. These modifications resulted in an 86.70% increase in endogenous melatonin content. Under drought stress, the comt snat asmt maize exhibited enhanced antioxidant enzyme activities, leading to a significant reduction in reactive oxygen species (ROS) accumulation compared to the control. Furthermore, endogenous levels of melatonin, abscisic acid (ABA), cytokinin (CTK), and auxin (IAA) were markedly elevated, whereas gibberellin (GA) content was significantly reduced. Consistently, the activities of SNAT, ASMT, and COMT were also enhanced in the mutant. Transcriptomic profiling further revealed that endogenous melatonin regulates ABA, IAA, CTK, and GA signaling pathways to enhance drought tolerance. In particular, ZmCOMT, ZmSNAT, and ZmASMT1 apparently modulated the expression levels of key regulatory genes such as ZmIAA2, ZmIAA23, ZmIAA7, ZmSAUR24, ZmPYL8, and ZmPIF3.1, associated with these hormone pathways. Collectively, endogenous melatonin reinforces drought tolerance by reducing ROS accumulation and reprogramming phytohormone homeostasis through regulation of hormone-related gene expression. Our findings provide important insights into the regulatory mechanisms by which endogenous melatonin enhances drought resistance in crops.},
}
@article {pmid41826557,
year = {2026},
author = {Chao, K and Dietrich, ML and Covey, SC and Momoh, M and Gutt, EG and Sandi, JD and Kamara, MS and Fofanah, IU and Rogers, MM and Kallon, TMPS and Samuels, RJ and Grant, DS and Sabeti, PC and Garry, RF},
title = {Adaptable, quantitative CRISPR/Cas12a-based assay for cytomegalovirus DNA in infant saliva.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41826557},
issn = {2045-2322},
mesh = {Humans ; *Cytomegalovirus/genetics/isolation &amp; purification ; *DNA, Viral/genetics/analysis ; *Saliva/virology ; *Cytomegalovirus Infections/diagnosis/virology/genetics ; *CRISPR-Cas Systems/genetics ; Infant ; Sensitivity and Specificity ; Infant, Newborn ; Polymerase Chain Reaction ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Congenital cytomegalovirus (CMV) infection is the leading non-genetic cause of infant hearing loss worldwide, and a significant cause of neurodevelopmental disabilities. Reliance on polymerase chain reaction (PCR) for CMV DNA testing hampers diagnostic and research efforts in low-resource settings and universal screening implementation in high-resource settings. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) detection and recombinase polymerase amplification (RPA) can be used together for low-cost viral detection. Here we describe an adaptable RPA-Cas12a assay for CMV DNA quantification based on the WHO international standard. Adequate quantification accuracy was achieved with contrived CMV samples but performance with Sierra Leonean infant saliva remains suboptimal. While improved quantification accuracy will require further optimization, our assay achieves screening test requirements, including > 80% sensitivity/specificity, quicker and more economically than PCR. This work highlights RPA-Cas12a-based assays for DNA quantification and suggests a path towards increased congenital CMV screening using PCR and RPA-Cas12a synergistically.},
}
@article {pmid41826615,
year = {2026},
author = {Fernandes, LGV and Nally, JE},
title = {CRISPR-based mutagenesis of lipopolysaccharide biosynthesis genes in Leptospira interrogans reveals gene essentiality and confirms the role of an O-antigen polymerase.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41826615},
issn = {2045-2322},
mesh = {*Lipopolysaccharides/biosynthesis/genetics ; *Leptospira interrogans/genetics/metabolism/enzymology ; Animals ; *CRISPR-Cas Systems ; Leptospirosis/microbiology ; Mutagenesis ; *Genes, Essential ; O Antigens/genetics ; *Bacterial Proteins/genetics/metabolism ; Cricetinae ; Hexosyltransferases ; },
abstract = {Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira. Lipopolysaccharide (LPS) is an immunodominant and protective antigen for Leptospira, but its biosynthesis remains poorly understood. In this study, we employed CRISPR/Cas9-non-homologous end-joining and CRISPR-Prime Editing to mutate key genes within the rfb locus of L. interrogans, including those involved in core oligosaccharide assembly, and the biogenesis, polymerization, and ligation of O-antigen. Mutants were successfully generated in LIC11312 (waaF, heptosyltransferase II) and LIC12137 (wcaJ, undecaprenyl-phosphate glycosyltransferase) but yielded only in-frame deletions suggesting their essentiality. Mutants were also successfully generated in LIC12143, a putative O-antigen polymerase, which exhibited truncated LPS that failed to induce acute leptospirosis in hamsters but retained the ability to colonize kidneys. Mutation of LIC_RS09320, an O-antigen ligase, did not display a change in LPS phenotype. Bacterins prepared with either control wild-type or LIC12143 mutant cells conferred complete homologous protection with sterile immunity, though failed to protect against heterologous challenge. These findings confirm LIC12143 as a functional O-antigen polymerase and underscore the challenges in generating knockout mutants to understand LPS biosynthesis in leptospires.},
}
@article {pmid41955743,
year = {2026},
author = {An, X and Tian, W and Wang, Y and Ren, Y and Xu, C and Chai, Q and Ai, P},
title = {Enhancing tomato fruit sweetness by CRISPR/Cas9-mediated SlVIF gene editing.},
journal = {Plant physiology and biochemistry : PPB},
volume = {233},
number = {},
pages = {111270},
doi = {10.1016/j.plaphy.2026.111270},
pmid = {41955743},
issn = {1873-2690},
mesh = {*Solanum lycopersicum/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Fruit/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Sucrose/metabolism ; },
abstract = {In tomato plants, vacuolar invertase inhibitor (VIF) is a negative regulator of sucrose (SUC) degradation into fructose (FRU) and glucose (GLU), suggesting knockout of SlVIF promotes the accumulation of FRU and GLU. A dual-target vector of pKSE402-SlVIF was constructed and introduced into the tomato breeding parent '1912' via Agrobacterium-mediated transformation. The editing efficiency was as high as 47%. Two Cas9-free T1 lines exhibited delayed fruit ripening, significantly increased FRU and GLU levels, and reduced SUC content at the red ripe stage compared to wild-type (WT) plants, while fruit size and yield were unchanged. Additionally, in the SlVIF knockout mutants, the VIF protein structure was significantly altered, and VIN enzyme activity was markedly increased, which may underlie the enhanced degradation of sucrose in tomato fruits. The findings indicate that the sweetness of tomato fruit can be improved by CRISPR/Cas9-mediated SlVIF gene editing, which provides valuable resources for high-quality breeding in tomato.},
}
@article {pmid42033211,
year = {2026},
author = {Aliciaslan, M and Erbasan, E and Erendor, F and Sanlioglu, S},
title = {Advances in CRISPR Base Editing: From Molecular Evolution to Therapeutic Applications in Genomic Medicine.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {8},
pages = {e71159},
doi = {10.1111/jcmm.71159},
pmid = {42033211},
issn = {1582-4934},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Genomics/methods ; Animals ; *Evolution, Molecular ; *Genetic Therapy/methods ; },
abstract = {CRISPR-Cas9 systems revolutionized gene editing, but inherent drawbacks, namely DNA double-strand breaks (DSBs) and the difficulty of achieving precise repairs (due to low HDR efficiency), led researchers to invent new, more accurate gene editing tools. Base editing represents a significant leap forward, enabling targeted single-nucleotide conversions directly on the DNA without DSBs or donor templates. The core technology involves fusing catalytically dead or nickase Cas proteins to DNA deaminase enzymes. Cytosine base editors (CBEs) convert C•G to T•A pairs, while adenine base editors (ABEs) change A•T to G•C. These editors exploit the deaminase function within the R-loop structure formed by Cas binding and co-opt endogenous DNA repair mechanisms for precision. While offering improved efficiency and editing precision, base editing faces persistent challenges, such as off-target effects, bystander edits, delivery and ethical concerns. Continuous engineering efforts have refined these tools, enhancing accuracy, expanding targetability and reducing unwanted edits. The base editing arsenal has also broadened to include C-to-G base editors (CGBEs), dual A&amp;C editors and versions targeting organelles. Successful preclinical studies demonstrating the correction of mutations responsible for the disease have paved the way for clinical trials, which are now testing therapies for conditions like sickle cell disease, β-thalassaemia and hypercholesterolemia using various delivery systems. This review explores CRISPR base editing's origins, mechanisms of action, potential therapies and current restrictions, pointing to its broadening impact on medical genetics.},
}
@article {pmid42033509,
year = {2026},
author = {Çelik, B and Kiraz, Y and Şahin, Y and Tezcanlı Kaymaz, B},
title = {Targeting STAT5A via CRISPR/Cas9 restores TKI sensitivity in resistant chronic myeloid leukemia cells.},
journal = {Medical oncology (Northwood, London, England)},
volume = {43},
number = {6},
pages = {},
pmid = {42033509},
issn = {1559-131X},
mesh = {Humans ; *STAT5 Transcription Factor/genetics/metabolism ; *Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics ; *CRISPR-Cas Systems ; *Protein Kinase Inhibitors/pharmacology ; K562 Cells ; Apoptosis/drug effects/genetics ; Gene Knockout Techniques ; Tumor Suppressor Proteins ; },
abstract = {Therapeutic resistance to tyrosine kinase inhibitors (TKIs) remains a major challenge in the clinical management of chronic myeloid leukemia (CML). The transcription factor STAT5A, a principal downstream effector of BCR::ABL1, has emerged as a key transcriptional regulator implicated in the development of TKI resistance. This study aims to functionally validate the role of STAT5A in TKI-resistant CML by employing CRISPR/Cas9-mediated gene knockout and assessing the downstream molecular and phenotypic alterations. We hypothesized that selective disruption of STAT5A would restore apoptotic sensitivity and TKI responsiveness in resistant CML models. Additionally, we sought to integrate bioinformatic transcriptional network analyses to confirm whether STAT5A directly regulates the genes modulated by its deletion, thus reinforcing its mechanistic relevance as a therapeutic target. STAT5A was knocked out using CRISPR/Cas9 in K562 cells and their TKI-resistant derivatives (K562/Ima-Res, K562/Pon-Res). Western blot analysis confirmed effective depletion of STAT5A protein following CRISPR/Cas9 editing, validating that the observed phenotypic and transcriptional changes were attributable to successful STAT5A knockout. Post-editing, XTT assays were performed to assess cell viability, followed by Annexin V/PI staining for apoptosis and PI-based flow cytometry for cell cycle analysis. RT-qPCR was used to quantify the expression of key genes involved in the JAK/STAT pathway (JAK2, STAT3, CISH) and apoptosis/DNA damage responses (TP53, ATM, CASP3, CASP8). In silico analyses were conducted using TRRUST and Harmonizome/ChEA3 to confirm whether the genes modulated by STAT5A deletion were direct transcriptional targets. For additional validation, expression matrices from GSE207627 and GSE208314 were reanalyzed to confirm STAT5A-centered pathway alterations in resistant CML datasets. STAT5A knockout significantly reduced cell viability and induced apoptosis across all CML cell models, accompanied by G0/G1 cell cycle arrest. RT-qPCR revealed altered expression of both JAK/STAT components (JAK2, STAT3, CISH) and apoptosis-related genes (TP53, ATM, CASP3, CASP8). Transcriptional target analysis confirmed that several of these genes-such as CDKN2B, BCL2L1, and CCND1-are direct STAT5A targets, reinforcing the functional consequences of STAT5A loss. Integration of these findings suggests that STAT5A knockout reprograms both intrinsic (CASP3, TP53, ATM) and extrinsic (CASP8, BCL2L1) apoptotic pathways, thereby restoring chemosensitivity. CISH dysregulation further suggested compensatory feedback within the signaling network. CRISPR/Cas9-mediated STAT5A disruption effectively reverses TKI resistance in CML cells by reprogramming apoptotic and proliferative signaling. These findings identify STAT5A as a mechanistically validated and clinically actionable target, supporting its potential for combination strategies with TKIs or STAT5 inhibitors such as pimozide. Integration of transcriptional network analysis supports the mechanistic basis of these effects. STAT5A emerges as a compelling therapeutic target, meriting further investigation in preclinical models and patient-derived samples to evaluate its translational potential. Future validation in patient-derived CD34[+] CML models may advance STAT5A-based therapeutic design.},
}
@article {pmid42033623,
year = {2026},
author = {Askri, SMH and Li, F and Wang, J and Shahwar, D and Zhang, X},
title = {Toward sustainable control of phyto-nematodes: integrating lessons from crops to advance genetic modification in tomato.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {5},
pages = {},
pmid = {42033623},
issn = {1432-2242},
support = {ZR2024MC063//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Solanum lycopersicum/genetics/parasitology ; Animals ; *Plants, Genetically Modified/parasitology/genetics ; *Disease Resistance/genetics ; *Plant Diseases/parasitology/genetics/prevention &amp; control ; *Tylenchoidea/physiology ; Crops, Agricultural/genetics/parasitology ; Plant Breeding ; },
abstract = {Emerging molecular approaches offer promising avenues to enhance tomato defense, demonstrating potential to effectively suppress root-knot nematode infestation and improve crop resilience under diverse environmental conditions. Root-knot nematodes (RKN, Meloidogyne spp.) continue to challenge tomato production by disrupting root architecture, impairing nutrient uptake, and reducing yields. Recent advances in plant biotechnology provide multiple avenues to enhance nematode resistance beyond conventional resistance (R)-gene utilization. This review discusses molecular and transgenic strategies investigated for RKN resistance across diverse plant systems, including the transfer or engineering of R-genes, expression of anti-feedant proteins and nematotoxic peptides, host-induced gene silencing targeting essential nematode genes, and CRISPR/Cas-mediated modification of host susceptibility factors, with an emphasis on their relevance for future use in tomato. Each approach interferes with distinct stages of nematode infection-from host recognition and feeding-site establishment to reproduction-and has demonstrated measurable reductions in gall formation, egg production, or nematode fitness, primarily in controlled-environment studies and selected field evaluations conducted in non-tomato or model crop systems. The review further evaluates progress in translating these cross-species advances into field-relevant tomato production contexts, emphasizing considerations for tissue-specific expression, stacking of complementary traits, and regulatory compliance affecting practical implementation. By highlighting the mechanistic insights gained from these molecular interventions across multiple crops, this work identifies key opportunities for integrating complementary genetic strategies into tomato breeding programs. Collectively, these advances underscore the potential of combining precision genetics and biotechnology to develop durable, broad-spectrum nematode resistance, reducing dependence on chemical nematicides and supporting sustainable horticultural production.},
}
@article {pmid40850686,
year = {2026},
author = {Wang, J and Yang, W and Fang, J and Xu, H and Li, J and Zhang, Y and Liang, Q and Xu, B and Li, H and Niu, E},
title = {Field-parallel six-sample microfluidic detection of plant viruses via raffinose-assisted one-pot LAMP-CRISPR/Cas12b.},
journal = {Journal of advanced research},
volume = {83},
number = {},
pages = {81-89},
doi = {10.1016/j.jare.2025.08.030},
pmid = {40850686},
issn = {2090-1224},
mesh = {*Plant Diseases/virology ; *Plant Viruses/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Raffinose/chemistry ; *Molecular Diagnostic Techniques/methods/instrumentation ; Lab-On-A-Chip Devices ; *Microfluidics/methods ; },
abstract = {INTRODUCTION: Plant viral diseases threaten food security globally. Rapid, accurate and early detection of viral pathogens is critical for proactive surveillance and effective control.<br><br>OBJECTIVES: This study aimed to develop a platform for the diagnosis of plant viruses in the field.<br><br>METHODS: We developed an ALERT platform (AapCas12b coupled RT-LAMP Extraction-field RNA testing) that integrates RT-LAMP and CRISPR/Cas12b-based lateral flow system for the diagnosis of plant viruses. The portable metal incubator, coupled with the integrated microfluidic chip, enables parallel detection of six test samples in the field. Additionally, a raffinose additive solution effectively achieved the phase separation between the LAMP and CRISPR/Cas12b system through promoting dynamic diffusion of LAMP products amplicons.<br><br>RESULTS: The ALERT platform exhibited comparable sensitivity to conventional RT-PCR, and field detection showed 100% diagnostic agreement with RT-PCR.<br><br>CONCLUSIONS: This platform exhibits outstanding performance with high sensitivity and portability, and has great potential for rapid plant virus detection, especially in environments with resource-limited settings.},
}
@article {pmid41101730,
year = {2026},
author = {Zhang, H and Tonini, MR and Martires, LCM and Jenkins, HN and Pratt, CB and Gordon, EN and Zhao, S and Choi, AH and Meier, SR and Khendu, T and Liu, S and Shen, B and Stowe, H and Pashiardis, K and Pan, X and Bandi, M and Zhang, M and Yu, Y and Min, C and Huang, A and Andersen, JN and Nicholson, HE and Teng, T},
title = {Tumor suppressor collateral damage screens reveal mRNA homeostasis protein HBS1L as a novel vulnerability in ch9p21 driven FOCAD deleted cancer.},
journal = {Biochimica et biophysica acta. Molecular cell research},
volume = {1873},
number = {1},
pages = {120070},
doi = {10.1016/j.bbamcr.2025.120070},
pmid = {41101730},
issn = {1879-2596},
mesh = {*GTP-Binding Proteins/genetics/metabolism ; *RNA, Messenger/metabolism ; Chromosome Deletion ; Chromosomes, Human, Pair 9 ; Genes, Tumor Suppressor ; Synthetic Lethal Mutations ; *Endonucleases/genetics/metabolism ; *Nuclear Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; Humans ; *Tumor Suppressor Proteins/genetics/metabolism ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Animals ; Mice ; Female ; },
abstract = {Chromosomal deletion of tumor suppressor genes often occurs in an imprecise manner, leading to co-deletion of neighboring genes. This collateral damage can create novel dependencies specific to the co-deleted context. One notable example is the dependency on PRMT5 activity in tumors with MTAP deletion, which co-occurs with CDKN2A/B loss, leading to the development of MTA-cooperative PRMT5 inhibitors. To identify additional collateral damage context/target pairs for chromosome 9p and other common loci of chromosomal deletions, we conducted a combinatorial CRISPR screen knocking out frequently co-deleted genes in combination with a focused target library. We identified the gene encoding the ribosome rescue factor PELO as synthetic lethal with the SKI complex interacting protein FOCAD, which is frequently co-deleted alongside MTAP and CDKN2A/B on chromosome 9p. A genome-wide screen in FOCAD isogenic cells further identified the ribosome rescue GTPase and PELO binding partner HBS1L as the top synthetic lethal target for FOCAD loss. Analysis of publicly available data and genetic manipulation of HBS1L using orthogonal modalities validated this interaction. HBS1L dependency in FOCAD-deleted cells was rescued by FOCAD re-expression, and FOCAD intact cells could be rendered HBS1L-dependent by FOCAD knockout, demonstrating the context specificity of this interaction. Mechanistically, HBS1L loss led to translational arrest and activated the unfolded protein response in FOCAD-deleted cells. In vivo, HBS1L deletion eliminated growth of FOCAD-deleted tumors. Here we propose a model where the FOCAD/SKI complex and HBS1L/PELO work together to resolve aberrant mRNA-induced ribosomal stalling, making the HBS1L/PELO complex an intriguing novel target for treating FOCAD-deleted tumors.},
}
@article {pmid41298833,
year = {2026},
author = {Kim, SH and Kim, MM},
title = {Functional analysis of AKT1 knockout in fibrosarcoma cells using CRISPR/Cas9 technology.},
journal = {Journal of human genetics},
volume = {71},
number = {5},
pages = {309-313},
pmid = {41298833},
issn = {1435-232X},
mesh = {*Proto-Oncogene Proteins c-akt/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Humans ; Cell Line, Tumor ; *Gene Knockout Techniques ; Forkhead Box Protein O1/genetics/metabolism ; Signal Transduction/genetics ; Gene Editing ; TOR Serine-Threonine Kinases/genetics/metabolism ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; INDEL Mutation ; },
abstract = {AKT1 (Protein Kinase B alpha) is a serine/threonine kinase that plays a pivotal role in regulating various cellular processes. To elucidate the role of the AKT1 gene in signaling pathways, this study generated AKT1 knockout (KO) HT-1080 cells using the CRISPR/Cas9 system. Gene-editing efficiency was validated through Sanger DNA sequencing and insertion/deletion (InDel) analysis. Quantitative real-time PCR and Western blot analyses were performed to evaluate the expression levels of AKT1 mRNA and protein, as well as to examine the expression of AKT1 downstream effectors: mTOR, BCL-2, and FOXO1. The AKT1 single-guide RNA sequence was successfully cloned into the CRISPR/Cas9 vector, leading to the establishment of AKT1 KO cells. InDel analysis identified eight editing types, with two dominant populations. The expression levels of AKT1 mRNA and protein were significantly reduced in the KO cells. The expression levels of mTOR, BCL-2, and FOXO1 were significantly altered in the KO cells compared to normal cells. These findings highlight the impact of AKT1 disruption on signaling pathways and provide fundamental insights into the regulatory role of the AKT1 gene.},
}
@article {pmid41545752,
year = {2026},
author = {Chen, ZY and Guo, R and Wang, M and Ji, SJ and Zhang, JW and Zheng, HX and Wang, ST and Xie, X},
title = {PLN-L31A/I40A for the treatment of inherited heart disease caused by PLN-R14del mutations.},
journal = {Acta pharmacologica Sinica},
volume = {47},
number = {5},
pages = {1191-1203},
pmid = {41545752},
issn = {1745-7254},
mesh = {Animals ; Humans ; *Calcium-Binding Proteins/genetics/metabolism ; Myocytes, Cardiac/metabolism ; Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism ; *Genetic Therapy/methods ; Mice ; Gene Editing ; Mutation ; Disease Models, Animal ; CRISPR-Cas Systems ; Dependovirus/genetics ; },
abstract = {Phospholamban (PLN) is a regulatory protein of the SERCA2α calcium transporter, which plays an important role in maintaining calcium homeostasis in cardiomyocytes. Deletion of the 14th arginine of PLN (PLN-R14del) leads to dysregulation of SERCA2α and PLN aggregation, and is a common cause of dilated cardiomyopathy. In this study, by using CRISPR-Cas9 gene editing technology, we constructed the PLN-R14del mouse model and hESCs. The PLN[R14del/R14del] mice developed severe ventricular dilation, cardiac fibrosis, and PLN aggregation, as well as premature death due to heart failure. Reduced cardiomyocyte functions and PLN aggregation were also observed in the human PLN[R14del/WT] cardiomyocytes differentiated from gene-edited hESCs. AAV delivery of PLN-L31A/I40A, which blocks PLN-R14del and SERCA2α interaction but without blocking the function of the latter, provided a therapeutic effect in both mice and human cardiomyocytes. These results not only suggest that PLN-L31A/I40A gene therapy is practical, but also suggest that blocking the interaction between PLN-R14del and SERCA2α with other modalities, such as small molecules, might also be beneficial.},
}
@article {pmid41634394,
year = {2026},
author = {Harada, M and Fu, G and Badis, Y and Cock, JM and Coelho, SM and Nagasato, C and Motomura, T},
title = {Functional roles of mastigonemes in Ectocarpus gamete swimming revealed by CRISPR-Cas9 mutagenesis.},
journal = {Protoplasma},
volume = {263},
number = {3},
pages = {943-954},
pmid = {41634394},
issn = {1615-6102},
mesh = {*CRISPR-Cas Systems/genetics ; *Germ Cells/metabolism ; *Mutagenesis/genetics ; *Phaeophyceae/genetics ; *Flagella/metabolism ; Mutation/genetics ; },
abstract = {Mastigonemes on the anterior flagellum (AF) of flagellated Stramenopiles (which includes diverse organisms such as diatoms, brown algae, oomycetes and others) are tripartite tubular structures. We investigated the functions of mastigonemes in gametes of the brown alga Ectocarpus species 7 strain Ec32 using a mas1 mutant generated by CRISPR-Cas9. Loss of mastigonemes in the mas1 mutant gametes could be confirmed by immunofluorescence microscopy using a specific anti-MAS1 antibody and transmission electron microscopy, showing complete loss of mastigonemes from the AF. High-speed video analysis revealed a drastic reduction in swimming speed in the mas1 mutant gametes compared to wild type gametes, despite an increase in the AF beat frequency. Additionally, waveform analysis indicated larger AF double amplitudes in the mas1 mutant gametes. These results suggested that mastigonemes enhance the AF thrust. The mas1 mutant male gametes fertilized female gametes (wild type strain Ec25). A mas1 mutant female strain was established from the heterozygous sporophyte that developed from such a zygote. Both wild type and the mas1 mutant male gametes could fertilize the mas1 mutant female gametes. Mastigonemes are therefore dispensable for gamete recognition and fusion in the brown alga Ectocarpus.},
}
@article {pmid41644117,
year = {2026},
author = {Kovooru, L and Zhang, J and Monni, FG and Dutta, T and Gongye, X and Asiedu, B and Infelise, P and Barreby, E and Jamialahmadi, O and Mahlapuu, M and Mancina, RM and Romeo, S},
title = {MTARC1 p.A165 ablation reduces hepatocellular carcinoma aggressiveness in vitro and in vivo.},
journal = {Clinical and molecular hepatology},
volume = {32},
number = {2},
pages = {829-842},
doi = {10.3350/cmh.2025.1261},
pmid = {41644117},
issn = {2287-285X},
support = {//SciLifeLab/ ; //BioMS/ ; //Wallenberg Foundation via the Wallenberg Centre for Molecular and Translational Medicine/ ; 22 2270 Pj//Swedish Cancerfonden/ ; 2023-02079//Swedish Research Council/ ; ALFGBG-965360//Agreement between the Swedish government and the county councils/ ; 20220334//Swedish Heart Lung Foundation/ ; NNF23OC0082114//Novonordisk Distinguished Investigator Grant - Endocrinology and Metabolism/ ; NNF24OC0091535//Novonordisk Project grants in Endocrinology and Metabolism/ ; },
mesh = {Humans ; *Carcinoma, Hepatocellular/pathology/genetics/metabolism ; Animals ; *Liver Neoplasms/pathology/genetics/metabolism ; Cell Proliferation ; Mice ; Cell Line, Tumor ; Cell Movement ; CRISPR-Cas Systems ; Lipid Metabolism ; RNA, Small Interfering/metabolism ; Gene Knockdown Techniques ; },
abstract = {BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide and is driven by metabolic reprogramming that supports tumor growth and progression. A common missense genetic variant (rs2642438, p.A165T) in mitochondrial amidoxime reducing component 1 (MTARC1), identified as protective against liver disease, has been recently associated with lower prevalence of steatosis, cirrhosis, and HCC. However, the mechanistic role of MTARC1 in HCC is unclear. Therefore, we sought to decipher the role of MTARC1 in HCC.<br><br>METHODS: We investigated the role of MTARC1 in HCC by performing siRNA-mediated knockdown across human immortalized HCC cell lines (Hep3B2, HuH7, HepG2 and HepaRG) homozygous for the risk allele (p.A165) and by generating stable CRISPR-Cas9 knockout (KO) models. Next, we assessed the effect of MTARC1 loss on cell proliferation, migration, lipid metabolism, and fatty acid oxidation in vitro, as well as tumor aggressiveness in a subcutaneous xenograft mouse model. Additionally, we performed global proteomics in both in vitro and xenograft models.<br><br>RESULTS: Transient knockdown of MTARC1 p.A165 reduced proliferation in HCC cell lines. CRISPR-Cas9-mediated stable MTARC1 p.A165 KO in Hep3B2 cells led to decreased neutral lipid intracellular accumulation, enhanced &#x3b2;-oxidation and reduced cell migration. An MTARC1 KO xenograft model had reduced tumor volume. Proteomic analyses of both in vitro HCC cells and xenograft tumors revealed inhibition of oncogenic pathways and activation of anti-proliferative proteins.<br><br>CONCLUSIONS: Downregulation of MTARC1 p.A165 inhibits lipid accumulation, dampens tumor-promoting pathways and restricts tumor growth, highlighting MTARC1 as a promising therapeutic target for HCC.},
}
@article {pmid41714766,
year = {2026},
author = {Taha, BA and Addie, AJ and Haider, AJ and Jasim, AJ and Ahmed, NM and Arsad, N},
title = {Perspective of smart nanocapsule swallowable laser-guided for integrated sensing and crispr-mediated cancer gene editing.},
journal = {Cancer gene therapy},
volume = {33},
number = {3},
pages = {351-365},
pmid = {41714766},
issn = {1476-5500},
mesh = {Humans ; *Gene Editing/methods ; *Nanocapsules/chemistry/administration &amp; dosage ; *Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Lasers ; Biosensing Techniques/methods ; Animals ; *Genetic Therapy/methods ; },
abstract = {Current therapeutic techniques for cancer often lack specificity. They also cause systemic toxicity and lack genetic control. Thus, cancer ranks among the most complex and crucial global health issues. The novel concept of smart nanocapsules is discussed in this Perspective. These oral medications modify genes using CRISPR technology and integrate biosensing and laser-guided activation to enable more personalized cancer therapies. The creation of these versatile nanocapsules is driven by three objectives. First, they aim to enable controlled gene editing in the gastrointestinal tract. Second, they deliver treatments to specific target areas. Third, they detect tumors in real time. Nanocapsules equipped with biosensing components provide microenvironmental input. An external laser can trigger the release of light-absorbing agents. Moreover, these features reduce off-target effects and allow spatiotemporal precision, thhe enteric-coated architecture ensures oral stability. Surface functionalization enhances selective tumor accumulation. AI-guided control algorithms can manage diagnostic interpretation and activation. The CRISPR-based cancer medicines offer the potential for improved safety, specificity, and translational use in the future. Combining advanced nanotechnology, gene editing, and AI-guided control could create innovative solutions.},
}
@article {pmid41941439,
year = {2026},
author = {He, Y and Shen, Y and Duan, M and Shen, J and Zhang, X and Chen, J and Liu, Z and Jia, F},
title = {An Environmentally Resilient, Metal-Organic Framework-Armored CRISPR/Cas12a Sensing Reactor (ENCASE) for the Ultra-Stable and On-Site Detection of Salmonella typhimurium in the Food Supply Chain.},
journal = {ACS sensors},
volume = {11},
number = {4},
pages = {3051-3062},
doi = {10.1021/acssensors.5c03704},
pmid = {41941439},
issn = {2379-3694},
mesh = {*Salmonella typhimurium/isolation &amp; purification/genetics ; *Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Food Microbiology ; Food Contamination/analysis ; Temperature ; },
abstract = {The development of reliable and field-deployable detection technologies of Salmonella typhimurium (S. typhimurium) throughout the food supply chain is crucial for the early warning and effective control of salmonellosis outbreaks. CRISPR-based biosensors offer excellent specificity, high sensitivity, and portability; however, their practical applications are significantly limited by the poor environmental stability of Cas enzymes, which are highly susceptible to temperature fluctuations and organic solvent interference. Here, a metal-organic framework (MOF) material, named ZIF-L, was employed as a sensing reactor to encapsulate the whole CRISPR sensing system, effectively enhancing its stability against variable external conditions such as temperature fluctuations and organic solvents encountered along the food supply chain. The feasibility of the conventional and encapsulated anti-S. typhimurium CRISPR sensor was confirmed through CLSM imaging, PAGE testing, and fluorescent verification. Importantly, the protective ability of the fabricated sensing reactor was precisely regulated by optimizing the pore size, ligand ratio, and dimensions of the MOFs and then evaluated under extreme detection conditions: (i) different external temperatures (4, 37, 50, 60, and 70 °C) and (ii) different organic solvents (methanol, acetone, and isopropyl alcohol). An impressive sensing performance of over 75% of its bioactivity, with a detection limit of 33 CFU/mL for S. typhimurium, was retained, confirming its detection ability under variable detection conditions. Moreover, recovery rates of 93.2-105.7% in spiked food samples, even when subjected to typical environmental interferences, including low temperatures (4 °C), high temperatures (60 °C), and organic solvent (methanol) exposure were obtained, showcasing its potential for the ultra-stable, on-site detection of S. typhimurium, particularly under variable external conditions.},
}
@article {pmid41996462,
year = {2026},
author = {Gu, Y and Hart, T and Leon-Novelo, L and Shen, JP},
title = {Double-CRISPR Knockout Simulation (DKOsim): A Monte-Carlo randomization system to model cell growth behavior and infer the optimal library design for growth-based double knockout screens.},
journal = {PLoS computational biology},
volume = {22},
number = {4},
pages = {e1013510},
doi = {10.1371/journal.pcbi.1013510},
pmid = {41996462},
issn = {1553-7358},
mesh = {Monte Carlo Method ; *Gene Knockout Techniques/methods ; *CRISPR-Cas Systems/genetics ; Computer Simulation ; Humans ; Computational Biology ; Gene Library ; *Cell Proliferation/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Models, Genetic ; },
abstract = {Advances in functional genomic technology, notably CRISPR using Cas9 or Cas12, now allow for large-scale double perturbation screens in which pairs of genes are inactivated, allowing for the experimental detection of genetic interactions (GIs). However, as it is not possible to validate GIs in high-throughput, there is no gold standard dataset where true interactions are known. Hence, we constructed a Double-CRISPR Knockout Simulation (DKOsim), which allows users to reproducibly generate synthetic simulation data where the single gene fitness effect of each gene and the interaction of each gene pair can be specified by the investigator. We adapted Monte-Carlo randomization methods to extend single knockout simulation methods to double knockout designs, which simulate the gene-gene interactions between all possible combinations of the input genes. Using DKOsim, we generated simulated datasets that closely resemble real double knockout CRISPR datasets in terms of Log Fold Change (LFC), GI distribution, and replicate correlation. We further inferred optimal CRISPR library designs by systematically investigating critical experimental parameters including depth of coverage, guide efficiency, and the variance of initial guide distribution. This simulation scheme will help to identify optimal computational methods for GI detection and aid in the design of future dual knockout CRISPR screens.},
}
@article {pmid42027081,
year = {2026},
author = {Hou, Q and Ren, J and Wu, Y and Zhao, P and Yue, S and Bi, S},
title = {Nucleic Acid Nanotechnology-Empowered CRISPR-Cas12a Systems for Biosensing and Bioimaging Applications.},
journal = {Small methods},
volume = {},
number = {},
pages = {e70666},
doi = {10.1002/smtd.70666},
pmid = {42027081},
issn = {2366-9608},
support = {ZR2024QB071//Natural Science Foundation of Shandong Province/ ; 22474067//National Natural Science Foundation of China/ ; tstp20230623//Taishan Scholar Foundation of Shandong Province/ ; 2025SHFXTD001//Open Project of Shandong Provincial Key Laboratory for Tumor Imaging Equipment Development and Tumor Diagnosis &amp; Treatment Integration Technology/ ; X2025104521145//Innovation and Entrepreneurship Training Program for College Students/ ; },
abstract = {The development of highly sensitive and simple bioanalytical platforms is crucial for advancing disease diagnostics and biomedical research. In recent years, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) protein systems have emerged as the "next-generation molecular technology" that profoundly impacts the field of genome editing and molecular diagnosis. In particular, CRISPR-Cas12a, an important CRISPR-Cas family member, has gained prominence as a robust tool in biosensing and bioimaging due to its easy design and high target specificity. Notably, the integration of nucleic acid nanotechnology, including nucleic acid amplification strategies and engineered functional nucleic acids, with CRISPR-Cas12a systems significantly improves detection sensitivity and specificity, enabling the analysis of low-abundance analytes. This review introduces the fundamentals of CRISPR-Cas12a and key nucleic acid-based toolboxes. Next, we systematically summarize the advantages of nucleic acid nanotechnology-empowered CRISPR-Cas12a platforms in detail and highlight recent advances in their applications in biosensing and bioimaging. Finally, current challenges and future perspectives of such nucleic acids-assisted CRISPR-Cas12a systems for disease diagnostics are discussed.},
}
@article {pmid42029722,
year = {2026},
author = {Gong, Z and Chen, M and Zhang, H and Mortimer, JC and Botella, JR},
title = {Evaluation of computational tools for the prediction of CRISPR/SpCas9 gRNA activity in plants.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {42029722},
issn = {1432-203X},
support = {CE230100015//P4S/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Nicotiana/genetics ; *Computational Biology/methods ; Genome, Plant/genetics ; Machine Learning ; *CRISPR-Associated Protein 9/genetics/metabolism ; Algorithms ; },
abstract = {CRISPR/Cas9 technologies are now routinely used in plant research, with guide RNA (gRNA) design being a critical determinant of genome editing success. However, rational design of highly active gRNAs is challenging due to complex sequence and biochemical factors affecting activity. While numerous computational prediction tools have been developed, they are predominantly trained on animal cell or microbial data and their performance in plants remains controversial or untested. In this study, using two independent Nicotiana benthamiana experimental datasets comprising a total of 52 gRNAs, we systematically evaluated over 20 freely accessible, Web-based in silico tools for predicting gRNA on-target efficiency. We identified several machine learning-based tools that showed strong correlation with experimental editing efficiency across both datasets. Importantly, gRNAs in the top quartile by prediction score produced significantly higher InDel frequencies than those in the lowest quartile for all tools tested. Furthermore, several algorithms available through CRISPOR, a platform containing a large number of non-model plant genomes, also showed good predictive performance. This may enable better integration of on-target and off-target predictions in gRNA design. Our findings provide practical guidance for improving gRNA design in plant genome editing applications.},
}
@article {pmid42029959,
year = {2026},
author = {Sarki, YN and Keot, AK and Marwein, R and Singha, DL and Gogoi, DJ and Velmurugan, N and Chikkaputtaiah, C},
title = {Genotype-specific optimization of in vitro regeneration and Agrobacterium-mediated transformation in indica rice with 35S:RUBY and CRISPR/LbCas12a system.},
journal = {Planta},
volume = {263},
number = {6},
pages = {},
pmid = {42029959},
issn = {1432-2048},
support = {MMP025301//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; CRG/2022/007073//Science and Engineering Research Board/ ; },
mesh = {*Oryza/genetics/physiology ; *Transformation, Genetic ; Plants, Genetically Modified/genetics ; Genotype ; Regeneration/genetics ; CRISPR-Cas Systems/genetics ; *Agrobacterium/genetics ; Seeds/genetics ; 2,4-Dichlorophenoxyacetic Acid/pharmacology ; },
abstract = {This study establishes a genotype-specific transformation system for indica rice cultivars Ranjit, Mahsuri, and Kon Joha using 35S:RUBY and CRISPR/LbCas12a constructs, enabling functional genomics studies and genetic improvement. The indica rice subspecies generally faces challenges in functional genomics and genetic improvement due to its recalcitrance to tissue culture and Agrobacterium-mediated transformation. Three indica rice varieties, Ranjit, Mahsuri, and Kon Joha, cultivated in Assam (India) were selected to optimize callus induction, regeneration, and genetic transformation. Ranjit and Mahsuri are high-yielding cultivars, whereas Kon Joha is an indigenous aromatic landrace of high commercial value. Initially, key steps, such as callus induction and regeneration, were optimized for Ranjit and Kon Joha using mature seed. Thereafter, for transformation, immature embryos were selected as explants because of their competence for agro-infection. The highest callus induction frequency of 70.33% and 90% was achieved with 3.0 and 3.5 mg L[-1] of 2,4-dichlorophenoxyacetic acid (2,4-D) in Ranjit and Kon Joha, respectively. The best regeneration, in Ranjit (77%) and Kon Joha (54.5%), was achieved with 4.0 mg L[-1] 6-benzylaminopurine (BAP), 1.0 mg L[-1] kinetin, and 0.5 mg L[-1] naphthalene acetic acid (NAA). Transformation efficiencies with CRISPR/LbCas12a (OD660 = 0.8) were 5.6% (Ranjit), 7.5% (Mahsuri), and 15.33% (Kon Joha). The optimal in vitro regeneration conditions previously identified for Ranjit were adopted for Mahsuri transformation. Furthermore, a visual non-invasive reporter, RUBY, was employed to investigate the transformation of immature embryos in Ranjit and Kon Joha. The vivid red coloration in the early callusing stage indicates successful transformation events. Kon Joha plants regenerated from red calli showed a distinct phenotype with pink leaves and red roots. These findings confirm RUBY's effectiveness as a non-invasive reporter for rapid monitoring of rice transformation and underscore the importance of immature embryos in indica rice transformation.},
}
@article {pmid42030398,
year = {2026},
author = {Henard, JM and Lee, SA and Yu, YC and Shao, D and Azad, RK and Henard, CA},
title = {CRISPRi-based functional genomic screening identifies genes essential for CH4-dependent growth in a methanotrophic bacterium.},
journal = {Science advances},
volume = {12},
number = {17},
pages = {eaed4234},
doi = {10.1126/sciadv.aed4234},
pmid = {42030398},
issn = {2375-2548},
mesh = {*Methane/metabolism ; *CRISPR-Cas Systems ; *Genomics/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genes, Essential ; *Methylococcus capsulatus/genetics/growth &amp; development/metabolism ; },
abstract = {Methanotrophic bacteria are the primary organisms that consume atmospheric methane (CH4) and have potential to mitigate climate-active gases. However, a limited understanding of the genetic determinants of methanotrophy hinders the development of biotechnologies leveraging these unique microbes. Here, we developed and optimized a CRISPR interference (CRISPRi) system to enable functional genomic screening in methanotrophic bacteria. We built a genome-wide single guide RNA (sgRNA) library in the industrial methanotroph, Methylococcus capsulatus, consisting of ~45,000 unique sgRNAs mediating inducible, CRISPRi-dependent transcriptional repression. A selective screen during growth on CH4 identified genes associated with essential methanotrophic bacterial processes and previously unidentified essential gene candidates, highlighting the utility of CRISPRi for functional genetic screening in methanotrophs. The CRISPRi screen also led to nitrate mineral salts medium optimization to improve methanotroph biomass productivity from CH4. Collectively, our results show that the CRISPRi system and sgRNA library developed here can be used for facile gene-function analyses and genomic screening to identify genetic determinants of methanotrophy and isolation of improved methanotroph biocatalysts.},
}
@article {pmid42031757,
year = {2026},
author = {Ly, D and Jang, H and Goel, A and Singh, A and Raguram, A},
title = {Genome-wide screening reveals producer-cell modifications that improve virus-like particle production and delivery potency.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42031757},
issn = {2041-1723},
support = {DP5OD037342//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; },
mesh = {Animals ; Mice ; Humans ; *Gene Editing/methods ; *Virion/genetics/metabolism ; HEK293 Cells ; *Gene Transfer Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Engineered virus-like particles (eVLPs) are promising vehicles for transient delivery of gene editing agents. While extensive particle engineering has yielded efficient eVLPs, it remains underexplored whether engineering the cells used to produce eVLPs could further improve eVLP properties. We report an unbiased genome-wide screening approach to systematically investigate how genetic perturbations in producer cells influence eVLP production. This approach generates eVLPs loaded with guide RNAs that identify the genetic perturbation in the cell that produced a particular particle; the abundance of each guide RNA in eVLPs therefore reflects how the corresponding genetic perturbation influences eVLP production or cargo loading. We apply this approach to identify several genes that regulate eVLP cargo expression and loading into particles during the production process. Leveraging these insights, we engineer producer cells that support increased eVLP cargo packaging and a 2- to 9-fold increase in eVLP delivery potency across several cargo, particle, and target-cell types in cultured cells and in mice. Our findings suggest the potential of producer-cell engineering as a useful strategy for improving the utility of eVLPs and related delivery methods.},
}
@article {pmid41525452,
year = {2026},
author = {Traxler, EA and Hotan, Q and Shao, Y and Komar, CA and Chen, Q and Saari, M and Thrasher, AJ and Yang, S and Qin, K and Wang, M and Peslak, SA and Abdulmalik, O and Giardine, BM and Keller, CA and Hardison, RC and Minn, AJ and Khandros, E and Shi, J and Blobel, GA},
title = {An optimized CRISPR-Cas12a genome-wide screen reveals PTPA phosphatase pathway in fetal hemoglobin silencing.},
journal = {Blood},
volume = {147},
number = {17},
pages = {1999-2010},
doi = {10.1182/blood.2025030137},
pmid = {41525452},
issn = {1528-0020},
mesh = {Humans ; *Fetal Hemoglobin/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Silencing ; Repressor Proteins/genetics ; Erythroid Cells/metabolism/cytology ; gamma-Globins/genetics ; Carrier Proteins/genetics/metabolism ; Nuclear Proteins/genetics/metabolism ; Protein Phosphatase 2/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Reactivating the fetal globin genes HBG1 and HBG2 in adult erythroid cells represents a validated therapeutic approach for hemoglobinopathies. Central mediators of the fetal-to-adult hemoglobin transition include the direct transcriptional HBG1/2 repressors BCL11A, LRF, and NFIA/X. Limited-scale screens have expanded the regulatory circuity surrounding fetal globin silencing, but systematic genome-wide dissection of such pathways is lacking. We used a 2-tiered genetic screening strategy, a novel CRISPR-Cas12a-based screening platform followed by a domain-focused CRISPR-Cas9 screen, to interrogate all known human protein-coding genes for their impact on HBG1/2 regulation and erythroid cellular fitness, generating a comprehensive resource for the field. Among the top new hits was protein phosphatase 2A (PP2A) phosphatase activator (PTPA), an activator of the serine-threonine phosphatase PP2A whose loss elevates HBG1/2 levels while preserving erythroid differentiation. Phenotypic rescue experiments revealed that PTPA silences HBG1/2 expression primarily by regulating BCL11A expression. To our knowledge, this study represents the most comprehensive CRISPR dissection of HBG regulation to date, highlighting the power of Cas12a-based genome-scale screening for uncovering disease-relevant pathways.},
}
@article {pmid41724384,
year = {2026},
author = {Burnham, CM and Kurilung, A and Wanchai, V and Regenberg, B and Delgado-Calle, J and Basnakian, AG and Nookaew, I},
title = {An enhancement of extrachromosomal circular DNA enrichment and amplification to address the extremely low overlap between replicates.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {4},
pages = {111302},
pmid = {41724384},
issn = {1083-351X},
mesh = {*DNA, Circular/genetics ; Humans ; Animals ; Mice ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; },
abstract = {Extrachromosomal circular DNA (eccDNA) of chromosomal origin is present in all eukaryotic organisms and tissues that have been tested. Populations of eccDNA exhibit immense diversity and a characteristically low degree of overlap between samples, suggesting low inheritance of eccDNA between cells or a deficiency in the methods by which eccDNA is detected. This study revisits the Circle-Seq approach for enrichment of eccDNA to address these limitations, hypothesizing that experimental procedures significantly contribute to the observed low eccDNA overlap. We optimized the protocol by reducing the time needed to complete the procedure. Linear DNA is digested by increasing Exonuclease V activity. We employed CRISPR-Cas9 for mitochondrial linearization, which proved superior to using restriction enzymes. A key finding is the critical role of random hexamer primer concentration and genomic DNA input in rolling circle amplification (RCA) for generating high-quality long concatemeric tandem copy amplicons from eccDNA, essential for confident de novo eccDNA construction from long-read sequencing data. Lower primer concentrations substantially increased the percentage of concatemer-derived eccDNA and improved the overlap of identified eccDNAs in technical replicates. Applying this revised approach to human myeloma and breast cancer cell lines, as well as xenograft models, demonstrated >50% overlap in detected eccDNA, a substantial improvement over the <1% overlap observed in previous studies. Additionally, the oncogenic signature of eccDNAs can be identified across all replicates. These findings provide guidelines for developing standardized procedures for eccDNA profiling, advancing our understanding of eccDNA biology, and its potential clinical applications.},
}
@article {pmid41807709,
year = {2026},
author = {Jansson-Fritzberg, L and Chica, B and Latrick, C and Olland, A and Dementiev, A and White, A and Kutter, S and Lemercier, JN and Wolk, S},
title = {Mechanistic basis for improved activity of Engineered AsCas12a.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41807709},
issn = {2399-3642},
mesh = {*CRISPR-Associated Proteins/genetics/metabolism/chemistry ; *Protein Engineering/methods ; Gene Editing/methods ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/genetics/metabolism/chemistry ; *Bacterial Proteins/genetics/metabolism/chemistry ; DNA/metabolism ; DNA Cleavage ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {CRISPR-associated proteins (Cas) are central to gene editing, forming nuclease complexes with guide RNA to enable precise genome modification. Among numerous Cas variants, Cas9 and Cas12a are the most extensively studied. While much is known about the genomic substrates for these enzymes, less is known about the determinants of the DNA cleavage activity. Wild-type Cas12a exhibits higher intrinsic specificity than Cas9, minimizing off-target activity, but lower overall potency. Recent protein engineering has sought to improve both parameters. Here, we shed light on the structural and mechanistic basis by which an engineered AsCas12a variant achieves high potency while retaining its hallmark specificity. We show that reduced protein-DNA interactions facilitate more rapid R-loop formation, thereby enhancing cleavage activity. These results provide mechanistic insight into Cas12a function and highlight strategies for designing genome-editing nucleases with optimal balance between efficiency and specificity.},
}
@article {pmid41933721,
year = {2026},
author = {Zhang, M and Guan, Z and Guo, Y},
title = {NAA25 as a regulator of insulin signaling: Integration of FOXO1 imaging CRISPRi screen and Mendelian randomization.},
journal = {Life sciences},
volume = {395},
number = {},
pages = {124363},
doi = {10.1016/j.lfs.2026.124363},
pmid = {41933721},
issn = {1879-0631},
mesh = {*Forkhead Box Protein O1/metabolism/genetics ; Humans ; Signal Transduction ; *Insulin/metabolism ; *Insulin Resistance/genetics ; Mendelian Randomization Analysis/methods ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Quantitative Trait Loci ; Mitochondria/metabolism ; },
abstract = {BACKGROUND: Insulin signaling dysfunction underlies multiple metabolic diseases, yet systematic identification of its molecular regulators remains challenging. Elucidating the mechanisms underlying insulin resistance (IR) may facilitate the discovery of potential therapeutic targets for metabolic disorders.<br><br>METHODS: CRISPR interference (CRISPRi) screening coupled with image-enabled cell sorting (ICS) was employed to identify mitochondrial function-related genes regulating insulin signaling, using Forkhead box protein O1 (FOXO1) subcellular localization as a phenotypic readout. Candidate genes were then evaluated by Mendelian randomization (MR) integrating expression quantitative trait loci (eQTL) data and IR genome-wide association study (GWAS) data to assess genetically inferred associations with IR. The role of NAA25 in insulin signaling was characterized through expression profiling in IR tissues and correlation analysis across insulin-sensitive (IS) tissues. The functional role of NAA25 was directly assessed by siRNA-mediated knockdown.<br><br>RESULTS: A total of 117 candidate regulators were identified from 12,300 single guide RNAs (sgRNAs) targeting mitochondrial function-related genes. MR analysis prioritized NAA25 as a high-confidence candidate with a significant negative genetic association with IR (OR&#xa0;=&#xa0;0.972, P&#xa0;=&#xa0;0.009). Expression analysis revealed marked downregulation of NAA25 in adipose tissue from BMI-matched IR individuals. Correlation analysis demonstrated a strong association between NAA25 and FOXO1 (R&#xa0;=&#xa0;0.91, P&#xa0;<&#xa0;2.2e-308) across IS tissues. Furthermore, siRNA-mediated NAA25 knockdown significantly attenuated insulin-stimulated phosphorylation of AKT and FOXO1, providing direct functional evidence for its regulatory role in insulin signaling.<br><br>CONCLUSIONS: This study identifies NAA25 as a key regulator of FOXO1-mediated insulin signaling and establishes an efficient paradigm for translating functional genomics discoveries into promising validated therapeutic targets for metabolic diseases.},
}
@article {pmid42010707,
year = {2026},
author = {Milenkovic, A and Weber, BHF},
title = {Allele-specific suppression of pathogenic bestrophin-1 transcripts by CRISPR/Cas9-mediated genome editing.},
journal = {Genome medicine},
volume = {18},
number = {1},
pages = {},
pmid = {42010707},
issn = {1756-994X},
mesh = {Humans ; *Bestrophins/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Alleles ; Induced Pluripotent Stem Cells/metabolism ; Retinal Pigment Epithelium/metabolism/cytology ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mutation ; *Corneal Dystrophies, Hereditary/genetics/therapy ; },
abstract = {BACKGROUND: Treating autosomal dominant gene mutations remains challenging, particularly when mutations convey a gain-of-function or a dominant-negative effect, as standard gene supplementation strategies often fail to counteract the pathogenic allele.<br><br>METHODS: In this study, we employed human induced pluripotent stem cell-derived retinal pigment epithelium (hiPSC-RPE) to investigate allele-specific CRISPR/Cas9 genome editing as a potential treatment for Best disease (BD), an autosomal dominant macular dystrophy caused by over 250 distinct mutations in the bestrophin-1 (BEST1) gene. We designed and evaluated single guide RNAs (sgRNA) targeting three known BEST1 mutations (p.(R218C), p.(A243V), and p.(I295del)), assessing their impact on BD-associated hiPSC-RPE phenotypes and BEST1 channel function. Computationally predicted sgRNAs were rigorously tested for on-target efficiency, allele specificity and genome-wide off-target activities.<br><br>RESULTS: We found that shortening sgRNA length improved specificity in some cases, while introducing an additional mismatch generally compromised editing efficiency. Notably, only one of the three mutations yielded an sgRNA with both high cleavage efficiency and undetectable off-target effects in hiPSC-RPE cells. We then explored the consequences of allele-specific editing on BEST1 expression and function in clonal BD hiPSC-RPE lines. Eliminating the mutant BEST1 transcript led to enhanced BEST1 localization, improved protein stability and restoration of anion transport function.<br><br>CONCLUSIONS: Taken together, our findings support allele-specific gene editing as a viable therapeutic strategy for selected BEST1 mutations, while underscoring the necessity for rigorous testing of computationally designed sgRNAs, given their mutation- and context-dependent variability.},
}
@article {pmid42022672,
year = {2026},
author = {Edward, M and Owoicho, AW},
title = {Antimicrobial Resistance in Cancer Care: Challenge and Path Forward.},
journal = {Health science reports},
volume = {9},
number = {3},
pages = {e71976},
pmid = {42022672},
issn = {2398-8835},
abstract = {INTRODUCTION: Antimicrobial resistance (AMR) is a global health crisis that poses a devastating and specific threat to immunocompromised cancer patients, who are heavily reliant on antibiotics to navigate high-risk treatments. The rapid emergence of multidrug-resistant (MDR) pathogens compromises the efficacy of prophylaxis and empirical therapy, leading to increased morbidity, mortality, and economic burden. This article aims to critically analyze the unique challenges of AMR in oncology and evaluate the translational readiness of innovative therapeutic modalities as a sustainable path forward.<br><br>METHODS: We synthesize current literature to articulate the clinical and economic burden of AMR specifically within the oncology setting. The article goes beyond conventional antibiotic stewardship discussions to critically appraise non-traditional strategies, including the clinical and regulatory barriers facing bacteriophage therapy, antimicrobial peptides (AMPs), and CRISPR-Cas systems.<br><br>RESULTS: AMR threatens the safe delivery of systemic cancer therapies, as resistant infections necessitate dose modifications and treatment delays. Traditional antibiotic development is insufficient. The path forward requires a paradigm shift: coupling robust stewardship and rapid diagnostics with a critical investment in innovative therapies. While promising, these novel modalities are not "silver bullets" and face significant hurdles in standardization, manufacturing, and regulatory approval that must be addressed for clinical integration into cancer care.<br><br>CONCLUSION: The fight against AMR is inseparable from the fight against cancer. To safeguard the future of oncology, a concerted effort is required to advance non-traditional anti-infectives through the translational pipeline, while ensuring immediate, tailored infection management for this vulnerable population.},
}
@article {pmid42023266,
year = {2026},
author = {Merwaiss, F and García, A and Rogo, U and Querol-Martí, I and García-Sogo, B and de Paola, C and Rodriguez-Rodriguez, M and Pineda, B and Moreno, V and Vazquez-Vilar, M and Orzáez, D and Daròs, JA},
title = {Virus induced gene editing using potyviral vectors in Cas12a expressing plants.},
journal = {Horticulture research},
volume = {13},
number = {4},
pages = {uhag017},
pmid = {42023266},
issn = {2662-6810},
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas systems are revolutionizing precision genome editing and gene expression control in crop plants. While effective CRISPR-Cas applications traditionally rely on labor-intensive stable genetic transformation to deliver Cas nucleases and guide RNAs into plant cells, plant viruses have emerged as a faster and efficient alternative, a strategy known as virus-induced gene editing (VIGE). Cas12a, Class 2 Type V CRISPR nucleases, are an alternative to broadly used Cas9 for plant genome engineering. Both kind of nucleases offer precise editing, but some Cas12a unique features make them particularly well suited for VIGE. In this study, we first used a tobacco rattle virus vector to compare editing efficiency of various target sequences and CRISPR RNA (crRNA) architectures in Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a)-expressing Nicotiana benthamiana plants, evaluating results in infected tissues and seeds. Next, we developed a tobacco etch virus (genus Potyvirus)-derived vector efficiently delivering crRNAs throughout the plant. This approach enabled generation of plants with all four edited alleles in the allotetraploid N. benthamiana through in vitro regeneration from infected leaves, and to produce edited non-infected progeny, although at a very low frequency. We then demonstrated the successful application of the potyviral vector for VIGE in agronomically important crops, such as tomato or cultivated tobacco. Finally, we replicated this design using two other potyviral vectors, turnip mosaic virus, and lettuce mosaic virus. Given the conserved biological properties among potyviruses, we believe these findings are broadly applicable to the largest genus of plant RNA viruses, significantly expanding the host range of the VIGE technology.},
}
@article {pmid42024193,
year = {2026},
author = {Mahmoud, LM and Killiny, N},
title = {Crop biofortification for global food security: advances in genetic engineering and biotechnological approaches.},
journal = {Plant molecular biology},
volume = {116},
number = {3},
pages = {},
pmid = {42024193},
issn = {1573-5028},
}
@article {pmid42026322,
year = {2026},
author = {Li, Y and Zhao, W and Peng, X and Liang, Y and Xue, R and Liu, J and Xu, Z and Cao, D and Liu, B},
title = {TaGα knockout in wheat causes early heading and short organ length, with dose-dependent effects through various pathways.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {5},
pages = {},
pmid = {42026322},
issn = {1432-2242},
support = {2025-ZJ-947M//Qinghai Provincial Department of Science and Technology/ ; },
mesh = {*Triticum/genetics/growth &amp; development ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Plant Leaves/growth &amp; development/genetics ; Phenotype ; Gene Knockout Techniques ; Mutation ; Glutathione Transferase/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {TaGα regulates wheat development in a dose-dependent manner: a single mutant of TaGα solely accelerates heading, while a double mutant not only accelerates heading but also shortens organ length. The Gα subunit plays a crucial role in plant organ development and heading time; however, the exact functions in wheat are unknown. In the present study, knockout mutations in functional TaGα-7A and TaGα-7D were generated in the spring wheat cultivar 'Fielder' using the CRISPR/Cas9 system. Both single and double mutants exhibited early heading time. Reduced plant height, leaf length, and grain length were observed exclusively in the double mutant Gα[Δaadd], whereas leaf and grain width were unaffected. Histological sections revealed that cell length did not vary between the leaf and stem among 'Fielder,' Gα[Δaa], Gα[Δdd], and Gα[Δaadd], which indicated that cell number caused the leaf and stem length differences. Transcriptome analysis revealed dysregulated expression of kinesin and tubulin genes in double-mutant leaves, whereas altered oxidoreductase activity and differential expression of flowering-related genes were detected in the leaves of both single and double mutants. Immunoprecipitation coupled with mass spectrometry revealed that glutathione S-transferase (GST) physically interacted with TaGα directly, which was confirmed by luciferase complementation imaging and yeast two-hybrid assay. Furthermore, GST expression and enzyme activity were suppressed in both single and double mutants, resulting in elevated glutathione (GSH) content. The peak GSH content in wheat leaves during development corresponded to the heading time, which implied that GSH may have participated in regulation of heading time. According to the results of the present study, TaGα exerted regulatory effects on plant height, leaf length, grain length, and heading time in a dose-dependent manner via various pathways.},
}
@article {pmid40912997,
year = {2025},
author = {Xing, M and Su, H and Xiao, Z and Wang, Y and Ji, J and Yang, L and Xu, Y and Lu, Y and Yan, J and Fang, Z and Zhang, Y and Liu, L and Zeng, A and Lv, H},
title = {A one-bp deletion in BoDW1 encoding microtubule-associated kinase causes dwarfing in Brassica oleracea.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt E},
pages = {110429},
doi = {10.1016/j.plaphy.2025.110429},
pmid = {40912997},
issn = {1873-2690},
mesh = {*Plant Proteins/genetics/metabolism ; *Brassica/enzymology/genetics/growth &amp; development ; Gene Expression Regulation, Plant/physiology ; Sequence Deletion ; *Microtubule-Associated Proteins/genetics/metabolism ; Phenotype ; CRISPR-Cas Systems ; *Phosphotransferases/genetics/metabolism ; },
abstract = {Cabbage (Brassica oleracea var. capitata) is an important vegetable crop that is widely cultivated throughout the world. Plant height is a key agronomic trait in cabbage, influencing architecture and yield, and is mainly determined by cell division and stem expansion. Previously, we identified a natural cabbage dwarf mutant, 99-198dw, which displays compact plant architecture, shortened internodes, and small leaves with reduced cell numbers. However, the molecular mechanism underlying this trait remains unknown. In this study, we identified the causal gene Bodw1 using a map-based cloning approach. A 1-bp deletion in the eighth exon of BoDW1 resulted in the mutant phenotype. Knockdown and knockout of BoDW1 in wild-type cabbage lines via RNAi and CRISPR/Cas9 resulted in abnormal phenotypes observed in 99-198dw. BoDW1, homologous to the Arabidopsis gene RUNKEL, encodes a microtubule-associated kinase-like protein localized to microtubules and is mainly expressed in proliferating young tissues. Co-IP-MS, yeast two-hybrid, and BiFC showed that BoDW1 interacts directly with BoCDC48C, a key factor in membrane fusion at the cell division plane. Transcriptome analysis further identified differentially expressed genes associated with membrane fusion and cell plate formation. Our results suggest that BoDW1 probably regulates cabbage development by participating in cell division. Together, our study provides new insights into the molecular control of plant height.},
}
@article {pmid41813774,
year = {2026},
author = {Guo, J and Shi, S and Xie, S and Jing, L and Wang, C and Tao, D},
title = {An advanced rapid-visual CRISPR assay for detecting porcine reproductive and respiratory syndrome virus.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41813774},
issn = {2045-2322},
support = {(2022-021)//the SAAS Program for Excellent Research Team/ ; No.2023BBB170//the Key Research and Development Program of Hubei Province, China/ ; },
mesh = {*Porcine respiratory and reproductive syndrome virus/genetics/isolation &amp; purification ; Animals ; Swine ; *CRISPR-Cas Systems ; *Porcine Reproductive and Respiratory Syndrome/diagnosis/virology ; RNA, Viral/genetics ; Sensitivity and Specificity ; },
abstract = {The porcine reproductive and respiratory syndrome virus (PRRSV) remains a significant threat to the global swine industry, underscoring the urgent need for innovative diagnostic methods to detect and manage outbreaks effectively. We developed a novel CRISPR-based fluorescence assay for the highly sensitive detection of PRRSV-2. By combining reverse transcription-recombinase polymerase amplification (RT-RPA) with multiple-crRNA CRISPR/Cas13a system and single-stranded RNA-fluorescently quenched reporters (RQ-5U), our assay achieved a significant 28-fold increase in sensitivity compared to existed CRISPR/Cas13a-based PRRSV-2 detection methods. This multiple crRNA strategy allows detecting as low as 6 copies/µL of PRRSV-2 RNA, significantly improving the detection limit. Moreover, our method's accuracy in detecting simulated PRRSV-2 clinical samples matches that of quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings demonstrate that this visual, sensitive, and specific nucleic acid detection method holds great promise for enhancing the diagnosis and management of PRRS in the swine industry.},
}
@article {pmid41852146,
year = {2026},
author = {Mieritz, IK and Laustsen, C and Axelgaard, E and Dorset, SR and Bak, RO and Bertelsen, LB},
title = {Glycolytic alterations as biomarkers in polycystic kidney disease: A study using a PKD1 knockout model in NRK-52E rat kidney epithelial cells.},
journal = {Physiological reports},
volume = {14},
number = {6},
pages = {e70816},
pmid = {41852146},
issn = {2051-817X},
support = {R272-2027-4023//Lundbeckfonden (Lundbeck Foundation)/ ; R238-2016-534 3349//Lundbeckfonden (Lundbeck Foundation)/ ; 0077911//Novo Nordisk Fonden (NNF)/ ; R279-A16251//The Danish Cancer Society/ ; 500043//Karen Elise Jensens Fond (Karen Elise Jensen Foundation)/ ; 8056-00010B//Innovation Fund Denmark/ ; },
mesh = {Animals ; Rats ; *Glycolysis ; *Epithelial Cells/metabolism ; *Polycystic Kidney Diseases/metabolism/genetics ; Cell Line ; Biomarkers/metabolism ; Monocarboxylic Acid Transporters/metabolism/genetics ; *TRPP Cation Channels/genetics ; *Kidney/metabolism ; Pyruvic Acid/metabolism ; Gene Knockout Techniques ; L-Lactate Dehydrogenase/metabolism/genetics ; },
abstract = {Polycystic kidney disease (PKD) is a genetic disorder characterized by the formation of fluid-filled cysts in the kidneys, often resulting in progressive renal impairment. Mutations in the PKD1 gene represent the predominant genetic cause of autosomal dominant PKD. Here, we investigated how PKD1 knockout affects glycolytic metabolism in NRK-52E kidney epithelial cells using dynamic nuclear polarization (DNP)-enhanced magnetic resonance spectroscopy (MRS) with hyperpolarized [1-[13]C]pyruvate. PKD1 knockout NRK-52E kidney epithelial cells showed a significantly elevated pyruvate-to-lactate conversion as measured by hyperpolarized [1-[13]C]pyruvate (HP-MRS) and significantly increased lactate levels in culture medium, accompanied by upregulated lactate dehydrogenase (LDH) gene expression and enzymatic activity. Monocarboxylate transporter (MCT) expression was selectively altered (significant downregulation of MCT2 and MCT3; MCT1 not significantly changed). Pyruvate dehydrogenase (PDH) activity and transcript levels did not differ between groups. These results demonstrate glycolytic reprogramming associated with PKD1 deficiency and support hyperpolarized pyruvate MRS as a sensitive metabolic biomarker for detecting such alterations in real-time. These findings identify glycolytic remodeling as a robust metabolic consequence of PKD1 loss and demonstrate that HP-[1-[13]C]pyruvate MRS provides a flux-level biomarker suitable for real-time metabolic characterization.},
}
@article {pmid41885416,
year = {2026},
author = {Ellis, AL and Stauss, M and Barros Tiburcio, P and Emmen, IE and Edlefsen, PT and Kosmider, E and Barlow, S and Goss, M and Temte, JL and Stachler, E and McMahon, K and Sabeti, P and O'Connor, DH and O'Connor, SL},
title = {Adaptation of the multiplexed CRISPR-Cas13 CARMEN RVP assay for longitudinal detection of respiratory pathogens from air samples.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {4},
pages = {e0211725},
doi = {10.1128/aem.02117-25},
pmid = {41885416},
issn = {1098-5336},
support = {5R01AI170737/NH/NIH HHS/United States ; 5U01CK000542-02-00//Centers for Disease Control and Prevention Foundation/ ; 6U01CK000630-01-01//Centers for Disease Control and Prevention Foundation/ ; },
mesh = {Humans ; *SARS-CoV-2/isolation &amp; purification/genetics ; *COVID-19/virology/diagnosis ; *Air Microbiology ; *CRISPR-Cas Systems ; Wisconsin ; *Influenza A virus/isolation &amp; purification/genetics ; Influenza, Human/virology/diagnosis ; },
abstract = {UNLABELLED: Air sampling is a non-invasive alternative to individual testing for respiratory pathogens. Alternative methods to the "gold standard" quantitative reverse transcription-PCR (qRT-PCR) are required to enable higher throughput, lower cost, and more multiplexed detection of pathogens. The multiplexed CRISPR-Cas13 CARMEN Respiratory Viral Panel (RVP) was described previously for high-throughput detection of nine respiratory pathogens from nasal swab samples. Here, we modified and optimized the CARMEN RVP assay to overcome the unique challenges of air samples, including low biomass and environmental inhibitors. We monitored for SARS-CoV-2 and influenza A (Flu A) via qRT-PCR in air samples from 15 schools within Dane County, Wisconsin (USA), during the 2023-2024 school year. SARS-CoV-2 was detectable throughout the entire sampling period, while Flu A detection was seasonal from November 2023 to March 2024. We then analyzed a subset of samples from seven schools using an optimized CARMEN RVP assay for air surveillance (RVP_air) and compared the results to qRT-PCR. The RVP_air assay detected several additional pathogens beyond our primary targets. The frequencies and patterns of SARS-CoV-2 positivity, but not Flu A positivity, were similar between qRT-PCR and RVP_air across the 2023-2024 sampling period. We developed a secondary panel (RVP_air_flu) to better detect both H1N1 and H3N2 subtypes. Finally, we compared air sample results to clinical nasal swabs collected from the same school district. For several pathogens (SARS-CoV-2, HCoV-OC43, Flu A), positive air detections coincided with positive nasal swabs. These findings demonstrate that the RVP_air assay can effectively detect airborne pathogens from infected individuals within indoor spaces.<br><br>IMPORTANCE: Air sampling offers a cost-effective alternative to individual testing for respiratory pathogens within congregate settings. Optimization and use of multi-pathogen assays are especially valuable for capturing the breadth of pathogens that may be present simultaneously in the same space. The modified CARMEN RVP assays (RVP_air and RVP_air_flu) detected SARS-CoV-2 and Flu A during similar sampling time periods compared to qRT-PCR, while also detecting several additional respiratory pathogens (seasonal coronaviruses, respiratory syncytial virus). Importantly, pathogens detected from air samples corresponded to those detected from nasal swabs collected from individuals in the same spaces. Together, these findings highlight the utility of the RVP_air and RVP_air_flu assays as alternatives to qRT-PCR for environmental surveillance, with applications extending to other congregate spaces (hospitals, long-term care facilities) and high-risk settings, better informing communities and improving public health.},
}
@article {pmid41923628,
year = {2026},
author = {Ou, C and Huang, X and Huang, D and Luo, R and Chen, R and Li, X and Wu, X and Wu, Q and Gong, C},
title = {A harmless-to-harmful switchable and spatiotemporally activated nano-CRISPR hierarchically amplifies ferroptosis in melanoma.},
journal = {Cell reports. Medicine},
volume = {7},
number = {4},
pages = {102718},
doi = {10.1016/j.xcrm.2026.102718},
pmid = {41923628},
issn = {2666-3791},
mesh = {*Ferroptosis/genetics ; Humans ; *Melanoma/pathology/genetics/therapy/metabolism ; Animals ; Reactive Oxygen Species/metabolism ; Cell Line, Tumor ; Mice ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Lipid Peroxidation ; Lipid Peroxides/metabolism ; Iron/metabolism ; },
abstract = {Ferroptosis therapy faces challenges due to low lipid peroxide (LPO) levels. Herein, we develop a harmless-to-harmful switchable and spatiotemporally activated nano-CRISPR system (termed ARCHER) that sequentially amplifies ferroptosis sensitivity, iron ion levels, and reactive oxygen species (ROS) to amplify ferroptosis therapy efficiency. ARCHER targets cancer cells and releases CRISPR-Cas9 and Ce6Fe(III)Cl in response to hyaluronidase and tumor acidity. CRISPR-Cas9 sustains effective suppression of LPO-reducing protein GPX4. By persistently enhancing LPO accumulation through GPX4 downregulation, ARCHER primes cancer cells for ferroptosis and sensitizes them to subsequent interventions. Upon laser irradiation under acidic conditions, Ce6Fe(III)Cl undergoes spatiotemporal activation, transforming from inert form into cytotoxic Fe(III) ions and Ce6-generated ROS. Liberated Fe(III) and ROS synergistically amplify lipid peroxidation, driving LPO accumulation to trigger ferroptosis storm in sensitized cancer cells. In vivo studies demonstrated that ARCHER achieves 60% (3/5) tumor ablation with minimal off-target effects, validating its high therapeutic efficacy in ferroptosis-driven cancer treatment.},
}
@article {pmid42016307,
year = {2026},
author = {My, B and Lia, A and Rizzo, L and Maiorano, G and Galeone, A and Palamà, IE and Gigli, G},
title = {CRISPR-Cas9 engineering of CAR-T cells: Can non-viral nanoparticles unlock safer and scalable genome editing?.},
journal = {iScience},
volume = {29},
number = {5},
pages = {115422},
pmid = {42016307},
issn = {2589-0042},
abstract = {CAR-T cell therapy has revolutionized the treatment of hematologic malignancies. Still, durable activity in tumors remains limited by antigen heterogeneity and escape, immunosuppressive tumor microenvironment, and restricted persistence. Genome engineering with CRISPR-Cas systems offers a powerful route to reprogram CAR-T cells; however, translation increasingly depends on how editing payloads are delivered. Viral vectors remain a benchmark for efficient gene transfer, cargo constraints, insertional risk, immunogenicity, and manufacturing complexity motivate the development of safer, more scalable non-viral platforms. In this review, we provide an overview of CAR designs, clinical use, and current ex vivo manufacturing workflow; compare viral and non-viral delivery routes while distinguishing established ex vivo editing from emerging in vivo T cell programming; and outline genome-engineering strategies organized by therapeutic goals. We highlight feasibility trade-offs and discuss how nanoparticles could enable transient, non-viral delivery of genome editors, while noting that robust T cell targeting and standardized potency/safety assays remain key bottlenecks.},
}
@article {pmid42018092,
year = {2026},
author = {Zhao, R and Wang, C and Li, J and Liao, Y and Huang, C and Hu, T and Zhang, H and Zhang, W},
title = {DNA and RNA editing for the therapy of human diseases: current status, challenges, and future prospects.},
journal = {Molecular biomedicine},
volume = {7},
number = {1},
pages = {},
pmid = {42018092},
issn = {2662-8651},
support = {82302421//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Gene Editing/methods ; *RNA Editing ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; *DNA/genetics ; },
abstract = {The rapid development of DNA- and RNA-editing tools (collectively referred to as gene editing technologies) has caused a paradigm shift in the treatment of human diseases from symptomatic treatment to precision-based medicine. Both DNA-based and RNA-based editing systems, including Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived technologies and newly developed RNA editing tools, have pushed technological frontiers in terms of editing precision, hierarchical control, and reversibility; they have accumulated a growing body of preclinical and clinical evidence across diverse diseases ranging from inherited disorders to cancer, infectious diseases, and neurodegenerative diseases (ND). This review systematically summarizes the core principles and representative advances of DNA-based genome editing and RNA-based transcriptome editing technologies, comprehensively compares the two categories of technical strategies in terms of therapeutic potential, durability of effects, and risk profiles, and further explores the key challenges for achieving long-term safe and efficient in vivo applications, covering core bottlenecks such as delivery efficiency, tissue specificity, genotoxicity, and immunogenicity. Safety assessment has broadened to include tracking genotoxicity and genomic structural variations, whereas delivery systems and tissue specificity are determinant factors for in vivo therapeutic applications. Through the employment of both permanent and reversible editing strategies with high cargo-writing capacity and low integration risk, combined with programmable delivery systems, the therapeutic potential of hard-to-transfect tissues and complex diseases is anticipated to be broadened, opening new paths for clinical translation.},
}
@article {pmid42018449,
year = {2026},
author = {Cheung, CY and Samuels, I and Klaus, HR and Cook, GM and McNeil, MB},
title = {Metabolic perturbation reduces antibiotic tolerance in Mycobacterium tuberculosis.},
journal = {Microbiology (Reading, England)},
volume = {172},
number = {4},
pages = {},
doi = {10.1099/mic.0.001697},
pmid = {42018449},
issn = {1465-2080},
mesh = {*Mycobacterium tuberculosis/drug effects/metabolism/genetics ; Humans ; Macrophages/microbiology ; THP-1 Cells ; *Antitubercular Agents/pharmacology ; Microbial Sensitivity Tests ; *Drug Tolerance ; Gene Expression Regulation, Bacterial ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Drug Resistance, Bacterial ; Iron/metabolism ; Gene Knockdown Techniques ; },
abstract = {Mycobacterium tuberculosis is tolerant to many antibiotics, leading to impaired antibiotic killing. Using CRISPR interference (CRISPRi) transcriptional knockdowns, we generated a panel of metabolically compromised strains to identify tolerance pathways for pursuing in therapeutic development. Disrupting the regulation of intracellular iron storage, amino acid biosynthesis and redox defence mechanisms potentiated the lethality of multiple drugs and translated to infected THP-1 macrophages. This work reinforces the role of metabolism as a major contributor to drug tolerance in M. tuberculosis.},
}
@article {pmid42018671,
year = {2026},
author = {Umbach, A and Santini, A and Bulcaen, M and Guidone, D and Maule, G and Arosio, D and Carrozzo, I and Ciciani, M and Brugnara, E and Ramalho, A and Vermeulen, F and Galietta, LJV and Carlon, MS and Cereseto, A},
title = {Functional correction of the untreatable CFTR 1717-1G&gt;A mutation through mRNA- and sgRNA-optimized base editing.},
journal = {Science translational medicine},
volume = {18},
number = {846},
pages = {eadw8886},
doi = {10.1126/scitranslmed.adw8886},
pmid = {42018671},
issn = {1946-6242},
mesh = {*Cystic Fibrosis Transmembrane Conductance Regulator/genetics/metabolism ; Humans ; *Gene Editing/methods ; *RNA, Messenger/genetics/metabolism ; HEK293 Cells ; *Mutation/genetics ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cystic Fibrosis/genetics ; CRISPR-Cas Systems/genetics ; Organoids/metabolism ; Base Sequence ; },
abstract = {The 1717-1G>A is a prevalent splicing mutation causing cystic fibrosis (CF) for which no pharmacological treatments have been approved. This mutation disrupts a canonical 3' AG splice acceptor site in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, leading to severe RNA missplicing, which prevents the correct synthesis of the encoded protein. In this study, we developed an adenine base editing (ABE) strategy to efficiently correct the 1717-1G>A mutation. By using the ABE9 base editor with the protospacer adjacent motif-relaxed Streptococcus pyogenes clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) variant SpRY, we obtained up to 30% editing with limited bystander effects in a human embryonic kidney (HEK) 293-based cellular model. Through systematic optimizations of the ABE system, delivered by electroporation of base editor messenger RNA (mRNA) and single guide RNA (sgRNA), we demonstrated genetic repair of the 1717-1G>A mutation in airway epithelial cells and intestinal organoids derived from people with CF. Functional analysis was performed by measuring short-circuit current in air-liquid interface (ALI) culture and by assessing forskolin-induced swelling (FIS) in intestinal organoids, which revealed restoration of CFTR channel activity. These results highlight SpRY-ABE9 as a potential genome editing strategy to permanently correct the CFTR 1717-1G>A mutation and restore CFTR function.},
}
@article {pmid42020899,
year = {2026},
author = {Kumar, S and Zhao, D and Wong, VHY and Bui, BV and Liu, GS},
title = {Evaluation of CRISPR/CasRx-Mediated VEGF mRNA Knockdown in Mouse Retina.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3023},
number = {},
pages = {25-37},
pmid = {42020899},
issn = {1940-6029},
abstract = {Neovascular eye diseases (NEDs) are a group of diseases caused by the abnormal overgrowth of blood vessels in the eye. Normal vasculature is maintained through a dynamic balance of the vascular endothelial growth factor (VEGF). In diseases such as advanced diabetic retinopathy (DR) and neovascular age-related macular degeneration (AMD), an overexpression of VEGF leads to the formation of structurally weak and leaky blood vessels, resulting in vision impairment and, without intervention, legal blindness. In the clinic, NEDs are presently managed through anti-VEGF agents that specifically bind and neutralize VEGF signaling. While effective, this approach requires invasive intravitreal injections monthly and places a heavy burden on patients and healthcare providers. A flexible, long-lasting therapeutic that can reduce or eliminate frequent anti-VEGF treatment will significantly advance NED management.RNA editing with clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) is an emerging strategy to achieve reversible gene editing. The CRISPR-Cas13 system exclusively targets single-stranded RNA and allows gene silencing in a safe manner through RNA knockdown, as the genome is left intact. In addition, the compact CasRx enzyme (930aa) allows RNA silencing to be achieved through the delivery of a single adeno-associated virus (AAV), ideal for gene therapy applications. Herein, we outline methods to target VEGFA mRNA using CRISPR/CasRx in a mammalian cell line and humanized VEGFA transgenic mice.},
}
@article {pmid40958004,
year = {2026},
author = {Pham, DL and Cappabianca, D and Forsberg, MH and Weaver, C and Mueller, KP and Tommasi, A and Vidugiriene, J and Lauer, A and Sylvester, K and Lika, J and Bugel, M and Fan, J and Capitini, CM and Saha, K and Skala, MC},
title = {Label-free metabolic imaging monitors the fitness of chimeric antigen receptor T cells.},
journal = {Nature biomedical engineering},
volume = {10},
number = {4},
pages = {785-802},
pmid = {40958004},
issn = {2157-846X},
support = {P30 CA014520/CA/NCI NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; T32 GM140935/GM/NIGMS NIH HHS/United States ; P41 EB031772/EB/NIBIB NIH HHS/United States ; NSF-EEC 1648035//National Science Foundation (NSF)/ ; R01 CA278051/CA/NCI NIH HHS/United States ; S10 OD025225/OD/NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; R01 CA278051/CA/NCI NIH HHS/United States ; },
mesh = {*Receptors, Chimeric Antigen/metabolism/genetics ; Humans ; Animals ; *T-Lymphocytes/metabolism/cytology/immunology ; Mice ; Immunotherapy, Adoptive/methods ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Optical Imaging/methods ; Tumor Microenvironment ; Gene Editing ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapy for solid tumours is challenging because of the immunosuppressive tumour microenvironment and a complex manufacturing process. Cellular manufacturing protocols directly impact CAR T cell yield, phenotype and metabolism, which correlates with in vivo potency and persistence. Although metabolic fitness is a critical quality attribute, how T cell metabolic requirements vary throughout the manufacturing process remains unexplored. Here we use optical metabolic imaging (OMI), a non-invasive, label-free method to evaluate single-cell metabolism. Using OMI, we identified the impacts of media composition on CAR T cell metabolism, activation strength and kinetics, and phenotype. We demonstrate that OMI parameters can indicate cell cycle stage and optimal gene transfer conditions for both viral transduction and electroporation-based CRISPR/Cas9. In a CRISPR-edited anti-GD2 CAR T cell model, OMI measurements allow accurate prediction of an oxidative metabolic phenotype that yields higher in vivo potency against neuroblastoma. Our data support OMI as a robust, sensitive analytical tool to optimize manufacturing conditions and monitor cell metabolism for increased CAR T cell yield and metabolic fitness.},
}
@article {pmid41638392,
year = {2026},
author = {Zhang, P and Shen, M and Ding, L and He, L and Wu, Y and Yu, S},
title = {Controlled CRISPR/Cas12a activation via DNAzyme-mediated splitting of chimeric substrate for lead detection.},
journal = {Biochimie},
volume = {244},
number = {},
pages = {1-6},
doi = {10.1016/j.biochi.2026.02.001},
pmid = {41638392},
issn = {1638-6183},
mesh = {*DNA, Catalytic/metabolism/chemistry ; *Lead/analysis ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism ; *CRISPR-Associated Proteins/metabolism ; Humans ; *Bacterial Proteins/metabolism ; },
abstract = {Chronic lead exposure poses severe threats to human health, which demands a rapid detection strategy beyond conventional instrumentation-dependent approaches. While CRISPR/Cas12a systems offer promising alternatives through trans-cleavage activity, conventional Pb[2+] biosensors relying on DNAzyme-generated intact activators suffer from high background signals due to interference from uncleaved substrates. To address this limitation, we developed a steric-hindrance-controlled activation strategy by employing a chimeric DNAzyme substrate (Sub) that prevents Cas12a binding until Pb[2+]-dependent cleavage occurs. This DNAzyme-mediated splitting releases two fragments (A1/A2) that rearrange into split activators, triggering the CRISPR/Cas12a trans-cleavage of a quenched reporter (6-FAM/BHQ1). Under the optimal condition, the sensor achieved a linear detection range of 2.5-25 μM (R[2] = 0.998) with 2.18 μM LOD and high selectivity against interferents. Validation in tap water matrices demonstrated 98.6%-102.6% recovery (RSD 3.0%-7.5%), which showed robustness in real samples. This split-activator design paradigm eliminates background from uncleaved substrates without additional pretreatment steps to provide a versatile template for converting metal ions into CRISPR-detectable signals.},
}
@article {pmid41673138,
year = {2026},
author = {Dörr, J and Gregor, L and Lacher, SB and Oner, A and Sun, Y and Piseddu, I and Fertig, L and Spajic, S and Lesch, S and Michaelides, S and Seifert, M and Gottschlich, A and Samson, N and Majed, L and Briukhovetska, D and Simnica, D and Hartmann, V and Gabriel, K and Cohen, S and Boland, GM and Andreu-Sanz, D and Carlini, E and Stock, S and Holtermann, A and Müller, PJ and Strzalkowski, T and Trefny, MP and Endres, S and Jenkins, RW and Böttcher, JP and Kobold, S},
title = {Ablation of prostaglandin E2 signalling through dual receptor knockout in CAR T cells enhances therapeutic efficacy in solid tumours.},
journal = {Nature biomedical engineering},
volume = {10},
number = {4},
pages = {697-710},
pmid = {41673138},
issn = {2157-846X},
support = {101124203//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {*Dinoprostone/metabolism ; Humans ; Animals ; *Receptors, Prostaglandin E, EP4 Subtype/genetics/metabolism/deficiency ; Mice ; *Signal Transduction ; *Receptors, Prostaglandin E, EP2 Subtype/genetics/metabolism/deficiency ; *Receptors, Chimeric Antigen/metabolism/genetics ; Cell Line, Tumor ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/metabolism/immunology ; *Neoplasms/therapy/immunology/metabolism ; Gene Knockout Techniques ; Tumor Microenvironment ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Female ; },
abstract = {The efficacy of chimeric antigen receptor (CAR) T cell therapy in solid cancers is limited by immunosuppression in the tumour microenvironment (TME). Prostaglandin E2 (PGE2) is a key factor locally inhibiting T cell function. We hypothesized that targeted ablation of PGE2 signalling in CAR T cells may enhance their activity in PGE2-rich solid tumours. Here we generate knockout CAR T cells double deficient for the PGE2 receptors EP2 and EP4 (EP2[-/-]EP4[-/-]) by CRISPR-Cas9 engineering. EP2[-/-]EP4[-/-] CAR T cells expanded unabatedly in the presence of PGE2. Further, they effectively controlled syngeneic and human xenograft tumour models in vivo, which was accompanied by intratumoural accumulation and persistence of modified T cells. Improved anti-tumour activity was also observed against patient-derived tumour samples from patients with pancreatic ductal adenocarcinoma (PDAC), colorectal (CRC) and neuroendocrine (NET) cancer. Our data uncovers the detrimental impact of PGE2-mediated suppression on CAR T cell efficacy and highlights EP2 and EP4 targeting as a potential strategy.},
}
@article {pmid41794998,
year = {2026},
author = {Higuchi, R and Tatara, M and Horino, S and Katori, S and Sanbo, M and Hirabayashi, M and Watanabe, M and Konno, A and Hirai, H and Yoshioka, Y and Hirabayashi, T and Kobayashi, T and Kaneko, R and Yagi, T},
title = {Protocadherin γC4 regulates neuronal survival and dendritic self-avoidance.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41794998},
issn = {2399-3642},
support = {22H05498//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24H01237//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 25H02501//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {Animals ; *Cadherins/genetics/metabolism ; *Dendrites/metabolism/physiology ; Mice ; *Neurons/metabolism ; Cell Survival ; Cadherin Related Proteins ; Protocadherins ; CRISPR-Cas Systems ; Disease Models, Animal ; Purkinje Cells/metabolism ; },
abstract = {Animal models are indispensable for linking human genetic findings to disease mechanisms. Mutations in protocadherin gamma C4 (γC4), one of the 22 isoforms encoded by the protocadherin-γ (Pcdh-γ) gene cluster, cause a human neurodevelopmental syndrome with progressive microcephaly, seizures, and intellectual disability. Here, we established a γC4 mutant mouse model that exhibits motor dysfunction, seizures, reduced brain size, and increased embryonic neuronal apoptosis. Using DOMINO (Double Mutation-Induced Open Reading Frame Switch), a two-step CRISPR/Cas9-based genome-editing strategy, we also generated γC4fl-only mice that retain full-length γC4 while truncating the other 21 Pcdh-γ isoforms. Unlike Pcdh-γ cluster-deficient mice, γC4fl-only mice were viable and fertile. Furthermore, we show that the γC4 constant region (γCR) contributes to the regulation of Purkinje cell dendritic architecture and self-avoidance. Together, these findings indicate that γCR-containing γC4 is required for neuronal survival and dendritic patterning, supporting γC4 as a principal functional isoform within the Pcdh-γ gene cluster.},
}
@article {pmid41812962,
year = {2026},
author = {Yogi, D and Shashikala, T and Subramanian, G and Kumar, A and Kaninika, V and Manamohan, M and Jithesh, MN and Jha, GK and Asokan, R and Ashok, K},
title = {First report of CRISPR prime editing in a globally significant non-model organism, the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).},
journal = {Methods (San Diego, Calif.)},
volume = {250},
number = {},
pages = {36-45},
doi = {10.1016/j.ymeth.2026.03.001},
pmid = {41812962},
issn = {1095-9130},
mesh = {Animals ; *Spodoptera/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {The power of CRISPR/Cas9 mediated genome editing has been harnessed in different facets of entomological research, particularly useful in developing genetic pest management strategies. The edits thus obtained are robust and results in a loss-of-function of the target gene. Recently the development of newer editing approach called Prime editing is yet another addition in the insect editing tool-box. In this regard, the prime editing offers a transformative approach to precise genome manipulation by enabling targeted insertions, deletions, and nucleotide substitutions without double-strand break or donor template. While its application has been explored in mammalian system and plants, its deployment through the delivery of ribonucleoprotein complex (RNP) has been demonstrated for the first-time in the globally significant pest, Spodoptera frugiperda. Using a Cas9 (H840A)-reverse transcriptase fusion protein (PE2) and a customized prime editing guide RNA (pegRNA), we targeted exon 3 of the Tryptophan 2,3-dioxygenase (SfTO) gene to introduce a premature stop codon. Recombinant PE2 protein was expressed in E. coli, purified, and validated functionally through RT-PCR. The Ribonucleoprotein complex was microinjected into G0 eggs and subsequent genotyping revealed successful edits, including perfect and imperfect prime edits, as well as unintended mutations. Phenotyping revealed the mutants with altered eye pigmentation, and chromatographic analysis confirmed disruption in ommochrome biosynthesis, validating functional consequences of Prime editing. This study provides a foundational proof-of-concept for Prime editing in insect pests, opening new avenues for functional genomics and designing next-generation pest management strategies.},
}
@article {pmid41825300,
year = {2026},
author = {Low, YC and McKnight, CL and Elliott, DA and Thorburn, DR and Frazier, AE},
title = {Generation of a pluripotent embryonic stem cell TAFAZZIN hESC model (WAe009-A-3H) of Barth syndrome.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103948},
doi = {10.1016/j.scr.2026.103948},
pmid = {41825300},
issn = {1876-7753},
mesh = {Humans ; *Barth Syndrome/pathology/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism ; Female ; *Transcription Factors/genetics/metabolism ; Cell Line ; Acyltransferases/genetics ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Cell Differentiation ; },
abstract = {Barth syndrome is among the most common mitochondrial diseases presenting with cardiomyopathy. We have generated a human embryonic stem cell (hESC) model of Barth syndrome (TAFAZZIN[Δ3] C15) in a female background (H9 hESC) using CRISPR/Cas9 gene editing, with compound heterozygous variants in TAFAZZIN that result in exon 3 skipping in all stable transcripts. This cell line displayed characteristics consistent with pluripotent stem cells, including typical colony morphology, expression of pluripotency markers, trilineage potential, and a normal karyotype. This TAFAZZIN[Δ3] C15 line could be used for investigation of disease mechanisms in mitochondrial cardiomyopathy and preclinical therapeutic screening.},
}
@article {pmid41825380,
year = {2026},
author = {Wang, X and Zhang, X and Guo, Y and Zheng, Q and Hu, B and Liu, R and Cao, J},
title = {CRISPR/Cas12-driven portable paper-based electrochemical aptasensor based on 0D/2D Au@Ti3C2 MXene for AFB1 detection.},
journal = {Food chemistry},
volume = {511},
number = {},
pages = {148810},
doi = {10.1016/j.foodchem.2026.148810},
pmid = {41825380},
issn = {1873-7072},
mesh = {*Aflatoxin B1/analysis ; Food Contamination/analysis ; *Electrochemical Techniques/instrumentation/methods ; Gold/chemistry ; Paper ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/instrumentation/methods ; CRISPR-Cas Systems ; Limit of Detection ; Nitrites ; Transition Elements ; },
abstract = {Aflatoxin B1 (AFB1) is a widely distributed and potent carcinogenic small-molecule toxic contaminant, posing significant risks to food safety. Therefore, a portable, cost-effective, and field-deployable method is needed for sensitive detection of AFB1. This study developed a paper-based electrochemical aptasensor integrating CRISPR/Cas12a technology and 0D/2D Au@Ti3C2 MXene heterostructure for highly sensitive and rapid detection of AFB1. The aptasensor leveraged the Au@Ti3C2 MXene with excellent electron transfer property to amplify the electrochemical signals, while the CRISPR/Cas12a trans-cleavage activity, triggered by AFB1 specific aptamers, enabled high specificity. Utilizing a portable electrochemical workstation, the paper-based electrochemical sensing platform exhibited a linear detection range of 0.5 pg/mL-100 ng/mL with a limit of detection of 0.16 pg/mL and achieved recoveries of 93.0%-106.0% in spiked real samples, demonstrating promise as a rapid and portable tool for on-site analysis of mycotoxins in real samples.},
}
@article {pmid41826696,
year = {2026},
author = {Aird, EJ and Rabl, J and Knuesel, T and Groen, K and Awwad, SW and Korablev, B and Scherpe, L and Al-Herz, W and Hupfer, R and Recher, M and Jackson, SP and Hale, BG and Corn, JE},
title = {An SP110-SP100 axis is a critical regulator of promyelocytic leukaemia body dynamics and mitotic fidelity.},
journal = {Nature cell biology},
volume = {28},
number = {4},
pages = {684-695},
pmid = {41826696},
issn = {1476-4679},
support = {855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; 310030_188858//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 320030_232029//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; ALTF 144-2021//European Molecular Biology Organization (EMBO)/ ; Women's Postdoctoral Career Development Award in Science//Weizmann Institute of Science/ ; Outstanding Postdoctoral Women Fellowship//Council for Higher Education of Israel | Israeli Centers for Research Excellence (I-CORE)/ ; DRCPGM\100005//Cancer Research UK (CRUK)/ ; SEBINT-2024/100003//Cancer Research UK (CRUK)/ ; },
mesh = {Humans ; *Mitosis ; *Autoantigens/metabolism/genetics ; *Promyelocytic Leukemia Protein/metabolism/genetics ; *Antigens, Nuclear/metabolism/genetics ; *Leukemia, Promyelocytic, Acute/genetics/metabolism/pathology ; CRISPR-Cas Systems ; DNA Damage ; },
abstract = {Stimulation of the innate immune system by foreign RNA elicits a potent interferon response and can trigger cell death. The mechanisms by which cells balance a robust response with cell-intrinsic lethality are still being uncovered. Here, using genome-wide CRISPR-Cas9 genetic screens with triphosphorylated RNA stimulation, we discover that promyelocytic leukaemia (PML) nuclear body-localized speckled protein 110 (SP110) is a potent inhibitor of type 1 interferon-driven cell death. Death suppression by SP110 counteracts a toxic activity of SP100, a major constituent of PML bodies. Loss of SP110 leads to mitotic retention of SP100 and PML bodies, which associate with and perturb segregating chromosomes, leading to micronucleus formation, DNA damage and genotoxic cell death. A combination of cryo-electron microscopy, AlphaFold modelling and cellular biochemistry reveals that SP110 dissolves toxic SP100 oligomers via necessary and sufficient direct interactions between their caspase activation and recruitment domains. These data reveal the critical roles of SP100 and SP110 in governing the disassembly of PML bodies during mitosis, as well as the repercussions if this process is misregulated.},
}
@article {pmid41826830,
year = {2026},
author = {Zhang, H and Zhang, P and Bindels, E and Mulugeta, E},
title = {Insights from pooled CRISPRi single-cell screens in K562 cells reveal gene functions, regulatory networks, and highlight opportunities and limitations.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41826830},
issn = {1471-2164},
mesh = {Humans ; K562 Cells ; *Gene Regulatory Networks ; *Single-Cell Analysis/methods ; *CRISPR-Cas Systems ; Transcription Factors/genetics ; Gene Knockdown Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; LIM Domain Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Pooled CRISPR screening combined with single-cell RNA sequencing (scRNA-seq) has emerged as a powerful strategy for dissecting gene function and reconstructing gene regulatory networks (GRNs) in complex biological systems. This approach enables high-throughput, parallel perturbation of multiple genes while providing transcriptome-wide readouts at single-cell resolution, overcoming many limitations of traditional arrayed screens. However, its broader application remains limited by technical challenges, including variable perturbation efficiency and difficulties in accurately identifying perturbed cells.In this study, we adapted and applied a modified CRISPR droplet sequencing (CROP-seq) protocol using CRISPR interference (CRISPRi) in K562 cells to knockdown six transcription factors (TFs): LMO2, TCF3, LDB1, MYB, GATA2, and RUNX1. Our modified approach, which allows direct capture of sgRNAs from the cDNA library without a separate enrichment step, significantly improved sgRNA assignment per cell. We successfully achieved reproducible knockdown of three TFs (MYB, GATA2, and LMO2), captured the impact of these perturbations on the TF target genes, and enabled us to reconstruct their GRNs and identify key regulons and transcriptional targets. These networks revealed both previously established (such as LMO2 GATA2 interaction) and novel regulatory interactions, which we independently validated, providing new insights into hematopoietic transcriptional control. To assess the efficiency of CRISPRi based pooled perturbation, we additionally analyzed publicly available Perturb-seq CRISPRi datasets and found that only ~40-50% of targeted genes led to effective knockdown, underscoring the variability in perturbation efficiency across experiments.Together, our results demonstrate both the potential and the current technical limitations of pooled CRISPRi-based single-cell screens. While this integrated approach holds great promise for high-resolution functional genomics, further optimization and standardized benchmarking are essential to improve its reliability, scalability, and reproducibility.},
}
@article {pmid41834297,
year = {2026},
author = {Zhu, L and Xiong, W and Yang, S and Qi, Q and Liu, X and Zhou, X and Tian, T},
title = {Dynamic Control of RNA Structure and Function through Bioorthogonal Staudinger Chemistry.},
journal = {ACS chemical biology},
volume = {21},
number = {4},
pages = {835-843},
doi = {10.1021/acschembio.6c00067},
pmid = {41834297},
issn = {1554-8937},
mesh = {*RNA/chemistry/metabolism ; Azides/chemistry ; Humans ; CRISPR-Cas Systems ; Nucleic Acid Conformation ; *Imidazoles/chemistry ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/chemistry ; },
abstract = {Here, we report a reversible chemical strategy for regulating RNA function through a Staudinger reaction-mediated postsynthetic modification. We designed a bifunctional azide reagent, 1,3-diazidopropan-2-yl 1H-imidazol-1-carboxylate (DAPIC), which specifically modifies the 2'-hydroxyl of RNA, thereby disrupting RNA structure and function. Treatment with 2-diphenylphosphinoethylamine (DPPEA) reactivates the modified RNA through an efficient Staudinger reduction. This approach enables reversible modulation of RNA folding, hybridization, and protein-binding interactions, and can be applied to guide RNAs in the CRISPR-Cas9 system. DAPIC modification completely abrogates Cas9-mediated DNA cleavage, which is restored in a DPPEA concentration-dependent manner both in vitro and in living cells. Compared with monoazide derivatives, DAPIC exhibits enhanced reactivity and reduced reagent requirements. This Staudinger-based RNA regulation platform establishes a robust and generalizable chemical tool for conditional gene editing and studies of RNA function in complex biological environments.},
}
@article {pmid41851507,
year = {2026},
author = {Zheng, R and Lu, Z and Wei, R and Shin, YC and Du, J and Zhang, Q and Li, J and Wang, X and Wei, Y and Liu, B and Chen, Y and Ding, L and Zhang, H and Chen, H and Huang, J and Ma, L},
title = {Improving the efficiency of high-fidelity Cas9 by enhancing PAM-distal interactions.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {4},
pages = {590-602},
pmid = {41851507},
issn = {1545-9985},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics ; Cryoelectron Microscopy ; Humans ; Mutation ; Models, Molecular ; },
abstract = {Engineering CRISPR enzymes for high fidelity often impairs cleavage activity. Meanwhile, a mechanistic understanding of why high-fidelity mutations reduce Cas9's cleavage activity remains unclear, presenting a challenge in balancing nuclease specificity and efficiency for clinical applications. In this study, we show that extending the spacer region to 21 or 22 nucleotides restores the impaired cleavage activity of SuperFi-Cas9, a high-fidelity Cas9 variant with 7 mutations in the RuvC domain at the protospacer adjacent motif (PAM)-distal region. Cryo-electron microscopy structures and mutational analyses reveal that the negatively charged mutations in a protruding loop of the RuvC domain create repulsive forces that destabilize the nuclease-single guide (sg)RNA-DNA complex. Spacer extension enhances interactions in the PAM-distal region, effectively restoring cleavage activity and balancing editing efficiency with specificity. In addition, we develop a deep learning model, AIdit-SuperFi, to predict optimal sgRNA length for high-fidelity genome editing. Our findings introduce a straightforward strategy to enhance CRISPR complex stability and provide mechanistic insights into the impaired cleavage activity of engineered high-fidelity Cas9, presenting a pathway toward precise and efficient genome editing and clinical translation of CRISPR technologies.},
}
@article {pmid41882346,
year = {2026},
author = {Wang, F and Guo, R and Zhang, S and Cui, Y and Wang, J and Hu, T and Liu, K and Wang, Q and Liu, Y and Nam, KH and Zhao, ZW and Ji, Q and Xu, X and Wang, E and Zhu, Y and Yang, Y and Luo, M and Ma, P and Ma, S and Xu, C and Hu, C},
title = {Structural insight into IscB's RNA-lid-based inactivation mechanism.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {4},
pages = {603-614},
pmid = {41882346},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; Models, Molecular ; *Escherichia coli Proteins/chemistry/metabolism/genetics ; Nucleic Acid Conformation ; Escherichia coli/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/chemistry/genetics ; },
abstract = {IscB, a compact Cas9 ancestor from the obligate mobile element guided activity system, has attracted growing interest as a programmable genome editor because of its small size and therapeutic delivery potential. Despite its promise, structural insights into IscB's regulation remain limited, with only a target-bound R-loop structure previously reported. Here, we present the structural trajectory of an engineered IscB, capturing its transition from a resting state to activation. Using cryo-electron microscopy, we resolve four high-resolution structures: the apo resting state, two intermediate complexes with 6-nt and 10-nt guide-target pairing and a fully paired 16-nt primed cleavage state. These structures uncover a dual inactivation mechanism mediated by RNA lids; the ωRNA lid blocks HNH domain access, while the guide RNA lid occludes the RuvC active site. As guide-target pairing progresses, the guide RNA undergoes a stepwise displacement, mimicking a 'car pedal' motion that triggers activation at 11-nt pairing. The HNH domain also contributes to R-loop stabilization through a positively charged R-wedge motif and undergoes a ~90° activation-driven rotation mediated by two hinge regions. In variants IscBHig1 and IscBHig2, engineering these hinge motifs to enhance conformational flexibility notably improved genome-editing efficiency in cells. In summary, our study reveals the molecular basis underlying IscB autoinhibition and activation, identifies previously uncharacterized regulatory features and establishes hinge elements as a target region for engineering compact, efficient genome editors.},
}
@article {pmid41910274,
year = {2026},
author = {Qiao, W},
title = {From CRISPR functional genomics to synthetic interventions: engineering antiviral strategies.},
journal = {Journal of virology},
volume = {100},
number = {4},
pages = {e0005726},
doi = {10.1128/jvi.00057-26},
pmid = {41910274},
issn = {1098-5514},
support = {C1032633001//Shenzhen Bay Laboratory/ ; },
mesh = {Humans ; *Synthetic Biology/methods ; *Antiviral Agents/pharmacology ; *Genomics/methods ; *CRISPR-Cas Systems ; Animals ; Virus Replication ; *Viruses/genetics ; *Virus Diseases/virology/therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Host-Pathogen Interactions/genetics ; },
abstract = {Virus-host interactions govern infection outcomes and viral evolution, but host determinants that enable or restrict viral replication have been difficult to map comprehensively and in the right cellular contexts. Pooled CRISPR perturbation screens now provide scalable, mechanistic entry points into host dependency and restriction landscapes across diverse viruses. Recent extensions, including single-cell readouts, imaging and spatial phenotyping, organoid models, and in vivo selection, are shifting the field from static hit lists toward contextual maps that explain how host pathways and cell states shape permissiveness. In parallel, synthetic biology is translating these maps into programmable intervention classes, including receptor decoys and binders that intercept entry, conditional protein depletion systems that modulate host factors with temporal control, gene circuits that couple infection sensing to tailored responses, and engineered immune cells with tunable antiviral functions. This review highlights conceptual and technical advances that connect CRISPR functional genomics to synthetic antiviral design, summarizes emerging principles that generalize across viral families, and discusses constraints that will determine whether screen-nominated mechanisms can be engineered into effective and safe antiviral strategies.},
}
@article {pmid41922875,
year = {2026},
author = {Burr, SP and Auckland, K and Glynos, A and Dhawanjewar, A and Ryall, C and Wei, W and Hynes-Allen, A and Prater, M and Sczaniecka-Clift, M and Prudent, J and Chinnery, PF and van den Ameele, J},
title = {MitoPerturb-Seq identifies gene-specific single-cell responses to mitochondrial DNA depletion and heteroplasmy.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {4},
pages = {711-723},
pmid = {41922875},
issn = {1545-9985},
mesh = {*DNA, Mitochondrial/genetics ; Animals ; Mice ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems ; *Heteroplasmy/genetics ; *Mitochondria/genetics/metabolism ; DNA Polymerase gamma/genetics ; Mitochondrial Proteins/genetics ; DNA-Binding Proteins/genetics ; Mutation ; Activating Transcription Factor 4/metabolism/genetics ; Transcription Factors/genetics ; GTP Phosphohydrolases/genetics ; High Mobility Group Proteins ; },
abstract = {Mitochondria contain their own genome, mitochondrial DNA (mtDNA), which is under strict control by the cell nucleus. mtDNA occurs in many copies per cell and mutations often only affect a proportion of them, giving rise to heteroplasmy. mtDNA copy number and heteroplasmy level together shape the tissue-specific impact of mtDNA mutations, eventually giving rise to both rare mitochondrial and common neurodegenerative diseases. Here, we use MitoPerturb-Seq for CRISPR-Cas9-based, high-throughput single-cell interrogation of the nuclear genes and pathways that sense and control mtDNA copy number and heteroplasmy. We screened a panel of mtDNA maintenance genes in mouse cells with a heteroplasmic mtDNA mt-Ta mutation. This revealed both common and perturbation-specific aspects of the integrated stress response to mtDNA depletion caused by Tfam, Opa1 and Polg knockout. These responses are only partially mediated by ATF4 and cause cell-cycle stage-independent slowing of cell proliferation. MitoPerturb-Seq, thus, provides experimental insight into disease-relevant mitochondrial-nuclear interactions and may inform development of therapies targeting cell-type- and tissue-specific vulnerabilities to mitochondrial dysfunction.},
}
@article {pmid41928564,
year = {2026},
author = {Topfer, SK and Rutherford, M and Zewe, F and Strive, T and Oh, KP},
title = {Viability-Based Assessment Reveals True Efficiency of CRISPR-Cas9 Transfection Methods in Rabbit Spermatozoa.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {59-70},
doi = {10.1177/25731599261424870},
pmid = {41928564},
issn = {2573-1602},
mesh = {Animals ; Rabbits ; Male ; *Spermatozoa/metabolism ; *CRISPR-Cas Systems/genetics ; *Transfection/methods ; Electroporation/methods ; *Gene Editing/methods ; Cell Survival/genetics ; Plasmids/genetics ; Sperm Motility ; Lipids ; },
abstract = {Modern genome editing tools such as CRISPR-Cas9 have revolutionized mammalian genome engineering, yet translation to in vivo applications remains limited by low efficiency and frequent occurrence of mosaicism. Sperm-mediated delivery of editing reagents is one proposed alternative that may mitigate these issues. This method depends on efficient transfection of genome editing materials into viable spermatozoa, a critical yet frequently overlooked parameter. Using FACS, we compared electroporation (Neon NxT) and lipofection (CRISPRMAX) for introducing CRISPR-Cas9 ribonucleoproteins into viable rabbit spermatozoa. Electroporation, shown to enable Cas9 and plasmid transfection in spermatozoa from other species, performed poorly once dead spermatozoa were excluded. In contrast, Lipofectamine CRISPRMAX improved transfection efficiency with minimal effects on spermatozoa viability and motility. These findings emphasize the importance of distinguishing true transfection (transfection of viable spermatozoa) from total transfection and highlight lipofection as a promising alternative to electroporation for sperm-based genome editing, with potential applications in rabbit genome engineering.},
}
@article {pmid41928587,
year = {2026},
author = {Gibson, JR and Dhungana, A and Pokhrel, M and Arthur, BB and Adebayo, O and Hossack, D and Cottle, RN},
title = {Toward Next-Gen Cell Therapy for Pediatric Patients: Neonatal Hepatocytes Tolerate Electroporation-Mediated Gene Editing and Engraft in the Liver.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {103-114},
doi = {10.1177/25731599261430830},
pmid = {41928587},
issn = {2573-1602},
support = {R01 HL168093/HL/NHLBI NIH HHS/United States ; },
mesh = {*Hepatocytes/transplantation/metabolism/cytology ; *Gene Editing/methods ; Animals ; *Electroporation/methods ; Mice ; CRISPR-Cas Systems ; *Tyrosinemias/therapy/genetics ; Humans ; *Liver/metabolism ; Animals, Newborn ; *Cell- and Tissue-Based Therapy/methods ; Mice, Knockout ; Disease Models, Animal ; Hydrolases/genetics ; },
abstract = {Hepatocyte transplantation (HTx) offers a safer, less invasive alternative to orthotopic liver transplantation for inherited metabolic liver diseases, especially in high-risk pediatric patients. Combining HTx with ex vivo gene editing is a promising autologous therapeutic strategy using the patient's cells. We investigated the feasibility of this approach by applying CRISPR-Cas9 gene knock-out to neonatal mouse hepatocytes and comparing their engraftment potential with that of mature adult cells in the Fah[-/-] mouse model of hereditary tyrosinemia type I (HT1). Electroporation-mediated gene editing did not significantly impair the ability of neonatal hepatocytes to engraft in vivo. Quantitative histological analysis revealed comparable liver repopulation levels between recipients of gene-edited neonatal cells and adult cells after hepatoxicity-mediated selection, providing a benchmark for electroporation-mediated gene editing in neonatal hepatocytes, and supporting the development of genetically corrected neonatal hepatocyte products as a crucial long-term or bridge-to-transplant therapeutic strategy for pediatric liver disease.},
}
@article {pmid41928679,
year = {2026},
author = {Li, D and Feng, J and Wang, X and Zhao, E and Liang, Y and Li, K and Li, Y},
title = {An Anaerobic Fluorescent Reporter System and CRISPR-Cas12a Enable High-Throughput Metabolic Engineering of Clostridium butyricum.},
journal = {ACS synthetic biology},
volume = {15},
number = {4},
pages = {1669-1680},
doi = {10.1021/acssynbio.6c00158},
pmid = {41928679},
issn = {2161-5063},
mesh = {*Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; *Clostridium butyricum/genetics/metabolism ; Anaerobiosis ; Gene Editing/methods ; Plasmids/genetics/metabolism ; Promoter Regions, Genetic/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Genes, Reporter ; High-Throughput Screening Assays/methods ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Clostridium butyricum is an important probiotic and industrial organism with significant potential for anaerobic bioproduction. However, the lack of efficient genetic tools, particularly for high-throughput screening under strict anaerobic conditions, has hindered its metabolic engineering. To address this, we first established a highly efficient conjugation method, significantly improving exogenous DNA transformation efficiency. Leveraging time-resolved transcriptomic data, we then mapped the dynamic activity of native promoters and developed a robust anaerobic fluorescent protein reporter system. This system overcomes the oxygen-dependent limitation of traditional reporters like GFP, enabling high-throughput and quantitative screening of promoter strength in live anaerobes. Furthermore, we constructed a CRISPR-Cas12a-based genome editing platform for scarless gene manipulation and a two-plasmid curing strategy to generate markerless and genetically stable engineered strains. The power of this integrated toolkit was demonstrated by engineering the acetaldehyde metabolic pathway. Overexpression of aldehyde dehydrogenase (ALDH) resulted in a 79.29% increase in enzyme activity, indicating an enhanced catalytic capacity for acetaldehyde oxidation. This proof-of-concept module, together with the anaerobic fluorescent reporter and CRISPR-Cas12a platform, supports a streamlined workflow for genetic part characterization and metabolic engineering in C. butyricum under strict anaerobic conditions.},
}
@article {pmid41930529,
year = {2026},
author = {Pan, H and Zhao, J and Yang, J and Cao, W and Liu, G},
title = {A ∆ssaV deletion attenuates Salmonella Choleraesuis to generate a self-limiting, immunogenic vaccine candidate.},
journal = {Vaccine},
volume = {80},
number = {},
pages = {128546},
doi = {10.1016/j.vaccine.2026.128546},
pmid = {41930529},
issn = {1873-2518},
mesh = {Animals ; Vaccines, Attenuated/immunology/genetics/administration &amp; dosage ; *Salmonella Vaccines/immunology/genetics/administration &amp; dosage ; Mice ; *Salmonella enterica/immunology/genetics/pathogenicity ; Antibodies, Bacterial/blood ; Gene Deletion ; Female ; *Type III Secretion Systems/genetics ; Immunity, Humoral ; Virulence ; *Salmonella Infections, Animal/prevention &amp; control/immunology ; Macrophages/microbiology/immunology ; Mice, Inbred BALB C ; Bacterial Proteins/genetics/immunology ; Immunity, Cellular ; Immunogenicity, Vaccine ; CRISPR-Cas Systems ; },
abstract = {Live attenuated Salmonella enterica serovar Choleraesuis (S. Choleraesuis) strains, a host-adapted swine pathogen with well-characterized virulence and strong immunogenicity, represents a rational choice for constructing live bacterial vaccines. The ssaV gene, a key component of the SPI-2 T3SS, was knocked out to construct a vaccine candidate designed to strike an optimal balance between attenuation and immunogenicity using CRISPR-Cas9. This mutant retained the ability to adhere and invade epithelial cells but showed markedly reduced survival in macrophages, indicating impaired SPI-2-dependent persistence. The ∆ssaV mutant exhibited a > 1000-fold increase in LD50 and underwent rapid systemic clearance, collectively confirming its marked attenuation and efficient elimination in vivo. The ∆ssaV mutant vaccine elicited strong humoral and cellular immune responses in mice and achieved a 70% survival rate upon lethal challenge with the WT strain. These results demonstrate that ssaV deletion creates a rationally designed vaccine strain that is attenuated, immunogenic, and self-limiting, positioning it as a promising live vaccine candidate in the murine model.},
}
@article {pmid41934379,
year = {2026},
author = {Lin, WW and Su, J and Li, JX and Chen, YW and Chen, JY and Wu, B and Cai, NQ and Gan, LJ and Liu, ZJ},
title = {Enhanced SNV Detection of HLA-B*15:02TA by Integrating Blocking RPA and Inhibiting CRISPR-Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {15},
pages = {11234-11245},
doi = {10.1021/acs.analchem.5c08035},
pmid = {41934379},
issn = {1520-6882},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; *CRISPR-Associated Proteins/antagonists &amp; inhibitors/genetics ; *Endodeoxyribonucleases/antagonists &amp; inhibitors/genetics/metabolism ; Bacterial Proteins ; },
abstract = {With the introduction of the RPA-based CRISPR-Cas12a method, crRNA allostery and Cas12a protein engineering have been applied to the identification of single-nucleotide variants (SNVs). However, the complicated testing procedure often falls short of the intended simple and rapid objectives of CRISPR-based diagnostics (CRISPR-Dx), and the discrimination factor (DF) for SNV distinction remains suboptimal (DF = 2-5). In this study, we proposed a one-tube "Blocking RPA-coupled Inhibiting CRISPR-Cas12a" (BRIC) cascade strategy that features a dual recognition of SNVs. In the BRIC cascade, a reduction of RPA amplification products of wild-type target (WT) and WT-activated Cas12a cleavage lowered the WT detection signal to near-background levels, without compromising the efficiency of RPA or CRISPR-Cas12a. With HLA-B*15:02TA (rs3909184, G > C), a gene associated with drug-induced cutaneous rash, as the SNV target, the proposed one-tube BRIC strategy achieved a DF of 32.51, with the WT signal approximating the background level, thereby greatly enhancing SNV detection specificity. On the other hand, MT can be detected in a large number of WT (with a resolution of 0.1%). Within the HLA-B*15:02TA concentration (CMT) range of 5.0 × 10[-7] to 1.0 × 10[-3] nM (500 aM to 1 pM), a linear relationship was evident between the fluorescence signal (FMT) and lgCMT (FMT = 4146.55 + 578.09lgCMT; R[2] = 0.9904), with a detection limit of 67.6 aM (>3σ). Combined with a 3D-printed portable fluorimeter, the proposed method reported high consistency with next-generation sequencing in detecting HLA-B*15:02TA in 22 clinical plasma samples, validating the reliability of the one-tube BRIC cascade assay. These improvements in assay design, process, and DF highlight the promising potentials of the BRIC strategy in clinical settings.},
}
@article {pmid41937727,
year = {2026},
author = {Kriete, A and Basika, T and Novas, R and Mathieson, OL and Belikoff, EJ and Kleiner, M and Scott, MJ},
title = {Conditional Expression of Cas9 and dCas9 in Lucilia cuprina Reveals dCas9-Associated Lethality.},
journal = {The CRISPR journal},
volume = {9},
number = {2},
pages = {71-88},
doi = {10.1177/25731599261436373},
pmid = {41937727},
issn = {2573-1602},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Female ; Male ; *Diptera/genetics ; *CRISPR-Associated Protein 9/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Sex Determination Processes/genetics ; Lucilia cuprina ; },
abstract = {Conditional sex transformation systems could improve genetic control strategies against insect pests. Here, we developed and tested CRISPR-based, tetracycline-repressible sex transformation strains in the Australian sheep blowfly, Lucilia cuprina. Using Tet-Off-regulated expression of Cas9 and dCas9, we targeted the sex-determining gene transformer with the goal of converting females into males. Conditional Cas9 expression enabled knockout of a visual marker gene, confirming inducible genome editing. However, strains expressing transformer-targeting sgRNA arrays did not undergo sex transformation. Embryonic microinjection of transformer-targeting sgRNAs into Cas9-expressing embryos produced intersex individuals, indicating that sgRNA expression from the integrated arrays was insufficient to disrupt the sex determination pathway. In contrast, high-level dCas9 expression was associated with developmental delays, reduced body weight, and lethality. These findings establish the first conditional CRISPR expression system in L. cuprina and demonstrate that Cas9 is compatible with inducible gene editing, whereas dCas9 is poorly tolerated at high expression levels.},
}
@article {pmid41952066,
year = {2026},
author = {Chatla, K and Ayalew, L and Yim, M and Ko, P and Lippold, S and Hernandez, G and Chua, BA and Patil, DP and Camperi, J},
title = {Analytical Assessment of sgRNA Impurities and Their Impact on Functional Performance.},
journal = {Analytical chemistry},
volume = {98},
number = {15},
pages = {11438-11447},
doi = {10.1021/acs.analchem.6c00747},
pmid = {41952066},
issn = {1520-6882},
mesh = {*RNA, Guide, CRISPR-Cas Systems/analysis/genetics ; Gene Editing ; Humans ; CRISPR-Cas Systems ; Chromatography, Reverse-Phase ; Chromatography, Gel ; Mass Spectrometry ; },
abstract = {Single guide RNA (sgRNA) is a critical component of the clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system, guiding Cas9 to specific genomic loci for precise DNA modification. With its growing clinical potential, sgRNAs have emerged as a promising modality for gene editing-based therapeutics, underscoring the need for robust analytical characterization to ensure quality, safety, efficacy, and regulatory compliance. Here, we present an integrated strategy for deep profiling of sgRNA impurities, combining high-resolution ion-pairing reversed-phase liquid chromatography (IP-RPLC) and size-exclusion chromatography (SEC) with advanced technologies, namely, native mass spectrometry and nanopore direct RNA sequencing. The isolation and characterization of isolated chromatographic peaks revealed truncated, chemically modified, and deletion-prone species. Notably, early eluting fractions in IP-RPLC exhibited elevated deletion frequencies in the target-specific region, correlating with reduced gene-editing efficiency and increased variability in T cells when tested using the Cas-CLOVER system. In contrast, late-eluting fractions in IP-RPLC revealed polyphosphorylated variants with minimal functional impact, while off-target analyses suggested that early eluting impurities may paradoxically reduce off-target editing. Lastly, even at high levels, sgRNA aggregates showed only a limited impact on activity: fully aggregated preparations displayed ∼10% lower knockout efficiency; however, an increase in cumulative off-target frequencies was observed. Overall, this study highlights the value of combining advanced analytical tools to achieve deep profiling of sgRNA impurities. By linking specific impurity profiles to functional outcomes, these findings provide actionable insights for improving sgRNA quality control and advancing the development of safe and effective gene editing-based therapeutics.},
}
@article {pmid42007994,
year = {2026},
author = {Tiwari, P and Rathinasabapathi, P},
title = {Programmable CRISPR-Cas diagnostic platforms for rapid detection of uropathogens and antimicrobial resistance.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42007994},
issn = {1432-072X},
}
@article {pmid42007998,
year = {2026},
author = {Sharma, D and Khan, M and Khan, JA},
title = {Genome editing‑based strategies to combat geminiviruses: CRISPR/Cas9 and emerging high‑fidelity tools.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42007998},
issn = {1432-072X},
}
@article {pmid42008182,
year = {2026},
author = {Azhar, F and Mazhari, BBZ and Ibrahim, MN and Alanazi, A and Islam, F},
title = {CRISPR-based tools in food safety and microbiology: applications for pathogen detection and control.},
journal = {Archives of microbiology},
volume = {208},
number = {7},
pages = {},
pmid = {42008182},
issn = {1432-072X},
}
@article {pmid42009546,
year = {2026},
author = {Xu, T and Zhao, L and Dai, Y and Duan, J and Wang, Y and Shao, L and Chen, D and Zhu, L and Xu, Z},
title = {[A rapid visual detection method for porcine circovirus type 4 based on enzymatic recombinase amplification and CRISPR/EsCas13d].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {4},
pages = {1755-1768},
doi = {10.13345/j.cjb.250568},
pmid = {42009546},
issn = {1872-2075},
support = {2024YFD1800500 and 2024YFD1800102//the National Key Research and Development Program of China/ ; sccxtd-2024-08 and sccxtd-2024-18//the National Modern Agricultural Industrial Technology System/ ; },
mesh = {*Circovirus/isolation &amp; purification/genetics ; Animals ; Swine ; *Recombinases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Circoviridae Infections/diagnosis/veterinary/virology ; *Swine Diseases/virology/diagnosis ; },
abstract = {The emerging outbreaks of infectious diseases in humans and animals worldwide pose serious threats to public health and cause substantial economic losses. This has raised the demand for more efficient and sensitive diagnostic methods to strengthen disease surveillance and early warning. In this study, we developed a portable visual platform based on ERA-CRISPR/EsCas13d for the rapid detection of porcine circovirus type 4 under resource-limited conditions. We optimized the platform by integrating enzymatic recombinase amplification (ERA), T7 transcription, and CRISPR/EsCas13d cleavage in a single-tube reaction, thereby simplifying the workflow and shortening the total detection time to 30 min. In addition, a rapid nucleic acid release method was employed, eliminating the need for laboratory-based extraction and complex heating steps, which further improved the simplicity and usability. In addition, lyophilized reagents were modified to enhance stability, thus reducing cold-chain and storage requirements and enabling cost-effective transport and field deployment. The method achieved a visual limit of detection of 50 cp/μL for porcine circovirus type 4 and demonstrated no cross-reactivity with six other common swine pathogens. Moreover, the platform supported two visual readout formats-UV light (470 nm) and lateral flow assay (LFA)-providing flexible options for interpretation in different application scenarios. Validation with 60 clinical samples showed 100% concordance with quantitative PCR results, highlighting the practical utility and application potential of the ERA-CRISPR/EsCas13d-based portable visual platform for rapid on-site diagnostics.},
}
@article {pmid42009554,
year = {2026},
author = {Li, Y and Wen, D and Zhou, Z and Liao, C and Zhao, Q and Shuai, J and Zhang, X and Yu, X and Huang, J},
title = {[A CRISPR-Cas13a-based amplification-free electrochemical biosensor for rapid detection of bovine viral diarrhea virus].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {4},
pages = {1868-1880},
doi = {10.13345/j.cjb.250329},
pmid = {42009554},
issn = {1872-2075},
support = {2021YFF0600805 and 2021YFF0602801//the National Key Research and Development Program of China/ ; 2024SNJF044//the Zhejiang Provincial Department of Agriculture and Rural Affairs Project/ ; 20241203A23//the Key Research Program in the Field of Agriculture and Social Development of Hangzhou/ ; },
mesh = {*Biosensing Techniques/methods ; Cattle ; Animals ; *CRISPR-Cas Systems/genetics ; *Diarrhea Viruses, Bovine Viral/isolation &amp; purification/genetics ; *Electrochemical Techniques/methods ; Nucleic Acid Amplification Techniques ; Bovine Virus Diarrhea-Mucosal Disease/diagnosis/virology ; },
abstract = {Bovine viral diarrhea virus (BVDV), a major pathogen in the global bovine industry, causes diarrhea, fever, and reproductive disorders, leading to substantial economic losses. Developing the methods for rapid and accurate detection of BVDV is crucial for epidemic control. Current detection methods have notable limitations. PCR-based nucleic acid amplification techniques rely on sophisticated instruments and complex procedures. CRISPR-Cas13a systems, despite their high specificity, still require nucleic acid pre-amplification, which results in cumbersome workflows and contamination risks. To establish a simpler and more efficient on-site detection method for BVDV, this study integrated the CRISPR-Cas13a system characterized by specific recognition with electrochemical sensing praised for efficient signal transduction to establish a novel nucleic acid amplification-free method for the detection of BVDV. Through optimization of key parameters, including CRISPR RNA (crRNA) combination, buffer components, and Cas13a/crRNA concentration ratio, the biosensor achieved a detection limit of 3 090 copies/μL-representing a 4-5 order of magnitude improvement in sensitivity compared with conventional Cas13a fluorescence-based detection-and completed the entire process from sample loading to result output within 35 min. Specificity tests demonstrated that the sensor exclusively detected BVDV without cross-reactivity to other common bovine viruses (bovine parainfluenza virus type 3, bovine respiratory syncytial virus, bluetongue virus, and foot-and-mouth disease virus). Clinical validation with 22 samples demonstrated 100% specificity and sensitivity. The developed CRISPR-Cas13a-based electrochemical biosensor offers the advantages of being nucleic acid amplification-free and operationally simple, serving as a powerful new tool for rapid on-site BVDV detection with significant potential for veterinary diagnostics and epidemic prevention and control.},
}
@article {pmid42009663,
year = {2026},
author = {Batty, P and Beneder, H and Schätz, C and Onea, G and Zaczek, M and Kutschat, AP and Abele, M and Müller, S and Superti-Furga, G and Winter, GE and Seruggia, D},
title = {Disruption of the SAGA CORE triggers collateral degradation of KAT2A.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42009663},
issn = {2041-1723},
support = {947803//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 10.55776/P36302//Austrian Science Fund (Fonds zur F&#xf6;rderung der Wissenschaftlichen Forschung)/ ; },
mesh = {*Histone Acetyltransferases/metabolism/genetics ; Humans ; Proteolysis ; Proteasome Endopeptidase Complex/metabolism ; Acetylation ; Histones/metabolism ; Ubiquitin-Protein Ligases/metabolism/genetics ; HEK293 Cells ; Proteomics ; *Trans-Activators/metabolism/genetics ; CRISPR-Cas Systems ; TATA-Binding Protein Associated Factors/metabolism/genetics ; p300-CBP-Associated Factor ; },
abstract = {The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex regulates gene expression through histone acetylation at promoters, mediated by its histone acetyl transferase (HAT), KAT2A. While SAGA structure and function are well characterised, mechanisms controlling the stability of individual subunits, including KAT2A, remain unclear. Here, using a fluorescence-based KAT2A stability reporter, we systematically dissect the molecular dependencies controlling KAT2A protein abundance, and identify the non-enzymatic SAGA CORE module subunits-TADA1, TAF5L, and TAF6L- as necessary for KAT2A stability. Loss of these subunits disrupts SAGA complex integrity, leading to non-chromatin-bound KAT2A that is degraded by the proteasome and consequent reduced H3K9 acetylation. Proteomic profiling reveals progressive loss of components from the CORE and HAT modules upon acute SAGA CORE disruption, indicating that an intact CORE is required for the stability of numerous SAGA components. Finally, a focused CRISPR screen of ubiquitin-proteasome system genes identifies the E3 ligase UBR5, a known regulator of orphan protein degradation, and the deubiquitinase OTUD5, as regulators of KAT2A degradation when the SAGA CORE is perturbed. Together, these findings reveal a dependency of KAT2A protein stability on SAGA CORE integrity and define an orphan quality control mechanism targeting unassembled KAT2A, revealing a potential vulnerability in SAGA-driven malignancies.},
}
@article {pmid42009664,
year = {2026},
author = {Corazzi, L and Ing, A and Benito, E and Cosenza, MR and Hasenfeld, P and Weber, T and Marx, AJM and Ionasz, VS and Trausch, N and Benedetto, S and Di Muzio, G and Ding, B and Berlanda, J and Giaisi, M and Claudino, N and Höfer, T and Korbel, JO and Wei, PC},
title = {Recurrent DNA break clusters drive replication-stress-induced copy number variants and genome diversification.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {42009664},
issn = {2041-1723},
support = {949990//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 101098056//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; YIP-DKFZ//Helmholtz Association/ ; },
mesh = {*DNA Copy Number Variations/genetics ; Animals ; *DNA Replication/genetics ; Mice ; Neural Stem Cells/metabolism ; *DNA Breaks ; *Genome ; CRISPR-Cas Systems ; Whole Genome Sequencing ; Single-Cell Analysis ; DNA End-Joining Repair/genetics ; Humans ; DNA Repair ; },
abstract = {Copy number variants (CNVs) are strongly implicated in neurological and psychiatric disorders and brain cancer, yet the process by which replication stress generates CNVs-and why some recur while others remain rare-remains poorly understood. Here, we show that recurrent DNA-break clusters (RDCs) act as common initiating lesions that drive both recurrent and non-recurrent CNVs. In murine neural progenitor cells subjected to chemically induced replication stress, bulk whole-genome sequencing identifies recurrent CNVs enriched at late-replicating RDCs within actively transcribed genes. Single-cell genome sequencing further uncovers frequent, non-recurrent CNVs associated with RDCs that arise during the transition from early to late DNA replication. These CNVs represent stable, heritable structural variants with breakpoints consistently enriched at RDCs. CRISPR/Cas9-mediated transcriptional suppression abolishes both RDC formation and CNV generation, establishing RDC-associated breaks as a shared upstream source. Mechanistically, CNV formation depends on DNA repair context: CNVs are Pol θ-dependent in NHEJ-deficient cells but arise independently of Pol θ in NHEJ-proficient cells. Together, these findings define RDCs as central drivers of replication-stress-induced genome diversification.},
}
@article {pmid42011779,
year = {2026},
author = {Biczók, Z and Krausz, SL and Simon, DA and Tóth, E and Varga, &#xc9; and Annus, T and Huba, F and Varga, M and Bakos, &#xc9; and Fodor, E and Welker, E},
title = {Disrupting pegRNA intramolecular complementarity via PBS and spacer sequence alterations can enhance prime editing efficiency.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
doi = {10.1093/nar/gkag292},
pmid = {42011779},
issn = {1362-4962},
support = {K134968//Hungarian Scientific Research Fund/ ; K142322//Hungarian Scientific Research Fund/ ; RRF-2.3.1-21-2022-00015//PharmaLab/ ; ELKH-PoC-2023//National Research, Development, and Innovation Office of Hungary/ ; //Hungarian Research Network/ ; //Ministry of National Economy/ ; },
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; CRISPR-Cas Systems ; Binding Sites ; Plasmids/genetics ; Base Sequence ; Humans ; Base Pair Mismatch ; },
abstract = {The length and sequence of the primer binding site (PBS) are critical for efficient prime editing, and its intramolecular complementarity with the prime editing guide RNA (pegRNA) spacer is a major drawback. We investigated the effects of these factors by literature analyses and by testing over 300 modified pegRNAs with weakened PBS-spacer interactions. It has been suggested that the effective PBS length for plasmid-delivered pegRNAs without end protection is considerably longer than what efficient priming requires due to exonuclease digestion of the PBS ends; however, analysing literature data of over 3000 pegRNAs revealed no significant shift in the optimal PBS length for epegRNAs compared to conventional pegRNAs. We also found improvement in editing efficiency with up to seven-fold when mismatches were introduced in the spacer or PBS sequence disrupting complementarity, although this effect is more pronounced with non-optimal PBS lengths. A combination of spacer mismatches and PBS deletions led to further editing improvements, even compared to the optimal PBS, although finding the best combination requires extensive optimization. Here, we achieved near-optimal editing efficiency in the majority of cases without the need for prior pegRNA optimization by using SPELL (Streamlined Prime Editing with fixed-Length PBS Leverage), a prime editing approach that employs a 17-20 nucleotide-long PBS with a single nucleotide deletion.},
}
@article {pmid42001810,
year = {2026},
author = {Saleem, MS and Khan, SH and Rana, IA and Ahmad, A},
title = {CRISPR/Cas9-mediated editing of the GhJAZ2 gene improves fiber length and lint percentage in Gossypium hirsutum L.},
journal = {GM crops &amp; food},
volume = {17},
number = {1},
pages = {2660546},
doi = {10.1080/21645698.2026.2660546},
pmid = {42001810},
issn = {2164-5701},
mesh = {*Gossypium/genetics/metabolism/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing ; *Cotton Fiber/analysis ; Plants, Genetically Modified/genetics ; *Plant Proteins/genetics/metabolism ; },
abstract = {Cotton is regarded as a strategic agricultural commodity owing to its renewable and naturally derived fiber. With the escalating global demand for high-quality fiber, genetic improvement of fiber traits is a critical focus for sustaining and advancing the textile industry standards. The cotton GhJAZ2 gene encodes the Jasmonate ZIM-domain 2 protein, a known repressor in the jasmonic acid signaling pathway and negatively regulates fiber initiation. In this study, we designed a gRNA that simultaneously targets GhJAZ2 homologs and assembled it into the CRISPR vector (pHSE401). Subsequently, the construct (pHSE401-gRNA) was transformed into cotton (Gossypium hirsutum L.) using an Agrobacterium-mediated in planta transformation strategy, targeting the shoot apical meristem as the primary site of transformation. Sanger sequencing analysis revealed consistent single-base pair indels at the targeted site across both A and D sub-genomes, with edited T1 progenies showing both inherited and newly introduced indels at the targeted loci. Fiber analysis of edited lines compared to the control revealed a significant (p < .05) enhancement in lint percentage (≤13.74%) and fiber length (≤16.91%). This study demonstrated the effective application of CRISPR/Cas9 for targeted trait improvement in cotton, offering GhJAZ2-edited lines that can be advanced to develop transgene-free cultivars with improved fiber traits.},
}
@article {pmid42001897,
year = {2026},
author = {Li, Q and Cao, J and Deng, S and Yu, K},
title = {Precise Multi-Gene Editing Strategies for Xenotransplantation Donor Pigs: Overcoming Immune and Coagulation Barriers.},
journal = {Xenotransplantation},
volume = {33},
number = {2},
pages = {e70128},
doi = {10.1111/xen.70128},
pmid = {42001897},
issn = {1399-3089},
support = {32072722//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Gene Editing/methods ; Swine ; Humans ; Graft Rejection/immunology ; CRISPR-Cas Systems/genetics ; *Blood Coagulation/genetics/immunology ; *Heterografts/immunology ; Tissue Donors ; Organ Transplantation ; },
abstract = {Organ transplantation is the preferred treatment for end-stage organ failure, but the severe shortage of donors severely restricts its clinical application. Xenotransplantation, especially using pigs as donors, is considered an ideal source of alternative donors due to the high similarity between their organ structures and those of humans. However, significant differences in immune recognition and coagulation regulation between species can easily induce a series of rejection reactions, including hyperacute rejection, acute humoral rejection, T-cell-mediated rejection, and chronic vascular complications. It also carries risks such as physiological metabolic incompatibility and potential viral transmission. In recent years, with the development of tools such as CRISPR/Cas, precise multi-gene editing technology has become possible, enabling the simultaneous knockout of multiple xenoantigen genes (such as GGTA1, CMAH, and B4GALNT2) and the introduction of human genes regulating complement, coagulation, and immune responses (such as hCD55, hTBM (THBD), and hCD47), significantly improving the immune tolerance and physiological compatibility of donor organs. This article systematically reviews the immune and coagulation barriers in xenotransplantation, focusing on precise multi-gene editing strategies for pigs used in xenotransplantation. It highlights editing pathways such as tandem knock-in at the same site, simultaneous multi-site editing, stepwise modular editing, and homology-directed repair (HDR) enrichment. Combined with representative organ-specific examples (heart, kidney, liver, and lung), including key non-human primate studies and early human exploratory cases where available, it explores the application prospects of these strategies in creating safe clinical-grade donor pigs and promoting the clinical translation of xenotransplantation.},
}
@article {pmid42002179,
year = {2026},
author = {Guha, S and Tharmatt, A and Yadav, S and Siwakoti, P and Kumeria, T and Mittal, A and Chitkara, D},
title = {Lipid-polymer hybrid nanoplex loaded microneedle patches as a corneal delivery platform for CRISPR/Cas expressing plasmid.},
journal = {International journal of biological macromolecules},
volume = {},
number = {},
pages = {152092},
doi = {10.1016/j.ijbiomac.2026.152092},
pmid = {42002179},
issn = {1879-0003},
abstract = {Lipid-polymer hybrid (LPH) systems have evolved into a promising vehicle for delivering therapeutic agents. This study demonstrates cationic LPH nanoplexes composed of a biodegradable cationic mPEG-polycarbonate based cationic copolymer and cholesterol to enhance delivery efficiency of genetic materials. The optimized blank formulation, BNPX-3, exhibited a particle size of 93.60 nm, polydispersity index (PDI) of 0.116, and a zeta potential (ZP) of 21.9 mV. After complexation of pcDNA3-EGFP plasmid and CRISPRi plasmid at N/P ratios of 10 and 20, respectively, the nanoplexes exhibited particle sizes of 123.4 nm and 131.6 nm, with corresponding PDI values of 0.128 and 0.142 and zeta potentials of 12.2 mV and 10.5 mV, respectively. Cytocompatibility studies demonstrated >80% cell viability in HEK293 and SIRC cells over a wide concentration range. Transfection efficiencies using LPH nanoplexes for pcDNA3-EGFP (6159 bp) and CRISPRi plasmids (11,266 bp) were found to 60.09% and 48.02%, respectively, in HEK293 cells, and 56.04% and 40.34%, respectively, in SIRC cells. These efficiencies were comparable to Lipofectamine 3000 and superior to formulations prepared with cationic polymers alone. To enable efficient delivery of the nanoplexes, the developed nanoplexes were incorporated into dissolvable microneedle (MN) patches. The microneedle patch demonstrated successful loading of pDNA-complexed nanoplexes and efficient delivery ex-vivo.},
}
@article {pmid42002396,
year = {2026},
author = {Chen, L and Luo, J and Zhang, H and Zhao, P},
title = {RPA Combined With CRISPR/Cas12a for Rapid and Ultrasensitive Detection Dual-Gene of Methicillin-Resistant Staphylococcus aureus (MRSA).},
journal = {Journal of molecular recognition : JMR},
volume = {39},
number = {3},
pages = {e70035},
doi = {10.1002/jmr.70035},
pmid = {42002396},
issn = {1099-1352},
support = {2025A1515010579//Natural Science Foundation of Guangdong Province, China/ ; 211102114530659//Shaoguan Municipal Science and Technology Program, China/ ; 220610154531525//Shaoguan Municipal Science and Technology Program, China/ ; 220525096180441//Shaoguan Engineering Research Center for Research and Development of Molecular and Cellular Technology in Rapid Diagnosis of Infectious Diseases and Cancer Program, China/ ; KEYANSHEN (2023) 01//Research Fund for Joint Laboratory for Digital and Precise Detection of Clinical Pathogens, Yuebei People's Hospital Affiliated to Shantou University Medical College, China/ ; RS202001//Research Project for Outstanding Scholar of Yuebei People's Hospital Affiliated to Shantou University Medical College, China/ ; 2023B110008//Research Project for Guangdong&#x2009;Provincial&#x2009;Clinical&#x2009;Research&#x2009;Center&#x2009;for&#x2009;Laboratory&#x2009;Medicine, China/ ; },
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics ; Humans ; *Staphylococcal Infections/diagnosis/microbiology ; Penicillin-Binding Proteins/genetics ; Sensitivity and Specificity ; Microbial Sensitivity Tests ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The increasing issue of infections caused by methicillin-resistant Staphylococcus aureus (MRSA) necessitates rapid and reliable diagnostic methods. While existing RPA-CRISPR/Cas12a platforms have demonstrated potential for MRSA detection, most rely on single-gene targets or require multiple Cas enzymes. Here, we have developed a novel dual gene detection strategy that simultaneously detects the S. aureus specific femA gene and the methicillin-resistant mecA gene in a single RPA-CRISPR/Cas12a reaction. This integrated approach enables clear discrimination between MRSA and methicillin-sensitive Staphylococcus aureus (MSSA) in just 30 min, with results visualized via both fluorescence and lateral flow strips. The assay exhibited high specificity (no cross-reactivity with common pathogens) and a sensitivity of 10 copies/μL, comparable to qPCR. Validation with 39 clinical samples showed 100% concordance with antimicrobial susceptibility testing. Our dual-gene RPA-CRISPR/Cas12a platform represents a significant advancement in point-of-care MRSA diagnostics, offering enhanced accuracy and operational simplicity.},
}
@article {pmid42003550,
year = {2026},
author = {Marsic, T and Gundra, SR and Aouida, M and Masood, M and Salibi, A and Schmidt, F and Alquwayzani, R and Mahfouz, MM},
title = {Precise, specific gene editing via a compact GoCas12m-FokI chimeric nuclease.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
doi = {10.1093/nar/gkag342},
pmid = {42003550},
issn = {1362-4962},
support = {1/1035-01-01//BAS/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; *Deoxyribonucleases, Type II Site-Specific/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/genetics/metabolism ; HEK293 Cells ; *Endodeoxyribonucleases/genetics/metabolism ; Recombinant Fusion Proteins/genetics/metabolism ; Bacterial Proteins ; },
abstract = {CRISPR gene editing technologies have transformed functional genomics and biotechnology. Despite these advances, challenges such as limited delivery capacity and off-target activity continue to hinder their therapeutic translation. We developed a chimeric gene editing platform by fusing the compact, catalytically inactive Cas12m guiding module (GoCas12m) with the FokI nuclease domain. GoCas12m-FokI system integrates the programmable DNA-binding capability of Cas12m with the dimerization-dependent cleavage mechanism of FokI, enabling precise genome editing. Our engineered XTEN-fused GoCas12m-FokI editor exhibits robust activity on both surrogate reporters and endogenous human loci, achieving high-efficiency editing at clinically relevant targets-including CLTA1, HBB, AIFM1, and ABL with no detectable off-target activity at in silico-predicted sites, as confirmed by targeted deep sequencing. Notably, GoCas12m-FokI is nearly half the size of conventional Cas9- or Cas12a-based editors, facilitating delivery via adeno-associated virus and other cargo-limited vectors. This dual-guided editor showed comparable editing efficiency to previously reported FokI-dCas9 systems on endogenous loci, while possessing a different PAM requirement and domain orientation. By combining compact architecture, high specificity, and modular programmability, the GoCas12m-FokI editor offers a powerful alternative for therapeutic genome editing and a promising tool for in vivo gene therapy applications.},
}
@article {pmid42003552,
year = {2026},
author = {White, N and Hu, YT and Chalk, JA and Kurgan, G and Naseem, A and Schmaljohn, E and Sturgeon, M and Cavazza, A and Thrasher, AJ and Turchiano, G},
title = {DNA-PKcs inhibitor AZD7648 reveals sgRNA cross-contaminants and enhanced sensitivity of genome engineering off-target activity in HSPCs.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
doi = {10.1093/nar/gkag318},
pmid = {42003552},
issn = {1362-4962},
support = {217112/Z/19/Z/WT_/Wellcome Trust/United Kingdom ; 1160024//Great Ormond Street Hospital Children's Charity/ ; //Curing Rare Inherited Diseases Using Innovative Gene Therapies/ ; //European Union's Horizon 2020/ ; },
mesh = {Humans ; *DNA-Activated Protein Kinase/antagonists &amp; inhibitors/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Pyrazoles/pharmacology ; DNA End-Joining Repair/drug effects ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Therapeutic gene editing with designer nucleases can be compromised by undesired repair outcomes. DNA repair inhibitors are used to bias DSB repair toward HDR, but their impact on larger structural rearrangements, including large deletions and translocations, remains unclear. We quantify the mutational burden associated with end-joining inhibitor compounds. With a highly precise Cas9 nuclease, repair inhibition yields modest increases in aberrations, whereas promiscuous single guide RNAs (sgRNAs) amplify aberrant outcomes by orders of magnitude. Donor templates mitigate mutational burden at on-target sites, and in rare cases donor sequences bridge translocations between on- and off-target loci. Because DNA-PKcs inhibition does not itself induce instability over short intervals but increases the likelihood of capturing chromosomal aberrations postediting, we leveraged this to enhance assay performance. Compared to CAST-Seq, high-resolution CAST-Seq achieved a median ~12-fold increase in detected aberrations and, in this higher-sensitivity context, revealed unintended, target-specific sgRNA contaminants in GMP-like batches, underscoring direct genotoxicity risk and the need for stricter guide purity controls. A modified, translocation-quantitative rhAmpSeq reports all translocation combinations between two loci, enabling robust off-target validation beyond indel-only readouts. Finally, we evaluate AZD7648, finding limited aberration increases with precise nucleases and reconciling reports of extensive large deletions by quantifying assay- and design-dependent biases.},
}
@article {pmid41481841,
year = {2026},
author = {Jin, YM and Li, XD and Zhu, JK and Shao, CY and Huang, BB and Huang, HL and Wang, XW and Jiang, HB and Chen, W},
title = {Programmable adenine base editing in cyanobacteria using an engineered TadA-Cas9 fusion.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70655},
doi = {10.1111/tpj.70655},
pmid = {41481841},
issn = {1365-313X},
support = {32470092//National Natural Science Foundation of China/ ; 32170108//National Natural Science Foundation of China/ ; 2024QL060//Ningbo Youth Leading Talent Project/ ; //Ningbo University Startup Funding/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Adenine/metabolism ; *Synechocystis/genetics ; *Cyanobacteria/genetics ; Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Anabaena/genetics ; },
abstract = {Cyanobacteria are photosynthetic prokaryotes with great potential in green biomanufacturing and basic research. Despite decades of pioneering achievements, the application of advanced genome editing tools, particularly CRISPR-based systems, has remained limited in cyanobacteria. In this study, we developed pCyABE, a new adenine base editor for efficient and precise A·T to G·C editing in cyanobacteria. This system utilizes a TadA-Cas9 nickase fusion and functions without double-strand breaks or donor templates. We demonstrated its high editing efficiency in Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, highlighting its broad usability. pCyABE supports multiplex editing and enables start codon disruption for gene functional studies. Furthermore, this tool exhibits low off-target activity and can be effectively removed via sucrose counterselection. In conclusion, pCyABE provides a versatile and efficient genome editing platform that significantly expands the genetic toolbox for cyanobacterial research and biotechnology applications.},
}
@article {pmid41483428,
year = {2026},
author = {Deng, X and Gao, Q and Shen, K and Mu, W and Ge, T and Gu, J and Yang, X and Cheng, J and Wang, J and Zhang, W and Li, D and Zhou, J and Xiao, M},
title = {TNFRSF13B Variant-Induced TACI Dysregulation Underlies CAEBV Pathogenesis.},
journal = {Journal of clinical immunology},
volume = {46},
number = {1},
pages = {12},
pmid = {41483428},
issn = {1573-2592},
support = {82200259//National Natural Science Foundation of China/ ; 81830008//National Natural Science Foundation of China/ ; 82270203//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Transmembrane Activator and CAML Interactor Protein/genetics/metabolism ; *Epstein-Barr Virus Infections/genetics/immunology/metabolism ; *Herpesvirus 4, Human/physiology/immunology ; Frameshift Mutation ; Signal Transduction ; Exons/genetics ; CRISPR-Cas Systems ; },
abstract = {The tumor necrosis factor (TNF) receptor superfamily member, transmembrane activator and CAML interactor (TACI) encoded by TNFRSF13B, are extensively involved in immune responses. In our previous work, TNFRSF13B exon 2 variants were recurrently identified in chronic active Epstein-Barr virus disease (CAEBV). Here we aim to reveal the roles of TNFRSF13B variants in CAEBV, and investigate the feasibility of targeting TNFRSF13B/TACI as a new approach to control EBV infection. The lymphoblastoid cell lines (LCL) models carrying homozygous TNFRSF13B exon 2 frameshift mutations were constructed using CRISPR/Cas9. Immunological assays, transcriptomic analysis, and gene silencing experiments were performed on LCL models to measure the effect of TNFRSF13B exon 2 variants and explore the underlying mechanisms. TACI ligands and a TLR9 agonist were applied to modulate TACI signaling and EBV activities. Frameshift mutations in exon 2 of TNFRSF13B significantly up-regulated the short isoforms of TACI (TACI-S) at the expense of its long isoforms (TACI-L) in LCLs. The up-regulated TACI-S induced more intense activation of NF-κB, MAPK, and Rho signaling pathways, leading to the switch of EBV activities to lytic reactivation. The subsequent increased viral load and viral IL-10 provide a rational for the susceptibility of variant carriers to CAEBV. The BAFF trimer, an indirect TACI-signaling inhibitor, also significantly suppressed the EBV lytic program. Gene silencing experiments indicated that XBP-1 might be involved in the TACI-mediated regulation of EBV lytic activities in EBV-immortalized B cells. This study underscores the impact of TNFRSF13B variants on EBV infection and host immune responses, offering insights into CAEBV pathogenesis and potential therapeutic strategies.},
}
@article {pmid41483499,
year = {2026},
author = {Jin, G and Yang, C and Deng, Q and Wu, L and Chen, W and Chen, Z},
title = {Establishing a chimeric tRNA-sgRNA scaffold and computational basis for enhanced CRISPR interference.},
journal = {Biochemical and biophysical research communications},
volume = {798},
number = {},
pages = {153222},
doi = {10.1016/j.bbrc.2025.153222},
pmid = {41483499},
issn = {1090-2104},
mesh = {*RNA, Transfer/genetics/chemistry ; *CRISPR-Cas Systems/genetics ; Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; Humans ; Molecular Docking Simulation ; Gene Editing/methods ; Molecular Dynamics Simulation ; Nucleic Acid Conformation ; },
abstract = {The CRISPR/Cas9 system revolutionizes genome engineering, yet optimizing the stability and expression levels of single-guide RNA (sgRNA) is crucial for achieving more effective gene regulation. Transfer RNAs (tRNA), known for their inherent stability, present a valuable solution. In this study, we developed a chimeric tRNA-sgRNA (tgRNA) by integrating sgRNA into the anticodon stem of a Sephadex aptamer-human HBV ε tRNA (SeptRNA) scaffold, resulting in the formation of SeptgRNA. When applied to target the E. coli ampC and ompA genes, SeptgRNA exhibited significantly increased accumulation compared to conventional sgRNAs. To overcome potential steric hindrance from the tRNA scaffold, we utilized CRISPR interference (CRISPRi) by co-expressing SeptgRNA with deactivated Cas9 (dCas9), which effectively suppressed DNA transcription. This approach demonstrated superior gene expression suppression compared to traditional sgRNA-based CRISPRi. Molecular docking and molecular dynamics simulations revealed that the SeptRNA scaffold stabilizes the sgRNA stem-loop architecture and enhances the stability of the dCas9-tgRNA-DNA ternary complex. Our findings provide proof-of-concept for the use of chimeric tgRNAs in gene knockdown, highlighting their potential for increased expression levels and improved stability. This study advances the CRISPR/Cas9 toolkit and underscores the versatility of tRNA scaffolds in genetic engineering applications.},
}
@article {pmid41484149,
year = {2026},
author = {Kremer, N and Mueller, F and Nguyen, H and Schulz, L and Popp, T and Artes, E and Wolters, J and Renner, M and Vetter, I and Maffini, S and Robles, MS and Musacchio, A and Bange, T},
title = {CUL4A-DDB1-DCAF10 is an N-recognin for N-terminally acetylated Src kinases.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {132},
pmid = {41484149},
issn = {2041-1723},
support = {5041 140321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Cullin Proteins/metabolism/genetics ; Humans ; Acetylation ; *src-Family Kinases/metabolism/genetics ; HEK293 Cells ; *DNA-Binding Proteins/metabolism/genetics ; Proteolysis ; Protein Processing, Post-Translational ; Ubiquitination ; Ubiquitin-Protein Ligases/metabolism ; CRISPR-Cas Systems ; },
abstract = {Co-translational N-terminal modifications such as methionine excision, acetylation, and myristoylation govern protein stability, localization, and folding. Disruption can expose N-terminal degrons that trigger ubiquitin-mediated degradation, safeguarding the proteome. N-terminal acetylation usually protects proteins from degradation, but can also promote it through the Ac/N-degron pathway. Src-family kinases (SFKs), signaling enzymes implicated in tumorigenesis, require N-terminal myristoylation for function. Using peptide pull-downs, mass spectrometry, and AlphaFold 3 predictions, we identify DCAF10 as the E3 ligase substrate receptor for alternatively N-terminally acetylated SFKs. Combining siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout of endogenous Lyn with inducible Lyn-GFP variants confirms that DCAF10 regulates SFK levels by recognizing an N-terminal acetylated glycine residue. In vitro, a CUL4A-DDB1-DCAF10 complex ubiquitinates N-terminally acetylated SFKs. Thus, we define a novel N-degron pathway that monitors replacement of myristoylation by acetylation and activates degradation of SFKs upon acetylation. This mechanism may extend to other N-terminally myristoylated proteins beyond SFKs.},
}
@article {pmid41484984,
year = {2026},
author = {Forbes, CA and Shaw, NC and Chen, KG and Hedges, M and Er, TS and Hool, L and Ward, M and Poulton, C and Baynam, G and Lassmann, T and Fear, VS},
title = {A precision medicine approach to interpret a GATA4 genetic variant in a paediatric patient with congenital heart disease.},
journal = {Human genomics},
volume = {20},
number = {1},
pages = {29},
pmid = {41484984},
issn = {1479-7364},
support = {Telethon 7 Ball, Gift of Giving, 2020.//McCusker Charitable Foundation/ ; Perron People//Stan Perron Charitable Foundation/ ; Perron Programs and Partnership, 2025//Stan Perron Charitable Foundation/ ; /WT_/Wellcome Trust/United Kingdom ; Lassmann//Feilman Foundation/ ; UWA-VCCRI Chair in Cardiovascular Research//Wesfarmers/ ; APP2004282//National Health and Medical Research Council/ ; },
mesh = {Humans ; *GATA4 Transcription Factor/genetics ; Induced Pluripotent Stem Cells/metabolism/pathology ; Myocytes, Cardiac/metabolism/pathology ; *Heart Defects, Congenital/genetics/pathology ; *Precision Medicine/methods ; Gene Editing ; *Genetic Variation ; Cell Differentiation/genetics ; CRISPR-Cas Systems ; Male ; Child ; Female ; },
abstract = {BACKGROUND: Patients with congenital heart disease are identified in 1% of live births. Improved surgical intervention means many patients now survive to adulthood, the corollary of which is increased mortality in the over-65-year-old congenital heart disease (CHD) population. In the clinic, genetic sequencing increasingly identifies novel genetic variants in genes related to CHD. Traditional assays for interpreting novel genetic variants are often limited by gene-specificity, whereas animal models are cumbersome and may not accurately reflect human disease. This study investigates CRISPR gene editing in induced pluripotent stem cells and cardiomyocyte-directed differentiation as a human disease model to investigate novel genetic variants identified in association with CHD.<br><br>METHODS AND RESULTS: We identified a GATA4 p.Arg284His genetic variant in a paediatric patient. This genetic variant was introduced into induced pluripotent stem cells (iPSCs) using CRISPR gene editing with homology-directed-repair. GATA4 genetic variant and isogenic control iPSCs were selected and differentiated into cardiomyocytes. Expression of the GATA4 p.Arg284His variant resulted in altered calcium transients, indicative of CHD and consistent with the patient's clinical phenotype. Transcriptomics revealed cellular pathway changes in cardiac development, calcium handling, and energy metabolism that contribute to disease aetiology, mechanism and identification of potential treatments.<br><br>CONCLUSION: Directed differentiation of iPSCs harbouring the GATA4 p.Arg284His genetic variant recapitulated the CHD phenotype, indicated disease mechanisms, and pointed to potential sites for targeting with therapy. The study highlights the utility of transcriptomics for the functional interpretation of cardiac genetic variants and is an exemplar for precision medicine approaches for the investigation of CHD.},
}
@article {pmid41485050,
year = {2026},
author = {Wang, K and Li, X and Li, J and Yu, Z and Li, P and Xie, Y and Liu, J and Huang, H and Zhang, S and Zhang, M and Ma, W and Gao, F and Du, X and Wang, J and Capecchi, MR and Wu, S},
title = {Basic enables selection-free efficient knockin of large DNA in primary human T cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {2309-2323},
pmid = {41485050},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes/metabolism/immunology ; Genetic Vectors/genetics ; *Receptors, Chimeric Antigen/genetics/metabolism ; Animals ; *Gene Knock-In Techniques/methods ; Dependovirus/genetics ; Mice ; *DNA/genetics ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Efficient and precise insertion of large DNA fragments into primary human T cells has remained a bottleneck for gene and cell therapy. We present BaEVshort-AAV6 site-specific integration for CAR T (BASIC), a modular platform that combines BaEVshort-pseudotyped virus-like particles for Cas9 RNP delivery with AAV6 donor vectors for homology-directed repair. BASIC achieves >85% knockin efficiency without drug selection or electroporation, preserving cell viability while enabling multiplex genome engineering. Edited chimeric antigen receptor (CAR)-T cells show uniform CAR expression, enhanced cytotoxicity, and complete tumor clearance in vivo. BASIC offers a clinically scalable solution for next-generation cell therapies.},
}
@article {pmid41485557,
year = {2026},
author = {Li, D},
title = {Rising Star Engineering the Genome for Curative Futures.},
journal = {Journal of molecular biology},
volume = {438},
number = {5},
pages = {169618},
doi = {10.1016/j.jmb.2025.169618},
pmid = {41485557},
issn = {1089-8638},
mesh = {*Gene Editing/methods ; Humans ; Animals ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; *Genetic Engineering/methods ; Mice ; Neoplasms/therapy/genetics ; Disease Models, Animal ; },
abstract = {As a professor of biomedicine in the School of Life Sciences at East China Normal University (ECNU), I am dedicated to developing advanced genome editing technologies for disease modeling and precise gene therapy. My foundational training at Hunan Normal University and Texas A&amp;M University cultivated a deep interest in using engineered cellular and animal models to understand human diseases. Since 2013, my laboratory at ECNU has pioneered the use of TALEN and CRISPR/Cas9 for the rapid generation of knockout mouse and rat models for disease studies. Once stepped in genome editing field, I shifted my focus to advancing editing tools and developing gene therapy strategies for genetic disorders and cancer. My team has developed a suite of high-performance base editors for nuclear DNA, mitochondrial DNA, and RNA, broadening editing capabilities while enhancing precision and safety. Leveraging these technologies, we have designed several therapeutic strategies that have shown efficacy in cellular and animal models of genetic diseases. Through collaborative efforts, we have successfully translated genome editing into clinical applications, contributing to the treatment of patients with β-thalassemia. Additionally, we have developed a non-viral, site-specific CAR-T platform for lymphoma therapy. Looking forward, I aim to develop the next generation of long-fragment, site-specific integration technologies and accelerate clinical translation to bring transformative cures to more patients.},
}
@article {pmid41485979,
year = {2026},
author = {Shimizu, T and Okamoto, M and Kawamoto, K},
title = {Evaluation of a d-octaarginine-linked polymer for CRISPR-Cas9 ribonucleoprotein (RNP) delivery and genome editing in murine dendritic cells.},
journal = {The Journal of veterinary medical science},
volume = {88},
number = {3},
pages = {374-383},
pmid = {41485979},
issn = {1347-7439},
mesh = {Animals ; *Dendritic Cells/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Mice ; *Ribonucleoproteins/genetics ; *Oligopeptides/chemistry ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Transfection ; *Polymers/chemistry ; },
abstract = {We previously reported that poly (N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) efficiently introduces proteins and nucleic acids into cells. Based on these results, we hypothesized that VP-R8 can introduce a complex composed of guide RNA and Cas9 (RNP complex) into cells and induce genome editing mediated by the CRISPR-Cas9 system. We tested this hypothesis using a mouse dendritic cell line and mouse primary dendritic cells. The RNP complexes formed by guide RNA consisting of CRISPR RNA (crRNA), fluorescently labeled trans-activating crRNA (tracrRNA), and GFP-fused Cas9 were introduced into a mouse dendritic cell line using VP-R8 or control transfection reagents. Cells transfected using VP-R8 exhibited higher fluorescence than those transfected with other transfection reagents, indicating that VP-R8 efficiently introduced the RNP complex into the mouse dendritic cell line. Genome editing of the target DNA was detected in cells transfected with the RNP complex using VP-R8 and dominant relative to other transfection reagents. We also observed that VP-R8 effectively delivered RNP complexes consisting of single-guide RNA and Cas9 and induced genome editing in the dendritic cell line. Additionally, VP-R8 efficiently delivered RNP complexes into mouse primary dendritic cells and induced genome editing of the functional gene without producing early inflammatory cytokines. Thus, VP-R8 shows potential as a transfection tool to generate dendritic cells with specific gene regions deleted by genome editing via the CRISPR-Cas9 system. This approach aims to elucidate the detailed molecular mechanisms of dendritic cell function for its application to vaccines.},
}
@article {pmid41486484,
year = {2025},
author = {Boogari, M and Mohebbi, M and Hadidi, N},
title = {Genetically Engineered Probiotics: Design, Therapeutics, and Clinical Translation.},
journal = {Iranian biomedical journal},
volume = {29},
number = {6},
pages = {374-383},
doi = {10.61882/ibj.5197},
pmid = {41486484},
issn = {2008-823X},
mesh = {*Probiotics/therapeutic use ; Humans ; *Genetic Engineering/methods ; *Translational Research, Biomedical ; Animals ; },
abstract = {Genetically engineered probiotics (GEPs) aim to address transient colonization and the intra- and inter-subject variability that limit conventional probiotics. These strains utilize Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas editing, programmable gene circuits, and biosensors in chassis such as E. coli Nissle 1917 and L. lactis. This narrative review summarizes the current engineering toolkits and standards (e.g., SEVA), chassis selection criteria, biocontainment strategies, and translational requirements under CMC/GMP frameworks and discusses regulatory considerations for clinical translation. Representative examples include IL-10-secreting Lactococcus lactis and phenylalanine-metabolizing strains for phenylketonuria (SYNB1618/SYNB1934), which illustrate pharmacodynamic target engagement and short-term preclinical safety. We outline clinical advancements in predefined pharmacodynamics, durability of function, monitoring shedding and horizontal gene transfer, and genomic-microbiome-informed patient stratification. Systems modeling approaches (Genome-Scale Metabolic Model/ Agent-Based Model) are discussed as tools to guide rational design. GEPs offer programmable “sense-and-respond” therapeutics, with successful clinical adoption depending on durable efficacy, long-term safety, and clearly defined regulatory pathways.},
}
@article {pmid41486850,
year = {2026},
author = {Shi, M and Yu, P and Liu, L and Cheng, J and Shao, R and Sun, Y and Lv, J and Li, Y and Zheng, Z and Yu, J and Xu, B and Gan, L and Liang, Y and Zhang, Y and Fang, Y and Shen, W and Huang, J and Zhu, X and Hong, J and Ma, R and Wu, L and Cheng, Y and Zhao, C},
title = {Fluoropolymer-Mediated Delivery of a Dual TSHR/IGF1R-Targeting CRISPR-Cas9 System for Localized Therapy in Thyroid-Associated Ophthalmopathy.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {11},
pages = {e11078},
doi = {10.1002/adma.202511078},
pmid = {41486850},
issn = {1521-4095},
support = {DGF828030-3/005//Clinical Scientist Foundation of Fudan University/ ; pp25014//Clinical Scientist Foundation of Fudan University/ ; U25A20258//National Natural Science Foundation of China/ ; 82271126//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Receptor, IGF Type 1/genetics/metabolism ; *Graves Ophthalmopathy/therapy/genetics/pathology ; *CRISPR-Cas Systems/genetics ; Humans ; Mice ; *Receptors, Thyrotropin/genetics/metabolism ; Gene Editing ; *Polymers/chemistry ; Fibroblasts/metabolism ; Disease Models, Animal ; Adipocytes/metabolism ; Genetic Therapy ; Organoids/metabolism ; Antibodies, Monoclonal, Humanized ; },
abstract = {Thyroid-associated ophthalmopathy (TAO), a vision-threatening and disfiguring autoimmune orbital disorder, remains a therapeutic challenge due to the lack of therapies with orbital specificity, sustained efficacy, and minimal side effects. Herein, we present G4F7-CRISPR, a fluoropolymer-based CRISPR-Cas9 delivery platform engineered for localized and efficient disruption of thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor 1 receptor (IGF1R), two key mediators of TAO pathogenesis. G4F7-CRISPR achieved high insertion/deletion frequencies in primary orbital fibroblasts (Tshr: 37.2%; Igf1r: 42.8%) and mature adipocytes (Tshr: 22.4%; Igf1r: 24.3%), and maintained robust editing efficiency in orbital adipose tissue of TAO mouse models (Tshr: 30.7%; Igf1r: 32.4%). In both TAO mouse models and 3D human orbital organoids, dual-gene editing of Tshr and Igf1r via G4F7-CRISPR significantly suppressed orbital adipogenesis, inflammation, and fibrosis, demonstrating superior therapeutic efficacy over either single-gene approaches. Comprehensive off-target analyses in both TAO mouse models and orbital organoids revealed minimal off-target activity. Furthermore, G4F7-CRISPR exhibited excellent short- and long-term ocular and systemic safety in TAO mouse models. Notably, it outperformed teprotumumab-the FDA-approved therapy for TAO-in both therapeutic efficacy and safety, highlighting its potential clinical advantages. Collectively, these findings highlight the translational promise of G4F7-CRISPR as a safe, precise, and clinically viable gene therapy for TAO.},
}
@article {pmid41487293,
year = {2025},
author = {},
title = {Erratum: Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing [Corrigendum].},
journal = {Biologics : targets &amp; therapy},
volume = {19},
number = {},
pages = {745-746},
doi = {10.2147/BTT.S585961},
pmid = {41487293},
issn = {1177-5475},
abstract = {[This corrects the article DOI: 10.2147/BTT.S326422.].},
}
@article {pmid41488303,
year = {2025},
author = {Tsolakidou, PJ},
title = {CRISPR-Cas systems against carbapenem resistance: from proof-of-concept to clinical translation.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1725247},
pmid = {41488303},
issn = {1664-302X},
abstract = {Carbapenem-resistant Enterobacterales (CRE) pose a major global threat, driven by plasmid-borne carbapenemase genes such as bla KPC, bla NDM and bla OXA-48. CRISPR-Cas systems offer programmable strategies to selectively eliminate these resistance determinants. This mini-review summarizes recent advances in Cas9-based plasmid curing, RNA-targeting approaches such as Cas13a and Cas13d, and DNA-targeting Cas3-enhanced bacteriophage therapeutics that have entered early clinical evaluation. Particular attention is given to conjugative CRISPR-Cas9 plasmid systems, which enable targeted plasmid eradication without laboratory transformation and broaden the delivery toolbox beyond phage vectors. We further discuss major translational challenges, including delivery efficiency, phage host-range constraints, ecological risks of horizontal CRISPR dissemination, and off-target effects. Finally, we highlight emerging delivery platforms-outer membrane vesicles, lipid and polymeric nanoparticles, conjugative plasmids with containment circuits, and engineered live biotherapeutics-that may complement or overcome current limitations. Collectively, these developments illustrate the potential of CRISPR-based antimicrobials to augment traditional therapies through precise gene-level suppression of carbapenem resistance.},
}
@article {pmid41488985,
year = {2026},
author = {Kim, K and Lee, J and Lee, N and Cho, BK},
title = {CRISPR-Based Approaches to Engineer Nonmodel Bacteria for Bioproduction and Biotherapeutics.},
journal = {Biochemistry},
volume = {65},
number = {2},
pages = {166-194},
doi = {10.1021/acs.biochem.5c00613},
pmid = {41488985},
issn = {1520-4995},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Bacteria/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; Streptomyces/genetics/metabolism ; Humans ; },
abstract = {Microbial diversity encompasses vast genetic and functional capacities, with immense potential for biotechnological applications. Yet, most biotechnological advances have been confined to a narrow set of model organisms, leaving the broader repertoire of nonmodel microbes largely untapped due to species-specific barriers that hinder genetic manipulation. Over the past decade, the advent of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems has transformed microbial engineering by enabling precise, programmable, and scalable control of genomes and gene expression. Importantly, the relative independence of many CRISPR effectors from host cofactors has facilitated their use in microbes previously challenging to engineer, thus expanding opportunities to exploit their unique metabolic and biosynthetic traits. In this review, we summarize the major CRISPR-Cas toolkits and highlight recent innovations, with particular emphasis on translational applications in nonmodel organisms such as C1-gas-fixing acetogens, antibiotic-producing Streptomyces, and gut commensal Bacteroides. We emphasize three areas of emerging impact: engineering microbial cell factories for sustainable biomanufacturing, accelerating natural product discovery, and development of next-generation live biotherapeutics. Finally, we discuss current limitations and future opportunities, underscoring how the integration of genome editing, synthetic biology, and systems-level approaches is reshaping the landscape of microbial biotechnology.},
}
@article {pmid41489362,
year = {2026},
author = {Shin, J and Barrangou, R},
title = {Occurrence and applications of CRISPR-Cas systems in bifidobacteria.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {1},
pages = {e0170325},
pmid = {41489362},
issn = {1098-5336},
support = {internal grant//North Carolina Agricultural Foundation/ ; internal support//College of Agriculture and Life Sciences, North Carolina State University/ ; },
mesh = {*CRISPR-Cas Systems ; *Bifidobacterium/genetics ; *Gene Editing/methods ; Probiotics ; Genome, Bacterial ; Humans ; },
abstract = {Bifidobacterium is a key member of the human gut microbiota, and many strains are widely used as probiotics due to their health-promoting properties. Despite growing interest, genetic studies in Bifidobacterium have been relatively limited, primarily due to the lack of available genome editing tools. Recent advances in genomics and CRISPR-Cas systems provide opportunities for targeted genome modification in this genus. In this review, we provide an overview of the occurrence, diversity, and distribution of CRISPR-Cas systems across Bifidobacterium species and examine the editing tools developed and implemented to date. We also highlight practical challenges such as strain variability and low transformation efficiency and introduce future avenues of research such as large-payload insertion and in situ editing. Expanding the genetic toolbox for Bifidobacterium will broaden our understanding of this important genus and enable the development of next-generation probiotics.},
}
@article {pmid41489552,
year = {2026},
author = {Fan, R and Tong, Y and Luo, S and He, Y and Yang, C and Li, W and Liu, J and Pan, J and Zhu, Y and Zhang, X and Zhu, J and Zhu, Y and Guo, Y and Li, L and Situ, B and Yan, X and Ma, W and Chang, L and Zhang, Y},
title = {Integrated Electroporated-Lysis Electrochemical Platform Enables Sensitive and Rapid EV Protein and miRNA Profiling Based on Multiplex-Responsive CRISPR/Cas12a.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {13},
pages = {e13331},
doi = {10.1002/smll.202513331},
pmid = {41489552},
issn = {1613-6829},
support = {2024YFC2419100//National Key R&amp;D Program of China/ ; 82272424//National Natural Science Foundation of China/ ; 82572682//National Natural Science Foundation of China/ ; 82372343//National Natural Science Foundation of China/ ; 82402753//National Natural Science Foundation of China/ ; 22304006//National Natural Science Foundation of China/ ; 62471021//National Natural Science Foundation of China/ ; 2023A1515010909//Guang Dong Basic and Applied Basic Research Foundation of China/ ; JCYJ20230807142204008//Shenzhen Science and Technology Innovation Committee/ ; 2024J004//Southern Medical University/ ; 2024M750606//China Postdoctoral Science Foundation/ ; 2025T180607//China Postdoctoral Science Foundation/ ; T2425021//National Science Fund for Distinguished Young Scholars/ ; },
mesh = {*MicroRNAs/metabolism/genetics ; Humans ; *Extracellular Vesicles/metabolism ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *Electroporation/methods ; },
abstract = {Proteins and miRNAs in extracellular vesicles (EVs) have emerged as crucial biomarkers for tumor diagnosis. While CRISPR/Cas12a-based platforms have shown great promise in nucleic acid and protein detection, their susceptibility to off-target activation and structural instability remains a significant limitation. Here, we have developed an electroporation-lysis electrochemical platform integrated with DNA cube-cage-locked CRISPR/Cas12a (DC-Cas12a), termed EL-DC-Cas12a. This platform utilizes an electric field to rapidly lyse EVs, releasing their internal proteins and miRNAs. These released molecules then activate the DC-Cas12a system, thereby triggering the displacement of two distinct crRNA/Cas12a complexes that correspond to EV proteins and miRNAs, respectively. These complexes then specifically recognize and cleave electrochemical probes, generating quantifiable electrochemical signals that enable synchronous and accurate analysis of the two biomarkers. The integrated workflow for EV lysis and detection can be completed within 40 min, greatly simplifying the overall operation. The detection limits (LOD) of this platform for EV PD-L1 protein and miR-1246 were 5.44 × 10[4] particles/mL and 3.59 × 10[3] particles/mL, respectively. Moreover, by applying machine learning algorithms to analyze the EV-associated proteins and miRNAs profiling, the platform demonstrated a diagnostic accuracy of 98.3% in distinguishing healthy donors from early-stage GC patients, and 99% in differentiating early-stage from advanced-stage GC patients in a clinical gastric cancer cohort. Therefore, the proposed platform offers a promising strategy for multiplexed detection of EV biomarkers and precise discrimination of GC.},
}
@article {pmid41490267,
year = {2026},
author = {Carturan, A and Angelos, MG and Guruprasad, P and Patel, RP and Pajarillo, R and Lee, A and Espie, D and Zhang, Y and Chiang, YH and Xie, W and Rodriguez, JL and Harris, J and Devi, P and Afolayan-Oloye, OI and Xu, J and Sussman, JH and Elghawy, O and Yang, A and Barsouk, A and Cho, JH and Shaw, CE and Singh, E and Ugwuanyi, O and Paruzzo, L and Stella, F and Liu, S and Nason, S and Imparato, A and Rotolo, A and Lemoine, J and Barrett, DM and Posey, A and Rook, AH and Pillai, V and Bagg, A and Pileri, SA and Liu, D and Tan, K and Schuster, SJ and Teachey, DT and Porazzi, P and Ruella, M},
title = {Harnessing the CD2 axis to broaden and enhance the efficacy of CAR T-cell therapies.},
journal = {Blood},
volume = {147},
number = {16},
pages = {1842-1856},
doi = {10.1182/blood.2025031409},
pmid = {41490267},
issn = {1528-0020},
support = {R01 FD008168/FD/FDA HHS/United States ; R37 CA262362/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *CD2 Antigens/immunology/genetics/metabolism ; *Receptors, Chimeric Antigen/immunology/genetics ; Animals ; Mice ; *T-Lymphocytes/immunology ; *Hematologic Neoplasms/therapy/immunology ; CD58 Antigens/immunology ; CRISPR-Cas Systems ; },
abstract = {Patients with T-cell lymphomas and leukemias have overall poor outcomes because of the lack of targeted and effective treatments, particularly in the relapsed and refractory settings. The development of chimeric antigen receptor (CAR) T cells against T-cell neoplasms is limited by a lack of discriminating T-cell antigens that allow for effective antitumor responses while preventing CAR T-cell fratricide. We hypothesized that targeting CD2, a pan-T-cell antigen, with anti-CD2 CAR T cells engineered to lack CD2 expression (CART2) would improve manufacturability and preclinical efficacy. Optimized CD2-knockout CART2 cells, generated using CRISPR-Cas9, eradicated primary patient-derived CD2+ hematological neoplasms in vitro and in vivo, secreted effector cytokines, and exhibited adequate proliferative capacity. Nevertheless, CD2 has a key costimulatory function, and its deletion could lead to CAR T-cell dysfunction. Therefore, we tested the role of the CD2:CD58 axis in CAR T cells, using the anti-CD19 CART models. We demonstrate that CD2 loss attenuates CART19 efficacy by reducing avidity for tumor antigen, costimulation, and ultimately in vivo activity. Analogously, we show that tumor CD58 loss reduces CART19 efficacy. To overcome this issue, we developed a novel programmed cell death protein 1 (PD-1):CD2 switch receptor that rescues intracellular CD2 signaling, particularly when programmed death-ligand 1 is engaged, thereby improving in vivo outcomes. Collectively, we studied the role of CD2 both as a target for CAR T-cell therapy and as a critical costimulatory protein, whose signaling can be rescued using the PD-1:CD2 switch receptor. This receptor can be incorporated into CAR T cells and provides an effective strategy to overcome CD2-signaling deficiencies.},
}
@article {pmid41491239,
year = {2026},
author = {Amelan, A and Collins, SC and Damseh, NS and Hamada, N and Salim, A and Dvir, E and Monderer-Rothkoff, G and Harel, T and Nagata, KI and Yalcin, B and Shifman, S},
title = {CRISPR knockout screens reveal genes and pathways essential for neuronal differentiation and implicate PEDS1 in neurodevelopment.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {592-603},
pmid = {41491239},
issn = {1546-1726},
support = {466/21//Israel Science Foundation (ISF)/ ; 1863/24//Israel Science Foundation (ISF)/ ; ANR-18-CE12-0009//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Mice ; *Cell Differentiation/genetics ; *Neurons/physiology ; *Neurogenesis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *Neurodevelopmental Disorders/genetics ; Gene Knockout Techniques/methods ; Mice, Knockout ; CRISPR-Cas Systems ; Male ; },
abstract = {Neurodevelopmental disorders (NDDs) arise from disruptions in brain development, yet the underlying pathways remain incompletely understood. Here we demonstrate that genome-wide CRISPR knockout screens in mouse embryonic stem cells differentiating into neural lineages identify hundreds of essential genes, only a minority of which are currently implicated in NDDs. Dominant NDD genes were enriched for transcriptional regulators, whereas recessive NDD genes were predominantly involved in metabolic processes. Mouse models for eight genes (Eml1, Dusp26, Dynlrb2, Mta3, Peds1, Sgms1, Slitrk4 and Vamp3) revealed marked neuroanatomical abnormalities, including microcephaly in half of the cases. Focusing on PEDS1, a key enzyme in plasmalogen biosynthesis, we identified a bi-allelic variant in individuals with microcephaly, global developmental delay and congenital cataracts. In mice, Peds1 deficiency led to accelerated cell-cycle exit and impaired neuronal differentiation and migration. These pathways required for neural differentiation provide a genetic framework for discovering additional NDD genes.},
}
@article {pmid41491310,
year = {2026},
author = {Arena, KA and Kearns, CA and Ahmed, M and O'Rourke, R and Sagerström, CG and Franco, SJ and Appel, B},
title = {Gsx2 regulates oligodendrocyte precursor formation in the zebrafish spinal cord.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {30-44},
pmid = {41491310},
issn = {1095-564X},
support = {R01 NS124166/NS/NINDS NIH HHS/United States ; R35 NS122191/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Spinal Cord/embryology/cytology/metabolism ; *Zebrafish Proteins/metabolism/genetics ; *Oligodendroglia/metabolism/cytology ; Cell Differentiation/genetics ; Gene Expression Regulation, Developmental ; *Oligodendrocyte Precursor Cells/metabolism/cytology ; Neural Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Transcription Factors/metabolism/genetics ; *Homeodomain Proteins/genetics/metabolism ; Gene Regulatory Networks ; Cell Lineage ; },
abstract = {Nervous system development relies on sequential and coordinated formation of diverse neurons and glia from neural progenitor cells (NPCs). In the spinal cord, NPCs of the pMN domain produce neurons early in development followed by oligodendrocyte precursor cells (OPCs), which subsequently differentiate as oligodendrocytes (OLs), the myelinating glia of the central nervous system. The mechanisms that specify neural progenitor cells to the OL lineage are not yet well understood. Using zebrafish as an experimental model system, we generated single-cell RNA sequencing and single-nuclei ATAC sequencing data that identified a subpopulation of NPCs, called pre-OPCs, that appeared fated to produce OPCs. pre-OPCs uniquely express several genes that encode transcription factors specific to the OL lineage, including Gsx2, which regulates OPC formation in the mouse forebrain. To investigate Gsx2 function in zebrafish OPC specification, we used CRISPR/Cas9 genome editing to create gsx2 loss-of-function alleles. gsx2 homozygous mutant embryos initiated OPC formation prematurely and produced excess OPCs without altering OL differentiation. Using our single-nuclei multi-omics dataset, we predicted a gene regulatory network centered around gsx2 and identified genes that might be transcriptionally regulated by Gsx2. Taken together, our studies suggest that Gsx2 expression in pre-OPCs contributes to the timing of OPC specification.},
}
@article {pmid41492065,
year = {2026},
author = {Wang, Z and Wang, Y and Gao, H and Dai, J and Tang, N and Wang, Y and Ji, Q},
title = {Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA.},
journal = {Nature microbiology},
volume = {11},
number = {1},
pages = {81-93},
pmid = {41492065},
issn = {2058-5276},
mesh = {*Thioredoxins/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacteriophages/genetics/enzymology/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; Protein Binding ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; DNA Cleavage ; DNA/metabolism ; Escherichia coli/genetics/virology/metabolism ; },
abstract = {The evolutionary competition within phage-host systems led to the emergence of CRISPR-Cas defence mechanisms in bacteria and anti-CRISPR elements in bacteriophages. Although anti-CRISPR elements are well characterized, the role of bacterial factors that influence CRISPR-Cas efficacy has been comparatively overlooked. Type V CRISPR-Cas12 systems display striking functional and mechanistic diversity for nucleic acid targeting. Here we use a bioinformatic approach to identify Cas12p, a phage-associated nuclease that forms complexes with the bacterial thioredoxin protein TrxA to enable target DNA degradation. This represents an unexpected phage-bacteria interaction, in which the bacteriophage co-opts a bacterial factor to augment its own genome degradation machinery, potentially against competing phages. Biochemical characterization, cryo-EM-based structural analysis of the Cas12p-TrxA-sgRNA-dsDNA complex at 2.67 Å and bacterial defence assays reveal that TrxA directly binds and activates Cas12p, enabling its nuclease activity and subsequent CRISPR immunity. These findings expand our understanding of the multilayered intricacies of phage-bacteria molecular interactions.},
}
@article {pmid41494531,
year = {2026},
author = {Wan, W and Ji, X and Song, H and Zhang, Z and Kwok, CK and Fang, X and Li, X},
title = {Fluorogenic CRISPR for DNA imaging in live mammalian cells.},
journal = {Cell chemical biology},
volume = {33},
number = {1},
pages = {33-44},
doi = {10.1016/j.chembiol.2025.10.013},
pmid = {41494531},
issn = {2451-9448},
mesh = {Humans ; *DNA/genetics/analysis/metabolism ; *Fluorescent Dyes/chemistry ; Animals ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Optical Imaging ; },
abstract = {Spatiotemporal imaging of genomic DNA dynamics in live mammalian cells is essential for elucidating eukaryotic organization and processes relevant to health and disease. CRISPR systems greatly facilitate the development of live cell DNA imaging tools. However, conventional CRISPR imaging tools typically utilize constitutively fluorescent proteins, resulting in high background noise, nonspecific nucleolar signals, and low signal-to-noise ratios. To address this, fluorogenic CRISPR-based imaging tools have been developed. These tools remain non-fluorescent until they bind to the target DNA, thus significantly reducing the background and enhancing the sensitivity. This review summarizes four fluorogenic CRISPR strategies, each utilizing different fluorogenic reporters, including fluorogenic proteins, fluorogenic RNA aptamers, split fluorescent proteins, and molecular beacons. These fluorogenic CRISPR approaches successfully monitored the subnucleus gene loci localization, dynamics, and DNA breaks and repairs. We anticipate that this review can inspire researchers to expand the fluorogenic CRISPR for cellular DNA imaging and diverse bioapplications.},
}
@article {pmid41494573,
year = {2026},
author = {Pu, Z and Wang, X and Chen, Y and Li, J and He, X and Chen, W and Zhao, C},
title = {Application of CRISPR/Cas9 gene editing system in microalgal metabolic engineering and synthetic strategies of functional food ingredients.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108796},
doi = {10.1016/j.biotechadv.2026.108796},
pmid = {41494573},
issn = {1873-1899},
mesh = {*Microalgae/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Metabolic Engineering/methods ; *Functional Food ; *Food Ingredients ; },
abstract = {Microalgae are natural and sustainable biological resources rich in high-value nutrients such as lipids, proteins, and functional pigments, which show great potential in the fields of functional foods, dietary supplements, and natural colorants. However, the yields of target components in natural microalgae are often insufficient to meet commercialization demands. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing system, a revolutionary technology, provides a precise and effective means for targeted improvement of microalgae to enhance their nutritional value and yields. This review first outlines the basic principles of the CRISPR/Cas9 system, including its core components and gene editing mechanism. It then summarizes the application of this technology in microalgae, focusing on successful cases of modifying metabolic pathways to enrich specific nutrients, such as increasing the unsaturated fatty acid content of lipids, increasing the proportion of edible proteins, and enriching natural pigments with antioxidant properties. In addition, this review discusses the main challenges faced when applying this technology to microalgae, including delivery difficulties due to strong cell walls, low efficiency of genetic transformation, and the risk of off-target effects. Finally, the paper describes cutting-edge strategies to address these challenges, such as the development of high-fidelity Cas9 enzymes and the optimization of a single-guide RNA (sgRNA) design. Continued advances in these technologies are propelling microalgae into efficient and sustainable "cell factories", providing the food industry with more natural, healthy, and high-value functional ingredients.},
}
@article {pmid41494604,
year = {2026},
author = {Zeng, Y and Zhao, G and Wu, S and Hu, B and Forn-Cuní, G and Knol, R and El Ghalbzouri, A and Snaar-Jagalska, E and Kros, A},
title = {CD44-targeted lipid nanoparticles for enhanced CRISPR/Cas9 delivery in cancer gene editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114598},
doi = {10.1016/j.jconrel.2025.114598},
pmid = {41494604},
issn = {1873-4995},
mesh = {*Nanoparticles/administration &amp; dosage/chemistry ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Hyaluronan Receptors/metabolism/genetics ; Humans ; *Lipids/chemistry/administration &amp; dosage ; Cell Line, Tumor ; *Melanoma/genetics/therapy/pathology ; Polo-Like Kinase 1 ; *Skin Neoplasms/genetics/therapy/pathology ; Protein Serine-Threonine Kinases/genetics ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/genetics ; Mice ; Cell Cycle Proteins/genetics ; Peptides/chemistry ; Gene Transfer Techniques ; Female ; Liposomes ; },
abstract = {Skin cancer is the third most common malignancy, with melanoma being the most challenging due to its resistance to current therapies. Gene editing technologies like CRISPR/Cas9 offer a promising strategy for targeting cancer-specific genes, but the efficient delivery of these tools to tumor sites remains a significant challenge. Lipid nanoparticles (LNPs) have emerged as the leading platform for gene editing tools due to their ability to protect and transport large payloads. To enhance the precision of gene editing in melanoma, we developed CD44-specific peptide-modified LNPs for targeted delivery of CRISPR/Cas9 mRNA and guide RNA against polo-like kinase 1 (sgPLK1). Our approach led to enhanced targeting and gene editing efficacy by specifically delivering CRISPR/Cas9 and sgPLK1 to melanoma tumor cells, resulting in significant inhibition of tumor growth in both in vitro and in vivo skin melanoma models. Moreover, this platform showed the capacity to reach metastatic melanoma in the brain and resulting in substantial suppression of tumor growth in brain metastasis models. We envision that this peptide-modification strategy could be further employed to improve the targeting capabilities and therapeutic outcomes of LNPs for CRISPR/Cas9-based gene editing, paving the way for more precise and effective cancer treatments.},
}
@article {pmid41494883,
year = {2026},
author = {Mahmood, MA and Greenwood, JR and Millar, AA and Susila, H},
title = {Next-generation genome editing: no transgene, no tissue culture.},
journal = {Trends in plant science},
volume = {31},
number = {4},
pages = {383-385},
doi = {10.1016/j.tplants.2025.12.008},
pmid = {41494883},
issn = {1878-4372},
mesh = {*Transgenes/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; *Gene Editing/methods ; *Genetic Engineering/methods ; Tissue Culture Techniques ; CRISPR-Cas Systems ; },
abstract = {New approaches to engineering plant genomes have the potential to improve agriculture. However, transgenes insertion and tissue culture have become bottlenecks to genome-editing technology becoming widely adopted and achieving the promise of targeted editing. Recent developments in particle bombardment and viral vector-mediated delivery can open doors to overcome these limitations.},
}
@article {pmid41494892,
year = {2026},
author = {Wang, MR and Mu, W and Zhen, A and Kitchen, SG},
title = {CRISPR/Cas strategies to enhance CAR T-cell function and persistence via metabolic reprogramming.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.12.001},
pmid = {41494892},
issn = {1879-3096},
abstract = {While chimeric antigen receptor (CAR) T-cell therapy has become a standard of care in various blood cancers, its full curative potential for other diseases has yet to be maximized. One key limiting factor is progressive T-cell exhaustion and differentiation over time, leading to the loss of the CAR-expressing cells. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) gene manipulation to enhance CAR T-cell therapy has revolutionized the field in recent years. In this review, we will examine the application of CRISPR/Cas aimed at improving CAR T-cell function and persistence to combat the issues of exhaustion and dysfunction, with a focus on metabolic reprogramming. Understanding current preclinical CRISPR/Cas strategies for modulating CAR T-cell metabolism is critical in advancing CAR-T therapies to clinical applications.},
}
@article {pmid41495049,
year = {2026},
author = {Li, X and Shang, X and Liu, J and Zhang, Y and Jia, X and Li, H and Wang, Y and Gao, J and Ma, X and Zhang, X and Rong, X and Gan, W and Zhang, Y and Chen, J and Wang, L and Bao, Z and He, L and Yan, X and Liu, Y and Shao, J and Xiao, Z and Wang, Z and Zhu, H and Wang, Z and Wu, Y and Huang, Y},
title = {Intrathecal CRISPR-edited allogeneic IL-13Rα2 CAR T Cells for recurrent high-grade Glioma: preclinical characterization and phase I trial.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1362},
pmid = {41495049},
issn = {2041-1723},
mesh = {Humans ; Female ; Male ; *Immunotherapy, Adoptive/methods/adverse effects ; *Interleukin-13 Receptor alpha2 Subunit/genetics/immunology ; Middle Aged ; Adult ; *Glioma/therapy/immunology/pathology ; CRISPR-Cas Systems/genetics ; *Receptors, Chimeric Antigen/genetics/immunology ; *T-Lymphocytes/immunology/transplantation/metabolism ; *Brain Neoplasms/therapy/immunology/pathology ; Gene Editing ; Neoplasm Recurrence, Local/therapy ; Animals ; Graft vs Host Disease/prevention &amp; control/immunology ; Glioblastoma/therapy/immunology ; Mice ; Killer Cells, Natural/immunology ; Aged ; Treatment Outcome ; Cell Line, Tumor ; },
abstract = {Patients with recurrent high-grade glioblastoma have a median survival of 6-8 months, with limited therapeutic options. In recent years, interest has grown in applying chimeric antigen receptor T (CAR-T) cells to solid cancers, including advanced gliomas. Here we generated off-the-shelf CRISPR-Cas9-edited IL-13Rα2-specific allogeneic universal CAR-T cells (MT026) by disrupting the endogenous TCR to prevent graft-versus-host disease and knocking out HLA class I molecules to mitigate the host-versus-graft response, and observed minimal NK-cell-mediated rejection in preclinical studies. In a first-in-human, single-center, open-label investigator-initiated trial (ChiCTR2000028801) in patients with high-grade glioma with prior therapy failure and short life expectancy, intrathecal injection of MT026 via lumbar puncture (1.0-3.0×10^7 cells per dose) demonstrated favorable tolerability and safety (primary outcome), pharmacokinetic characteristics, and preliminary clinical activity (secondary outcomes). Among the five patients enrolled, one achieved a complete response and three achieved partial responses. No grade ≥3 adverse events were observed; the predominant treatment-related toxicities were grade 1-2 pyrexia, hypoxia, and vomiting. Trial enrolment was halted after enrolment of the first five patients, however these preliminary clinical data support the potential benefit of locally administered allogeneic universal CAR-T cell therapy for recurrent glioblastoma.},
}
@article {pmid41495894,
year = {2026},
author = {Ishihara, K and Matsumoto, S and Gerle, C and Gopalasingam, CC and Shigematsu, H and Shirai, T and Numata, T},
title = {Sequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495894},
issn = {1362-4962},
support = {20H02916//Japan Society for the Promotion of Science/ ; 24H00505//Japan Society for the Promotion of Science/ ; //Japan Foundation for Applied Enzymology/ ; //Naito Foundation/ ; //Institute for Fermentation, Osaka/ ; //Noda Institute for Scientific Research/ ; 23KJ1734//JSPS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *DNA Transposable Elements/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; Vibrio parahaemolyticus/genetics ; DNA/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; Models, Molecular ; },
abstract = {Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.},
}
@article {pmid41495909,
year = {2026},
author = {Derollez, E and Roson-Calero, N and Rouzé, P and Dedieu-Berne, A and Ballesté-Delpierre, C and Fraikin, N and Iorga, BI and Huang, TD and Bigot, S and Vila, J and Bogaerts, P and Lesterlin, C},
title = {Specific killing and resensitization of pathogenic Escherichia coli strains carrying blaCTX-M-15 β-lactamase using targeted-antibacterial-plasmids (TAPs).},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495909},
issn = {1362-4962},
support = {//Joint Programming Initiative on Antimicrobial Resistance/ ; JPIAMR2021-194//JPIAMR/ ; FRM-EQU202103012587//Foundation for Medical Research/ ; ANR-20-PAMR-0010//French Priority Research Program/ ; //Agence Nationale de la Recherches/ ; },
mesh = {*beta-Lactamases/genetics/metabolism ; *Escherichia coli/genetics/drug effects/enzymology ; *Plasmids/genetics ; CRISPR-Cas Systems ; Humans ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; Drug Resistance, Multiple, Bacterial/genetics ; Escherichia coli Infections/microbiology ; },
abstract = {Targeted-Antibacterial-Plasmids (TAPs) offer a precise approach to combat multidrug-resistant bacteria by selectively removing resistant strains while preserving commensals. Here, we assess TAPs that deliver CRISPR/Cas systems via conjugation to kill or resensitize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli carrying the blaCTX-M-15 gene. We systematically tested multiple variables in the TAP approach, including two commensal E. coli donors, three distinct helper plasmids encoding the transfer machineries, and six recipient strains harbouring the resistance gene either chromosomally or on a plasmid. Cas9-based TAPs induced double-stranded breaks in chromosomal blaCTX-M-15 genes, resulting in immediate bacterial death. When the target gene was plasmid-borne, Cas9 cleavage triggered plasmid loss and partial toxin-antitoxin-mediated killing. In contrast, dCas9-based TAPs inhibited blaCTX-M-15 expression without affecting cell viability, thereby restoring third-generation cephalosporin susceptibility. In mixed-culture experiments, TAPs specifically eliminated only blaCTX-M-15-carrying E. coli while sparing other non-targeted bacterial species. Conjugation assays in human faeces demonstrated substantial suppression of cefotaxime-resistant (CtxR) E. coli by both Cas9- and dCas9-based TAPs, underscoring their efficacy in complex microbial environments. These findings highlight TAPs' decolonization promise, paving the way for future microbiome-editing interventions against multidrug-resistant carriage or infection.},
}
@article {pmid41496278,
year = {2026},
author = {Bozaoglu, K and Massie, S and Irion, FE and Davies, KC and Kantor, I and Raabus, M and Haebich, KM and Vlahos, K and Howden, SE and Wright, J and Payne, JM and Lockhart, PJ},
title = {Simultaneous reprogramming and gene correction to generate six iPSC lines and isogenic controls from individuals with neurofibromatosis type 1.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103904},
doi = {10.1016/j.scr.2025.103904},
pmid = {41496278},
issn = {1876-7753},
mesh = {Humans ; *Neurofibromatosis 1/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cellular Reprogramming ; *Gene Editing ; CRISPR-Cas Systems ; Cell Line ; Male ; Female ; },
abstract = {Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder that affects ∼ 1: 2700 individuals (Lee et al., 2023) however the underlying pathogenic mechanisms are poorly understood. In this study, we performed simultaneous reprogramming and CRISPR-Cas9 genome editing to generate pluripotent stem cell (iPSCs) lines and their respective isogenic controls from six individuals with different pathogenic NF1 variants. All iPSC lines had a normal karyotype, were pluripotent and able to differentiate into the three embryonic germ layers. These iPSC lines are valuable pre-clinical models to investigate the pathomechanisms of NF1 and can be used for future screening to identify new therapeutic treatments for NF1.},
}
@article {pmid41496283,
year = {2026},
author = {Low, YC and McKnight, CL and Stojanovski, D and Thorburn, DR and Frazier, AE},
title = {Generation of a pluripotent human AGK knockout embryonic stem cell model (WAe009-A-3C) of Sengers syndrome.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103895},
doi = {10.1016/j.scr.2025.103895},
pmid = {41496283},
issn = {1876-7753},
mesh = {Humans ; *Human Embryonic Stem Cells/metabolism/cytology ; *Phosphotransferases (Alcohol Group Acceptor)/genetics/metabolism/deficiency ; Cell Line ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism ; Gene Knockout Techniques ; *Mitochondrial Diseases/genetics/pathology ; Gene Editing ; Cell Differentiation ; },
abstract = {Sengers syndrome is a rare mitochondrial disorder caused by the loss of a nuclear encoded mitochondrial protein, acylglycerol kinase (AGK). Here, we describe the generation of a novel in vitro stem cell model of Sengers syndrome (AGK[KO] C10) using CRISPR/Cas9 gene editing. This cell line displayed normal characteristics of pluripotent stem cells, including colony morphology, expression of pluripotency markers, trilineage potential, and no karyotypic abnormalities. Together with the parental H9 hESC control line, the AGK[KO] C10 line can ultimately be used for investigation of disease mechanisms and drug testing.},
}
@article {pmid41496284,
year = {2026},
author = {Fan, M and Zhao, M and Su, W and Tang, Z and Sun, W and Zhou, T and Liu, P},
title = {Generation of a PLIN2-GFP2-P2A-Puro human induced pluripotent stem cell line (SEUi001-A) via CRISPR/Cas9-mediated gene editing technology.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103896},
doi = {10.1016/j.scr.2025.103896},
pmid = {41496284},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Perilipin-2/genetics/metabolism ; Cell Line ; Green Fluorescent Proteins/metabolism/genetics ; Cell Differentiation ; },
abstract = {Perilipin 2 (PLIN2) dysregulation drives metabolic pathologies including non-alcoholic fatty liver disease (NAFLD). To enable real-time tracking of PLIN2 dynamics, we established a human induced pluripotent stem cell (hiPSC) line with endogenous GFP2 knock-in at the PLIN2 locus via CRISPR/Cas9-mediated non-homologous end joining (NHEJ). This PLIN2-GFP2 reporter line demonstrated synchronous fluorescence and transcriptional expression validated by flow cytometry. Genomic integrity was confirmed by normal diploid karyotype (46, XY). Pluripotency markers (POU5F1, SOX2, NANOG) were stably expressed. Furthermore, the cells possessed the ability to differentiate into three germ layers. As the first reported endogenous PLIN2 reporter in human stem cells, this model overcomes limitations of antibody-based detection and transgenic overexpression systems, preserving native regulatory mechanisms. The model provides a physiologically relevant platform for: (1) live monitoring of LD-mitochondria interactions, (2) high-throughput compound screening for metabolic disorders, and (3) modeling NAFLD pathogenesis in vitro, advancing precision therapeutics and mechanistic disease modeling.},
}
@article {pmid41496285,
year = {2026},
author = {Wang, J and Gao, Y and Jin, K and Jin, ZB and Xu, J},
title = {Generation of RB1 knockout human embryonic stem cell lines derived from H9 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103897},
doi = {10.1016/j.scr.2025.103897},
pmid = {41496285},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Gene Knockout Techniques ; *Retinoblastoma Binding Proteins/genetics/metabolism/deficiency ; Cell Line ; *Ubiquitin-Protein Ligases/genetics/deficiency/metabolism ; Cell Differentiation ; Animals ; Mice ; },
abstract = {RB1 is a tumor suppressor gene which plays a crucial role in cell cycle and cellular differentiation. Mutations or loss of RB1 are associated with retinoblastoma and a variety of other cancers. We generated RB1 knockout human embryonic stem cell (hESC) lines (H9) using CRISPR/Cas9-mediated gene targeting. These RB1-deficient cell lines maintain typical stem cell morphology, normal karyotype and expression of pluripotent marker genes. Furthermore, they retain their in vivo differentiation capacity, enabling the generation of multiple cell lineages. These RB1 knockout hESC lines provide valuable models for investigating the role of RB1 in tumorigenesis, neurodevelopment, and cell cycle regulation.},
}
@article {pmid41496287,
year = {2026},
author = {Chatrousse, L and Poullion, T and Mahiou, H and El-Kassar, L and Giraud-Triboult, K and Boissart, C and Benchoua, A},
title = {Knock-out of specific DMD gene isoforms in the parental hESC line SA001 using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103899},
doi = {10.1016/j.scr.2025.103899},
pmid = {41496287},
issn = {1876-7753},
mesh = {Humans ; *Dystrophin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Protein Isoforms/genetics/metabolism ; Cell Line ; Male ; Muscular Dystrophy, Duchenne/genetics ; *Gene Knockout Techniques ; },
abstract = {The DMD gene, which encodes the protein dystrophin, is involved in a group of diseases known as dystrophinopathies, which includes Duchenne Muscular Dystrophy (DMD). DMD is a progressive and lethal muscular disorder mainly affecting boys that results from the loss of function of the longer dystrophin isoform DP427 in skeletal muscles. Dystrophinopathies are also associated with poorly understood neurocognitive and neurodevelopmental disorders. To investigate the role of dystrophin isoforms in neural development, we specifically disrupted three dystrophin isoforms expressed in the brain, namely DP427, DP140 and DP71, in the male human embryonic stem cell line SA001 using the CRISPR/Cas9 system. (100 / 100 words).},
}
@article {pmid41496452,
year = {2026},
author = {Liu, J and Liu, Z and Qin, D and Qi, X and Chen, M and Cheng, D and Guo, S and Zhang, X and Zhang, L and Yan, T and Li, F and He, W and Lu, Z and Wang, H and Li, J and Yang, X and Shi, Y and Zhang, S and Zhao, H and Liu, C and Chen, S and Zhong, Y},
title = {An all-in-one visual selection system for male-sterile line production in maize and rice.},
journal = {Plant communications},
volume = {7},
number = {2},
pages = {101693},
pmid = {41496452},
issn = {2590-3462},
mesh = {*Zea mays/genetics/physiology ; *Oryza/genetics/physiology ; *Plant Infertility/genetics ; Seeds/genetics/growth &amp; development ; *Plant Breeding/methods ; CRISPR-Cas Systems ; Hybrid Vigor/genetics ; Plants, Genetically Modified ; },
abstract = {Heterosis has significantly improved crop yields, yet hybrid seed production remains hindered by labor-intensive manual emasculation. Although current male-sterility systems, such as cytoplasmic male sterility and environment-sensitive genic male sterility, have improved the efficiency of hybrid seed production, their limited genetic adaptability and high environmental dependence remain major challenges. Here, we report an all-in-one seed production technology (ASPT) that integrates CRISPR-Cas9, RUBY, and key seed production technology (SPT) components into a single vector, enabling efficient generation and propagation of male-sterile lines in both maize and rice. The engineered RUBY marker enables visual identification of male-sterile and maintainer lines, with an accuracy of 99.81% in automated seed sorting and 100% in secondary field screening. Notably, ASPT was successfully introduced into 21 genetically diverse elite maize inbred lines, demonstrating broad compatibility. ASPT enables scalable and precise propagation of male-sterile lines in both maize and rice, providing a broadly applicable strategy to advance hybrid seed production in crops.},
}
@article {pmid41496629,
year = {2026},
author = {Li, J and Wang, L and Yang, S and Zhou, X and Gou, Q and Cai, J and Yang, H and Wang, Q and Li, S},
title = {Chicken Shank Color Determined by Inhibition of Dermal Melanin (ID) is Mediated by a Structural Variation Regulating CDKN2A Expression.},
journal = {Pigment cell &amp; melanoma research},
volume = {39},
number = {1},
pages = {e70072},
doi = {10.1111/pcmr.70072},
pmid = {41496629},
issn = {1755-148X},
support = {2022hszd006//Major Project of Hubei Hongshan Laboratory/ ; 31772585//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Chickens/genetics ; *Melanins/metabolism ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; *Skin Pigmentation/genetics ; Melanocytes/metabolism ; *Gene Expression Regulation ; Polymorphism, Single Nucleotide/genetics ; Female ; Male ; CRISPR-Cas Systems/genetics ; Genome-Wide Association Study ; *Skin/metabolism ; },
abstract = {Shank color in chickens is a classic quantitative trait governed by four genetic loci. Among these, the Inhibition of dermal melanin (ID) locus, which suppresses dermal melanogenesis in the shank, is the sole sex-linked mutation and its molecular mechanisms remain elusive. To identify the causal mutation, we established a resource population segregating for shank colors. A genome-wide association study utilizing FarmCPU software identified a top-associated SNP on the Z chromosome. Linkage mapping subsequently narrowed the candidate region, within which we discovered a candidate structural variant associated with the yellow shank phenotype. This variant is characterized by a 143 bp deletion coupled with a 2 bp insertion. CDKN2A was the only gene within the same topologically associating domain to exhibit differential expression. Functional validation via CRISPR/Cas9-edited cells demonstrated that this mutation regulates CDKN2A transcription and is responsible for the ID shank color in chickens. We propose that the resulting absence of melanocytes is likely due to apoptosis. This work resolves the molecular basis of the ID locus, thereby completing the genetic puzzle of chicken shank color. This discovery enables the development of molecular markers for auto-sexing of day-old chicks, a tool with significant potential for the poultry industry.},
}
@article {pmid41496894,
year = {2026},
author = {Kantor, B and Duke, L and Bhide, PG},
title = {CRISPR-Cas editing technologies for viral-mediated gene therapies of human diseases: Mechanisms, progress, and challenges.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102786},
pmid = {41496894},
issn = {2162-2531},
abstract = {The gene therapy landscape has evolved substantially in recent years, beginning with the approval of the first adeno-associated virus-based gene therapy, Luxterna, in 2017. Since then, the US FDA has approved nearly 30 new viral gene therapy programs, with notable examples including Zolgensma, Spinraza, Hemgenix, Zynteglo, Lyfgenia, Kymriah, Skysona, and Tecelra. Remarkably, all these products rely on delivery via adeno-associated vectors (AAVs) and lentiviral vectors (LVs). Improvements in viral-mediated gene transfer efficiency and clinical-scale manufacturing, together with immense commercial interest, have greatly propelled the clinical adoption of gene therapy products. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and its related Cas proteins (CRISPR-Cas) have made significant advances in gene therapy, offering next-generation approaches for curative gene editing to treat genetic diseases and disorders. In this review, we examine the range of these therapeutics and their viral carriers, focusing primarily on LVs and AAVs. We provide a snapshot of the current status of the field and highlight some of the current challenges in the clinical application of gene therapy, with particular emphasis on viral CRISPR-Cas-based technologies and their future potential.},
}
@article {pmid41496934,
year = {2026},
author = {Khizar, M and Aminpoor, H and Zaib, M and Ali, Q and Karimi, H},
title = {Nanoparticle-enhanced CRISPR delivery: paving the path for in vivo tumor gene editing.},
journal = {Annals of medicine and surgery (2012)},
volume = {88},
number = {1},
pages = {1054-1055},
pmid = {41496934},
issn = {2049-0801},
abstract = {Nanoparticle-based delivery systems are redefining how CRISPR/Cas technology can be used in cancer treatment. By encapsulating CRISPR components within lipid, polymeric, or inorganic nanoparticles, researchers have improved their stability, circulation time, and tumor-targeting precision. The NTLA-2001 trial demonstrated the first successful use of lipid nanoparticles for in vivo CRISPR delivery in humans, paving the way for potential applications in oncology. Preclinical studies have shown promising results, with efficient gene knockout and tumor suppression across multiple models. Despite these advances, barriers remain, including limited delivery to solid tumors, potential off-target effects, and inconsistent nanoparticle formulations. Global research efforts spanning the United States, China, Europe, and India are now focused on refining delivery platforms and standardizing protocols. This letter highlights current progress, ongoing challenges, and the need for transparent, globally coordinated development. Nanoparticle-enhanced CRISPR delivery has the potential to bring genetic precision therapy from the laboratory to the clinic, offering a new avenue for durable and accessible cancer care.},
}
@article {pmid41497644,
year = {2025},
author = {Stringer, AM and Wade, JT},
title = {Identification of PAM Requirements for the Vibrio cholerae type I-E CRISPR-Cas System.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41497644},
issn = {2692-8205},
support = {R01 GM122836/GM/NIGMS NIH HHS/United States ; R35 GM144328/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that use RNA-guided protein complexes to target invading nucleic acid. A surveillance complex consisting of protein and a CRISPR-RNA (crRNA) binds target nucleic acid via base-pairing interactions, typically leading to processing of the target nucleic acid by a nuclease. CRISPR-Cas systems are classified based on their mechanism of action, with type I systems being the most prevalent in nature. Type I CRISPR-Cas systems target DNA, and require extensive complementarity between the crRNA and the target DNA. Moreover, type I systems require the presence of a "Protospacer Adjacent Motif" (PAM) sequence in the target DNA immediately adjacent to the expected region of base-pairing with the crRNA. Classical biotypes of the bacterial pathogen Vibrio cholerae have active type I-E CRISPR-Cas systems. While the optimal PAM sequence for this CRISPR-Cas system is known to be AAY, the activity of other sequences as possible PAMs has not been determined. Here, we quantify the effectiveness of all possible trinucleotide sequences in the PAM position for the V. cholerae type I-E CRISPR-Cas system. Our data indicate a hierarchy of PAM efficacy, with 15 of the 64 trinucleotide sequences functioning as a PAM.},
}
@article {pmid41497669,
year = {2025},
author = {Kumbara, A and Tognon, M and Carone, G and Fontanesi, A and Bombieri, N and Giugno, R and Pinello, L},
title = {CRISPR-HAWK: Haplotype- and Variant-aware guide design toolkit for CRISPR-Cas.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41497669},
issn = {2692-8205},
support = {R01 HG013618/HG/NHGRI NIH HHS/United States ; UM1 HG012010/HG/NHGRI NIH HHS/United States ; },
abstract = {MOTIVATION: Current CRISPR guide RNA design tools rely on reference genomes, overlooking how genetic variation impacts editing outcomes. As genome editing advances toward clinical applications, incorporating population diversity becomes essential for ensuring therapeutic efficacy across diverse populations.<br><br>RESULTS: We present CRISPR-HAWK, a framework integrating individual- and population-scale variants and haplotypes into gRNA design. Analyzing therapeutic targets across 79,648 genomes reveals that genetic variants substantially alter guide performance. For the clinically approved sickle cell disease therapeutic guide targeting BCL11A, we identify haplotypes that completely abolish predicted cutting activity. Across seven therapeutic loci, 82.5% of guides contain variants modifying on-target activity. Variants also create novel protospacer adjacent motif sites generating individual-specific guides invisible to reference-based design. These findings demonstrate that variant-aware selection is critical for equitable genome editing.},
}
@article {pmid41498549,
year = {2026},
author = {Gopalaswamy, R and Subbian, S},
title = {The power of resistance: mechanisms of antimicrobial resistance in Mycobacterium tuberculosis and its impact on tuberculosis management.},
journal = {Clinical microbiology reviews},
volume = {39},
number = {1},
pages = {e0019425},
pmid = {41498549},
issn = {1098-6618},
mesh = {*Mycobacterium tuberculosis/drug effects/genetics ; Humans ; *Antitubercular Agents/pharmacology/therapeutic use ; *Tuberculosis, Multidrug-Resistant/drug therapy/microbiology/diagnosis ; *Drug Resistance, Bacterial ; *Tuberculosis/drug therapy/microbiology ; },
abstract = {SUMMARYThe global resurgence of drug-resistant tuberculosis (DR-TB) presents a formidable challenge to public health, driven by a complex interplay of mycobacterial evolution, dynamics and outcomes of host-pathogen interactions and systemic gaps in diagnosis and treatment strategies. This comprehensive review delineates the multifactorial basis of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (Mtb), integrating molecular, immunological, and pharmacological perspectives to inform next-generation strategies for effective TB control. We reconceptualize TB as a dynamic clinical spectrum-ranging from asymptomatic infection to overt disease-shaped by granuloma biology and bacterial adaptation. This spectrum underpins both diagnostic ambiguity and therapeutic failure, particularly in the context of phenotypic drug tolerance/resistance to current anti-TB drugs. We discuss Mtb's intrinsic and extrinsic resistance mechanisms, including the lipid-rich cell envelope, efflux systems, and enzymatic drug modification, which are compounded by acquired mutations that disrupt drug activation, alter targets, and confer cross-resistance. These adaptations are further potentiated by granuloma-induced pharmacokinetic heterogeneity and host-induced metabolic quiescence. We highlight the emerging role of therapeutic drug monitoring and pharmacokinetic/pharmacodynamic modeling in optimizing individualized therapy, particularly for novel regimens incorporating bedaquiline, pretomanid, and linezolid. Moreover, we underscore the diagnostic limitations in detecting heteroresistance and early-stage disease, advocating for expanded deployment of advanced and targeted molecular diagnostic modalities. Finally, we propose a paradigm shift toward integrated, precision-based TB management, leveraging host-directed therapies, biofilm-disrupting agents, and real-time pharmacokinetics-guided dosing to preempt resistance emergence and improve clinical outcomes. This review provides a translational framework for addressing the biological and operational complexities of DR-TB in the era of AMR.},
}
@article {pmid41498761,
year = {2026},
author = {Fan, X and Zhang, R and Yang, L and Chen, S and He, M and Wang, Y and Huang, L and Zeng, J and Wu, S and Gao, M and Wang, H},
title = {CRISPR-Cas9-Loaded Theranostic Liposomes for Enhancing Radiosensitization of Prostate Cancer through POLD4 Gene Editing under Real-Time MRI Monitoring.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {16},
pages = {e19704},
pmid = {41498761},
issn = {2198-3844},
support = {SHDC22011310-A//Shanghai Municipal Hospital Development Center/ ; SHDC22025311-B//Shanghai Municipal Hospital Development Center/ ; 25ZR1401440//Science and Technology Commission of Shanghai Municipality/ ; 82271969//National Natural Science Foundation of China/ ; 202101070002E00085//Major Program of Shanghai Municipal Commission of Education/ ; },
mesh = {Male ; *Prostatic Neoplasms/genetics/radiotherapy/therapy/diagnostic imaging ; *CRISPR-Cas Systems/genetics ; Humans ; *Liposomes ; Animals ; *Magnetic Resonance Imaging/methods ; *Gene Editing/methods ; Mice ; Cell Line, Tumor ; Theranostic Nanomedicine/methods ; Genetic Therapy/methods ; },
abstract = {Radiotherapy is a fundamental treatment for prostate cancer; however, its therapeutic efficacy is frequently limited by radioresistance mediated through DNA repair mechanisms and other biological factors. Although gene therapy holds promise for overcoming such resistance, identifying effective radiosensitization targets and developing efficient gene delivery systems remain practically challenging. In this study, transcriptomic analysis of radiotherapy-treated prostate cancer cells revealed a marked upregulation of DNA polymerase delta subunit 4 (POLD4), a target that has not been systematically studied. To evaluate the potential of POLD4 for overcoming radioresistance, CRISPR-Cas9-based plasmids along with ultrasmall superparamagnetic iron oxide nanoparticles (USPIONs) were encapsulated within cationic liposomes for achieving an MRI-trackable gene delivery platform (plasmid and iron oxide co-loaded liposomes, termed PIO@Lipo). Comprehensive in vitro and in vivo studies demonstrated that PIO@Lipo enabled efficient POLD4 knockdown. Furthermore, PIO@Lipo synergized with radiotherapy to induce extensive DNA damage, promote tumor cell apoptosis, and remodel the immunosuppressive microenvironment. Notably, PIO@Lipo displayed superior MRI contrast enhancement capability and passive tumor targeting ability. In conclusion, this study has identified POLD4 as a potent target for radiosensitization, capable of disrupting DNA damage-repair homeostasis through MRI-monitored gene editing, thereby offering a promising strategy to enhance the efficacy of radiotherapy in prostate cancer.},
}
@article {pmid41498982,
year = {2026},
author = {Mangal, H and Mathur, S and Kumar, S and Chaurasia, A and Ranjan, R},
title = {Role of CRISPR in bioremediation of heavy metal(loid): a breakthrough in environmental biotechnology.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {1},
pages = {34},
pmid = {41498982},
issn = {1573-0972},
mesh = {*Metals, Heavy/metabolism ; *Biodegradation, Environmental ; *CRISPR-Cas Systems ; *Biotechnology/methods ; Gene Editing/methods ; Bacteria/genetics/metabolism ; Biosensing Techniques ; Plants/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {A number of technological advancements have made bioremediation an emerging and innovative technology, including its economic viability, increased competence, and natural environment friendliness. The efficiency, scalability, and specificity of conventional physical, chemical, and biological remediation techniques are still limited, despite their partial success. Recent developments in CRISPR-based genome engineering have made it possible to precisely manipulate metal transporters, detoxification enzymes, and stress-response pathways in microorganisms and plants, opening up new possibilities to improve bioremediation. This review offers a thorough and integrated examination of enzyme engineering, biosensing systems, microbial bioremediation, and CRISPR-enabled phytoremediation. This work is novel because it presents a unified roadmap for next-generation bioremediation technologies by integrating CRISPR editing with multi-omics, synthetic biology, and emerging CRISPR-based biosensors. We also go over ecological risks, current difficulties, legal issues, and potential field deployment scenarios in the future. These revelations collectively demonstrate the revolutionary potential of CRISPR in creating highly effective, sustainable, and scalable remedies for heavy metal pollution.},
}
@article {pmid41499124,
year = {2026},
author = {Wang, K and Wang, W and Hu, Y and Li, X and Yu, H and Xia, X and Kai, T and Wen, M},
title = {A Relay CRISPR/Cas12a System for Extraction-Free and Ultrasensitive Detection of Staphylococcus aureus in Complex Food Samples.},
journal = {Analytical chemistry},
volume = {98},
number = {2},
pages = {1657-1666},
doi = {10.1021/acs.analchem.5c06345},
pmid = {41499124},
issn = {1520-6882},
mesh = {*Staphylococcus aureus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Food Microbiology ; Electrochemical Techniques ; DNA, Bacterial/genetics/analysis ; Limit of Detection ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Staphylococcus aureus (SA) is a predominant foodborne pathogen, frequently associated with nutrient-rich matrices. However, rapid and ultrasensitive detection of SA in complex food samples remains a formidable challenge. In this work, we developed a relay biosensing system for detecting SA-16S rDNA that combines a magnetic bead-anchored APE1 DNA walker with an asymmetric CRISPR/Cas12a system. The magnetic bead-anchored DNA walker enables efficient separation of SA-targets from complex food matrices, while generating abundant activators through APE1-mediated catalytic cleavage. Employing spatially decoupled reactions with differential crRNAs, the asymmetric Cas12a system achieves programmable cascade amplification. Dual-mode fluorescence/electrochemical readout supports both laboratory analysis and on-site detection. The system demonstrates exceptional sensitivity, capable of detecting trace amounts of SA-16S rDNA (7.5 aM by fluorescence, 1.0 fM by electrochemistry) and SA (4 CFU/mL by fluorescence, 34 CFU/mL by electrochemistry). Overall, this work provides a modular and programmable biosensing strategy for bacterial identification, thereby facilitating advanced nucleic acid analysis within complex biological matrices.},
}
@article {pmid41499404,
year = {2026},
author = {Wan, X and Cohen, SM and Yu, Y and Hoan Le, H and Park, H and Groaz, A and Moreno, R and Tan, M and Schneider, J and Gronquist, MR and Shinya, R and Schroeder, FC and Sternberg, PW},
title = {O-acyltransferase genes involved in the production of volatile sex pheromones in Caenorhabditis elegans.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {2},
pages = {e2524778123},
pmid = {41499404},
issn = {1091-6490},
support = {R24OD023041//HHS | NIH (NIH)/ ; 00000//Chuck Lorre Research Scholars Program/ ; DGE 1745301//NSF | NSF Graduate Research Fellowship Program (GRFP)/ ; R24 OD023041/OD/NIH HHS/United States ; 00000//Tianqiao and Chrissy Chen Institute for Neuroscience/ ; },
mesh = {Animals ; *Sex Attractants/biosynthesis/metabolism/genetics ; *Caenorhabditis elegans/genetics/metabolism/enzymology ; Male ; *Acyltransferases/genetics/metabolism ; Female ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Gas Chromatography-Mass Spectrometry ; CRISPR-Cas Systems ; Sexual Behavior, Animal ; },
abstract = {Gene family expansions are critical for functional diversification, yet the contributions of paralogs to metabolic pathways are often unclear. In Caenorhabditis, the expanded O-acyltransferase (OAC) family-enzymes that transfer acyl groups to hydroxylated substrates-remains poorly characterized despite having been implicated in lipid metabolism. Using CRISPR-Cas9 mutagenesis, behavioral assays, gas chromatographic-mass spectral (GC-MS) analyses, and metabolomics, we systematically analyzed 59 OAC-family protein-coding genes to define their roles in regulating signaling molecules. We found that four adjacent paralogs (oac-13, oac-16, oac-25, and oac-28) on chromosome I are required for synthesizing volatile sex pheromones-airborne signals critical for male mate-searching. Specifically, oac-13 and oac-16 are necessary for producing both major pheromone components, while the identical tandem paralogs oac-25 and oac-28 regulate the production of the later-eluting component in gas chromatography. Disruption of these genes reduced production of key pheromone components and impaired male attraction. Metabolomics revealed that oac-16 and other OACs also modulate the synthesis and secretion of nonvolatile ascaroside pheromones, indicating dual roles in chemical signaling. This work uncovers functional specialization within an expanded gene family, illustrating how redundancy and divergence enable adaptive evolution of communication systems.},
}
@article {pmid41500123,
year = {2026},
author = {Zang, Z and Chen, J and Dong, Y and Chen, L and Yang, M and Mu, M and Zhou, L and Zhang, W and Zou, G and Liu, C},
title = {CRISPR-Cas12a biosensing technology advances and applications in precision diagnostics and cancer research.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129298},
doi = {10.1016/j.talanta.2025.129298},
pmid = {41500123},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Neoplasms/diagnosis/genetics ; *Precision Medicine/methods ; Biomarkers, Tumor/genetics ; Gene Editing ; },
abstract = {CRISPR-Cas12a has become a versatile biotechnology platform with important applications in biosensing, diagnostics, and precision genome editing. This system is activated by a single crRNA, exhibits target-responsive trans-cleavage activity, and recognizes programmable PAM sequences. These features provide a robust basis for accurate detection of diverse biomarkers. Its detection capabilities include nucleic acid targets such as viral RNA and cancer mutations, as well as non-nucleic acid molecules like exosomes and proteins. Recent advancements have shown significant advantages, including multi-temperature adaptability, rapid kinetics, and compatibility with both DNA and RNA targets. Technical improvements include machine learning-assisted crRNA design for enhanced prediction accuracy and engineered EnAsCas12a variants that overcome conventional PAM restrictions. Notable achievements involve entropy-driven circuits that achieve attomolar-level sensitivity, smartphone-compatible four-channel quantitative detection systems, and streamlined integrated workflows completed within 30 min. Advances in sensor design, such as metal-organic framework encapsulation and high-performance aptamer-based sensors, have further expanded detection capabilities. In oncology research, CRISPR-Cas12a technology provides powerful tools to comprehensively analyze complex molecular networks within the tumor microenvironment (TME) and facilitate ultrasensitive detection of early-stage cancer biomarkers. Additionally, in genome editing, CRISPR-Cas12a enables precise genomic modifications due to distinct repair pathways, versatile delivery methods, and efficient creation of transgenic models. Thus, it expands its functional scope beyond diagnostics. With ongoing development, this technology is expected to evolve into an integrated platform combining TME research, point-of-care cancer diagnostics, and programmable genome engineering, offering innovative solutions for both biomedical research and clinical translation.},
}
@article {pmid41500141,
year = {2026},
author = {Zheng, S and Tie, H and Chai, S and Wang, M and Wang, S and Zeng, YY and Wu, G and Zhang, TG},
title = {Molecular mechanisms and biotechnological advances in herbicide resistance: Insights into the development of herbicide-tolerant crops.},
journal = {Journal of plant physiology},
volume = {317},
number = {},
pages = {154690},
doi = {10.1016/j.jplph.2026.154690},
pmid = {41500141},
issn = {1618-1328},
mesh = {*Herbicide Resistance/genetics ; *Crops, Agricultural/genetics/drug effects ; *Herbicides/pharmacology ; *Biotechnology ; Gene Editing ; Plants, Genetically Modified/genetics ; *Plant Weeds/drug effects/genetics ; CRISPR-Cas Systems ; },
abstract = {Herbicides play a pivotal role in modern agriculture by controlling weed populations and safeguarding crop yields. However, the long-term and extensive use of herbicides has accelerated the evolution of herbicide-resistant weeds, thereby diminishing their efficacy and posing a serious threat to global food security. Recent advances in molecular biology and plant biotechnology have greatly expanded our understanding of herbicide resistance mechanisms and enabled the development of crops with enhanced herbicide resistance. Herbicide resistance genes function primarily by encoding detoxifying enzymes, modifying herbicide target sites, or activating specific metabolic pathways that mitigate herbicidal toxicity. Emerging genetic tools, including transgenic approaches and CRISPR/Cas-mediated genome editing, have further facilitated the precise introduction of resistance traits into major crops. It is noteworthy that this review offers novel insights into the latest CRISPR/Cas applications, including base editing and prime editing for developing novel, non-transgenic herbicide-resistant crops. Furthermore, it provides a systematic overview of advanced strategies for engineering multi-gene stacking traits to combat complex or evolving weed resistance. This review integrates recent progress in elucidating the molecular targets of herbicides and the underlying resistance mechanisms, and highlights the potential of modern biotechnological strategies for engineering herbicide-resistant crops to promote sustainable and environmentally responsible weed management.},
}
@article {pmid41500180,
year = {2026},
author = {Sheng, C and Wang, J and Tan, M and Zhang, J and Sun, M and Sun, J and Shao, Y and Tu, J and Zhu, L and Song, X},
title = {Establishment of detection method of chicken infectious anemia virus based on CRISPR/Cas12a system.},
journal = {Research in veterinary science},
volume = {201},
number = {},
pages = {106046},
doi = {10.1016/j.rvsc.2025.106046},
pmid = {41500180},
issn = {1532-2661},
mesh = {*Chicken anemia virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Animals ; Chickens ; *Poultry Diseases/diagnosis/virology ; *Circoviridae Infections/veterinary/diagnosis/virology ; Sensitivity and Specificity ; },
abstract = {Chicken Infectious Anemia Virus (CIAV) causes chicken infectious anemia, characterized by anemia and immune dysfunction. The rapid dissemination of this virus is generating substantial economic consequences for poultry producers. The CRISPR/Cas12a system is widely used for virus detection through crRNA-guided target recognition and the paracrine activity of Cas12a. To enable rapid and highly sensitive detection of Chicken Infectious Anemia Virus (CIAV), a CRISPR-Cas12a-based fluorescence assay was refined. Through optimization of the CRISPR/Cas12a system and integration of enzymatic recombinase amplification (ERA), the assay achieved a detection limit of 1 copy/μL, demonstrating its significant utility for CIAV diagnostics. In addition, a CRISPR/Cas12a lateral flow assay was developed and optimized, achieving a sensitivity of 10^3 copies/μL for the rapid and visual detection of target analytes. This technique exhibits high specificity for CIAV, showing no cross-reactivity with other chicken viruses. Overall, the system enables rapid CIAV detection with cost-effective equipment, making it suitable for virus monitoring.},
}
@article {pmid41501085,
year = {2026},
author = {Singh, P and D'Rozario, R and Chakraborty, B and Meher, S and Raychaudhuri, D and Tannir, AJ and Li, Y and Majumdar, A and Hawkins, J and Xiong, Y and Lorenzi, P and Sharma, P and Akdemir, K and Pilie, P and Jain, AK and Lee, BHL and Goswami, S},
title = {Loss of KDM6A-mediated genomic instability and metabolic reprogramming regulates response to therapeutic perturbations in bladder cancer.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1382},
pmid = {41501085},
issn = {2041-1723},
support = {P30 CA016672/CA/NCI NIH HHS/United States ; S10 RR029552/RR/NCRR NIH HHS/United States ; S10 OD012304/OD/NIH HHS/United States ; R37 CA279192/CA/NCI NIH HHS/United States ; R25 CA265800/CA/NCI NIH HHS/United States ; },
mesh = {*Urinary Bladder Neoplasms/genetics/drug therapy/metabolism ; Animals ; *Histone Demethylases/genetics/metabolism ; Humans ; Mice ; *Genomic Instability/genetics ; Cell Line, Tumor ; Cisplatin/therapeutic use/pharmacology ; Drug Resistance, Neoplasm/genetics ; CRISPR-Cas Systems ; DNA Repair/genetics ; Female ; Mice, Knockout ; Loss of Function Mutation ; Histones/metabolism ; Metabolic Reprogramming ; },
abstract = {Mutations in epigenetic regulators are common in bladder cancer, yet their impact on therapeutic responses remains unclear. Here, we identify that loss-of-function mutations in KDM6A, a histone demethylase altered in about 26% of advanced bladder cancers, are associated with poor survival after cisplatin chemotherapy, whereas they correlate with improved outcomes with anti-PD-1 therapy. Using CRISPR-Cas9-engineered murine and human bladder cancer models, we show that KDM6A deficiency increases formation of extrachromosomal circular DNA carrying chemoresistance loci, promoting cisplatin resistance. In parallel, KDM6A loss impairs DNA repair and rewires tumor metabolism, reducing glycolysis and lactate output. This metabolic shift diminishes histone lactylation in regulatory T cells, suppressing immunoregulatory genes and limiting expansion of PD-1[hi] regulatory T cells. Collectively, our findings establish KDM6A mutation as a key regulator of therapeutic responses, providing a foundation for its use in guiding precision therapy in advanced bladder cancer.},
}
@article {pmid41501459,
year = {2026},
author = {Dmytrenko, O and Yuan, B and Crosby, KT and Krebel, M and Chen, X and Nowak, JS and Chramiec-Głąbik, A and Filani, B and Gribling-Burrer, AS and van der Toorn, W and von Kleist, M and Achmedov, T and Smyth, RP and Glatt, S and Bravo, JPK and Heinz, DW and Jackson, RN and Beisel, CL},
title = {RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity.},
journal = {Nature},
volume = {649},
number = {8099},
pages = {1312-1321},
pmid = {41501459},
issn = {1476-4687},
support = {R35 GM138080/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages/physiology ; Catalytic Domain ; *CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; Cryoelectron Microscopy ; Models, Molecular ; *RNA Cleavage ; *RNA, Bacterial/metabolism/genetics/chemistry/immunology ; *RNA, Transfer/metabolism/chemistry/ultrastructure/genetics/immunology ; Bacteria/immunology/virology ; },
abstract = {In all domains of life, tRNAs mediate the transfer of genetic information from mRNAs to proteins. As their depletion suppresses translation and, consequently, viral replication, tRNAs represent long-standing and increasingly recognized targets of innate immunity[1-5]. Here we report Cas12a3 effector nucleases from type V CRISPR-Cas adaptive immune systems in bacteria that preferentially cleave tRNAs after recognition of target RNA. Cas12a3 orthologues belong to one of two previously unreported nuclease clades that exhibit RNA-mediated cleavage of non-target RNA, and are distinct from all other known type V systems. Through cell-based and biochemical assays and direct RNA sequencing, we demonstrate that recognition of a complementary target RNA by the CRISPR RNA triggers Cas12a3 to cleave the conserved 5'-CCA-3' tail of diverse tRNAs to drive growth arrest and anti-phage defence. Cryogenic electron microscopy structures further revealed a distinct tRNA-loading domain that positions the tRNA tail in the RuvC active site of the nuclease. By designing synthetic reporters that mimic the tRNA acceptor stem and tail, we expanded the capacity of current CRISPR-based diagnostics for multiplexed RNA detection. Overall, these findings reveal widespread tRNA inactivation as a previously unrecognized CRISPR-based immune strategy that broadens the application space of the existing CRISPR toolbox.},
}
@article {pmid41501880,
year = {2026},
author = {Zhu, M and Wu, Y and Ou, H and Liu, X and Wang, Y and Liu, X and Zou, C and Yang, G and Du, M and Yu, D and Zheng, D and He, L and Zhang, K and Zhang, W and Wang, S and Qin, H and Hao, Q and He, Y and Lin, W and Zhang, Y and Gu, J and Li, M and Qin, W and Cao, Z},
title = {Targeting TRIM25 as a therapeutic strategy to enhance ferroptosis in glioblastoma cells.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {20},
pmid = {41501880},
issn = {1477-3155},
support = {01-SWKJYCJJ17//the National Defense Biotechnology Fund for Outstanding Young Talents/ ; 2025GTKP008//the National Key Laboratory of Oncology/ ; },
mesh = {*Ferroptosis/drug effects ; Humans ; *Glioblastoma/metabolism/pathology/drug therapy ; Animals ; Cell Line, Tumor ; *Tripartite Motif Proteins/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; Mice ; *Brain Neoplasms/metabolism/pathology ; CRISPR-Cas Systems ; Gene Editing ; Mitochondria/metabolism ; Nanoparticles/chemistry ; Mice, Nude ; Ubiquitination ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis (< 7% 5-year survival) under current first-line treatment. While inducing programmed cell death (PCD) is a promising antitumor strategy, its effectiveness in GBM remains controversial. Ferroptosis emerged as the most enriched PCD process and was highly correlated with GBM malignant progression.<br><br>METHODS: We performed a CRISPR-Cas9 loss-of-function screen to identify critical ferroptosis contributors. Mechanistic studies involved assessing mitochondrial function and morphology. Protein interaction and degradation pathways were investigated using immunoprecipitation and ubiquitination assays. We developed a blood-brain-barrier-penetrating genome editing delivery system, Angiopep-2-modified nanoparticles with disulfide bonds (ANPSS), loaded with Cas9/sgRNA complexes.<br><br>RESULTS: Voltage-dependent anion channel 2 (VDAC2) was identified as a critical contributor to ferroptosis. VDAC2 overexpression induced mitochondrial dysfunction and characteristic ferroptotic mitochondrial morphology. The E3 ubiquitin ligase TRIM25 was identified as a key suppressor of VDAC2, directly interacting with it and inducing its K48-linked polyubiquitination and subsequent proteasomal degradation. In vivo, the ANPSS(sgTRIM25) system effectively targeted GBM cells, significantly promoted ferroptosis, and inhibited GBM progression.<br><br>CONCLUSIONS: Our findings demonstrate that TRIM25 is a critical negative regulator of VDAC2-dependent ferroptosis in GBM. Targeting TRIM25 using the ANPSS(sgTRIM25) genome editing system effectively overcomes ferroptosis resistance and suppresses tumor growth, representing a viable therapeutic approach for GBM.},
}
@article {pmid41502163,
year = {2026},
author = {Manojkumar, C and Limbola, M and Paul, S and Thangadurai, K and Rajendran, KV and Roy, A and Mandal, B and Jeena, K and Bedekar, MK},
title = {CRISPR-Cas12a-based lateral flow detection of white spot syndrome virus: a dual-target approach for detection of early and latent infection.},
journal = {Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc},
volume = {38},
number = {2},
pages = {192-204},
pmid = {41502163},
issn = {1943-4936},
mesh = {*White spot syndrome virus 1/isolation &amp; purification/genetics ; Animals ; *CRISPR-Cas Systems ; *Penaeidae/virology ; Sensitivity and Specificity ; Viral Envelope Proteins/genetics ; },
abstract = {White spot syndrome virus (WSSV; family Nimaviridae; taxon species White spot syndrome virus) is a major viral pathogen that poses a significant threat to the global shrimp industry, with early detection being the most effective strategy for disease control. We developed a CRISPR-Cas12a-based dual-target detection assay for WSSV, specifically targeting the VP28 gene (gene product is a major envelope protein) and WSSV366 (a latency-associated gene), optimized using Indian WSSV isolates. Our CRISPR RNAs for both targets had high efficiency, and we evaluated the assay using fluorescence-based and lateral flow strip (LFS) endpoint detection. In fluorescence assays, the Cr-WSSV assay (without recombinase polymerase amplification, RPA) detected WSSV at 3 × 10[5] copies/μL; RPA integration significantly enhanced sensitivity, allowing detection at as low as 20 and 200 copies for VP28 and WSSV366, respectively, with 100% specificity. We developed a CRISPR-based LFS assay with optimized FAM-biotin reporter concentrations of 100 nM and 250 nM, yielding robust and reproducible results for improved field applicability. Performance evaluation confirmed lack of cross-reactivity to other WOAH-listed shrimp pathogens, while maintaining detection limits of 20 and 200 copies of VP28 and WSSV366. Clinical validation further demonstrated that the RPA-Cr-WSSV-LFS assay successfully detected WSSV366 even in VP28-negative samples, underscoring the importance of detecting WSSV366 in latent infections. Our rapid, cost-effective, and highly sensitive CRISPR-Cas-based assay enhances WSSV surveillance and biosecurity in shrimp aquaculture by incorporating structural and latency-associated gene markers, making it a promising alternative to conventional molecular testing.},
}
@article {pmid41503690,
year = {2026},
author = {Ma, N and Zhang, M and Ghonaim, AH and Zhou, P and Wang, C and Zhou, J and Guo, G and Lebbink, RJ and Bosch, BJ and Zhu, H and Li, W and He, Q},
title = {The essential role of heparan sulfate in the entry of PDCoV and other porcine coronaviruses.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2614154},
pmid = {41503690},
issn = {2150-5608},
mesh = {Animals ; *Heparan Sulfate/metabolism ; Swine ; *Deltacoronavirus/physiology ; *Virus Internalization/drug effects ; Swine Diseases/virology ; Humans ; *Porcine epidemic diarrhea virus/physiology ; *Coronavirus Infections/virology/veterinary ; Cell Line ; CRISPR-Cas Systems ; Receptors, Virus/metabolism ; Transmissible gastroenteritis virus/physiology ; Alphacoronavirus ; },
abstract = {Porcine enteric coronaviruses, including porcine deltacoronavirus (PDCoV), porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and transmissible gastroenteritis coronavirus (TGEV), can cause acute diarrhea, vomiting, dehydration, and high mortality in suckling piglets. Recent studies revealing human PDCoV infections and the potential of SADS-CoV to penetrate human cell lines have heightened apprehensions about the zoonotic transmission risks of these viruses. While heparan sulfate (HS) serves as a receptor in PDCoV binding, the key host genes involved in HS biogenesis and the specific molecular mechanisms underlying this process have not been fully examined. Enzymes involved in HS biosynthesis, including SLC35B2, EXT1, and NDST1, were identified as critical host factors via the use of CRISPR-Cas9 knockout cells. Moreover, inhibition assays using heparin sodium, a competitive HS mimic, demonstrated dose-dependent reductions in PDCoV infection in vitro. Additionally, mitoxantrone, an HS-binding drug, reduced PDCoV infection. Furthermore, HS was confirmed to facilitate the entry of other porcine enteric coronaviruses (SeCoVs), including PEDV, SADS-CoV, and TGEV, underscoring the conserved role of HS in CoV pathogenesis. These insights contribute to the understanding of porcine coronavirus-host interactions and support the development of innovative antiviral interventions.},
}
@article {pmid41503845,
year = {2026},
author = {Li, MF and Zubair, A and Wdidi, S and He, S},
title = {Disrupting Viral Persistence: CRISPR/Cas9-Based Strategies for Hepatitis B and C Treatment, and Challenges.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {1},
pages = {e70986},
pmid = {41503845},
issn = {1582-4934},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Hepatitis B virus/genetics ; *Hepatitis B/therapy/virology/genetics ; Gene Editing/methods ; *Hepatitis C/therapy/virology/genetics ; *Hepacivirus/genetics ; Animals ; Antiviral Agents/therapeutic use/pharmacology ; Genome, Viral ; DNA, Circular/genetics ; DNA, Viral/genetics ; Genetic Therapy ; Virus Replication/genetics ; },
abstract = {Hepatitis B and C viruses (HBV and HCV) remain among the leading causes of liver disease worldwide. Current antiviral drugs, such as nucleotide analogues (NAs), can reduce the replication of new HBV and HCV infections but cannot completely eliminate chronic infections. This is primarily because a stable form of viral DNA, known as covalently closed circular DNA (cccDNA), persists in liver cells and continues to sustain the infection. In recent years, the CRISPR/Cas9 gene-editing system has emerged as a powerful tool for precisely cutting and inactivating specific DNA sequences. Due to its efficiency and ease of use, researchers have applied CRISPR/Cas9 in numerous studies to directly target and disrupt the HBV genome, demonstrating promising antiviral effects in both cell cultures and animal models. Targeting multiple sites within the HBV genome has been shown to further enhance its effectiveness, paving the way for potential combination therapies aimed at disabling both cccDNA and HBV and HCV DNA integrated into the host genome. Despite its potential, CRISPR/Cas9 still faces significant challenges before clinical application, most notably the risk of off-target effects-unintended cleavage of non-target DNA sequences-and the difficulty of delivering the system efficiently into liver cells in vivo. Future progress will depend on improving the tool's precision, efficiency, flexibility and delivery methods. In this review, we explore recent advances in designing guide RNAs (gRNAs) for targeting HBV and HCV, as well as the delivery systems used to transport CRISPR/Cas9 into cells. We also discuss the remaining challenges and potential strategies for advancing CRISPR/Cas9 from the laboratory toward a viable clinical cure for HBV and HCV.},
}
@article {pmid41504765,
year = {2026},
author = {Osei, EK and O'Mahony, AK and O'Hea, R and Moriarty, J and O'Doherty, &#xc1; and Wilson, M and Garcia Manzanilla, E and Mahony, J and Kenny, JG},
title = {Genomics of Irish swine-derived Streptococcus suis: population structure, prophages and anti-viral defence mechanisms.},
journal = {Microbial genomics},
volume = {12},
number = {1},
pages = {},
pmid = {41504765},
issn = {2057-5858},
mesh = {Animals ; *Streptococcus suis/genetics/virology/classification/isolation &amp; purification ; Swine/microbiology ; *Prophages/genetics ; Ireland ; Phylogeny ; *Streptococcal Infections/microbiology/veterinary ; Whole Genome Sequencing ; Genomics ; *Swine Diseases/microbiology ; Multilocus Sequence Typing ; Genome, Bacterial ; Genetic Variation ; Serogroup ; },
abstract = {Streptococcus suis is a major pig pathogen with zoonotic potential, posing an occupational risk to farmers and meat handlers. We characterized 110 S. suis strains from diseased pigs in Ireland (2005-2022) using whole-genome sequencing to investigate population structure and phage-host dynamics. We identified 15 distinct serotypes, with serotypes 9 and 2 being the most dominant. In silico multi-locus sequence typing revealed high diversity within the collection, identifying several sequence types (STs), including 26 novel STs. Investigation of strain-level genomic clustering using PopPUNK against global S. suis genomes showed that the Irish isolates were phylogenetically dispersed across the broader global S. suis population rather than clustering in a single clonal group. The majority of Irish isolates fall within the ten established pathogenic lineages, including the highly virulent zoonotic lineage 1. A locally persistent clonal lineage was identified among Irish isolates, showing minimal genetic variation over a decade.Prophage analysis revealed novel viral taxa that were interspersed among known streptococcal phages, rather than clustering distinctly. Restriction-modification systems were the predominant anti-viral defence systems identified across genomes. CRISPR-Cas systems were present in limited strains but showed substantial targeting bias toward full-length prophages, indicating ongoing phage pressure. CRISPR spacers matched non-S. suis streptococcal phages, and phylogenomic analysis revealed that Vansinderenvirus phages clustered with S. suis rather than other Streptococcus thermophilus phages, suggesting evolutionary connections between phage lineages infecting different streptococci.This study presents the first comprehensive genomic characterization of S. suis in Ireland, revealing a diverse population with significant implications for animal and human health.},
}
@article {pmid41505087,
year = {2026},
author = {Di Pede, AC and Bagheri, N and Belforte, E and Palone, A and Rossetti, M and Porchetta, A},
title = {Triplex DNA clamp regulates Cas12a activation for ssDNA and RNA sensing.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505087},
issn = {1362-4962},
support = {MFAG 2022-ID. 27151//AIRC/ ; //National Recovery and Resilience Plan/ ; //Italian Ministry of University and Research/ ; D53D23009090001//European Union/ ; 2022FPYZ2N//European Union/ ; PNRR M4C2-Investimento 1.4- CN00000041//Italian Ministry of Ministry of University and Research/ ; //Fondazione Umberto Veronesi/ ; },
mesh = {*DNA, Single-Stranded/metabolism/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA/metabolism/genetics/chemistry ; *DNA/chemistry/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {We present a molecular strategy that enables the programmable activation of the CRISPR-Cas12a system in response to triplex DNA formation triggered by single-stranded DNA (ssDNA) or RNA inputs. Our triplex-controlled Cas12a assay leverages the high specificity of clamp-like triplex structures to control a toehold-based strand displacement reaction within a rationally designed DNA hairpin (PAM-Switch). Upon displacement and protospacer adjacent motif (PAM) complementation, the Cas12a ribonucleoprotein (RNP) is activated, initiating trans-cleavage and producing a concentration-dependent fluorescent signal. By decoupling target recognition (via triplex formation) from direct hybridization with the Cas12a-crRNA complex, the assay eliminates the need for target-specific crRNAs. This design also allows multiple detection of distinct nucleic acid (NA) targets using a single Cas12a reaction mix. Through the use of triplex-based clamps, the proposed platform achieves enhanced specificity for single-nucleotide variants and supports the detection of both ssDNA and RNA targets across a broad range of lengths (10-20 nucleotides), addressing key limitations in current Cas12a-based diagnostics and opening new avenues for NA sensing.},
}
@article {pmid41505088,
year = {2026},
author = {Lee-Yow, YC and Valbuena, RC and Richter, CS and Chang, HY and Engreitz, JM},
title = {Junction-targeting designs limit the application of CRISPR-Cas13d in circular RNA perturbation studies.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505088},
issn = {1362-4962},
support = {//Lucile Packard Children's Hospital at Stanford University/ ; NNF21SA0072102//Novo Nordisk Foundation/ ; R35 HG011324/HG/NHGRI NIH HHS/United States ; DGE-1656518//GRFP/ ; /HHMI/Howard Hughes Medical Institute/United States ; //Stanford Interdisciplinary Graduate Fellowship/ ; T32 GM007790/GM/NIGMS NIH HHS/United States ; R35HG011324/HG/NHGRI NIH HHS/United States ; 5T32GM007790/GF/NIH HHS/United States ; },
mesh = {RNA, Circular/genetics ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA/genetics/metabolism ; K562 Cells ; Gene Knockdown Techniques/methods ; Exons ; },
abstract = {Circular RNAs (circRNAs) are RNA molecules formed through the backsplicing of linear exons. Several thousand have been identified, yet relatively few are functionally characterized due to challenges in distinguishing effects of circular from linear RNA targets. Recently, CRISPR-Cas13 systems have been utilized to directly target unique junctions formed through backsplicing, potentially allowing for selective degradation of circular isoforms. Applying this approach in pooled screens has indeed identified circRNAs proposed to affect viability in several cancer cell lines. However, the design limitations of applying Cas13d to study circRNAs are not fully characterized. Here, we assessed the limitations of Cas13d-mediated circRNA knockdowns by performing essentiality screens on 900 highly expressed circRNAs in K562, an ENCODE tier 1 cell line. We observed consistent off-target knockdown of linear isoforms by certain circRNA-targeting single-guide RNAs (sgRNAs). Re-analysis of existing Cas13d screens in other cell types revealed similar off-target effects. Using machine learning models that predict Cas13d sgRNA efficacy, we further found that most circRNA-targeting sgRNAs are unlikely to induce strong knockdown. After accounting for these design constraints, 0 of 346 circRNAs testable in our screens had detectable effects on proliferation. Our findings highlight key limitations of junction-targeting strategies, with implications for future circRNA perturbation studies.},
}
@article {pmid41505097,
year = {2026},
author = {Wang, L and Xiang, X and Yin, G and Shu, H and Wu, Y and Chen, H and Na, R and Gijzen, M and Hou, Y and Dong, S},
title = {Effector gene silencing coordinated by histone methylation and small RNAs enhances host adaptation in a plant pathogen.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505097},
issn = {1362-4962},
support = {32400164//National Natural Science Foundation of China/ ; 32488302//National Natural Science Foundation of China/ ; 25ZR1401177//Natural Science Foundation of Shanghai/ ; //Shanghai Jiao Tong University/ ; },
mesh = {*Histones/metabolism/genetics ; *Gene Silencing ; *Phytophthora/genetics/pathogenicity ; Plant Diseases/microbiology/genetics/parasitology ; Methylation ; *Host-Pathogen Interactions/genetics ; Epigenesis, Genetic ; CRISPR-Cas Systems ; Virulence/genetics ; *RNA, Small Untranslated/genetics/metabolism ; },
abstract = {Pathogen adaptability driven by epigenetic processes remains poorly understood and poses a significant challenge to sustainable disease management. Histone 3 lysine 27 trimethylation (H3K27me3) and small RNA (sRNA)-mediated silencing of avirulence (Avr) genes are two major strategies that pathogens employ to evade recognition by host resistance (R) proteins. Here, we demonstrate that these two epigenetic mechanisms operate in a coordinated manner to silence Avr genes in the oomycete Phytophthora sojae. CRISPR/Cas9-mediated editing of PsSu(z)12, a core component of the Polycomb repressive complex 2 (PRC2), abolished H3K27me3 deposition at Avr1b and Avr3a, leading to transcriptional reactivation and loss of avirulence. Complementation with PsSu(z)12 restored H3K27me3 and silencing at Avr1b, but not at Avr3a. This prompted sRNA profiling at both loci, revealing differential co-enrichment of sRNA and H3K27me3. Integrated analysis of H3K27me3-enriched chromatin immunoprecipitation, RNA and sRNA sequencing data uncovered a strong locus-specific co-silencing pattern, with 11 out of 12 H3K27me3-regulated arginine-X-leucine-arginine effectors also targeted by sRNAs. Notably, epigenetic variation among field isolates indicated regulatory heterogeneity and plasticity in effector control. Together, our findings establish PsSu(z)12 as a central hub coordinating H3K27me3 and sRNA-mediated effector gene silencing, revealing a dual-layered epigenetic mechanism that enables immune evasion and promotes pathogen adaptation.},
}
@article {pmid41505099,
year = {2026},
author = {Xu, B and Li, S and Li, Y and Zhao, S and Li, X and Han, J and Wu, D and Li, S and Chen, L and Xie, S and Han, X and Zhao, C},
title = {Enhanced SfaTnpB enables single-base-specific, one-pot nucleic acid detection for high-sensitivity diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505099},
issn = {1362-4962},
support = {2023YFF1001000//National Key Research &amp; Developmental Program of China/ ; SH23YCKY01//Basic Research Project of Yazhouwan National Laboratory/ ; 32202634//Natural Science Foundation of China/ ; //Yazhouwan National Laboratory/ ; },
mesh = {Humans ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; *Transposases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Papillomavirus Infections/diagnosis/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR/Cas12-based nucleic acid detection has revolutionized molecular diagnostics but shows limited single-nucleotide specificity, limited high-fidelity subtype discrimination, and limited compatibility with one-pot assays, restricting its broader clinical application. Here, we report a transposon-associated transposase B (TnpB) ortholog, SfaTnpB, with high trans-cleavage activity, robust single-base mismatch discrimination, and broad temperature tolerance. By stepwise engineering of its guide RNA (ωRNA), we developed an enhanced SfaTnpB (enSfaTnpB) system with markedly improved trans-cleavage efficiency. In combination with a TAM-independent split-activator strategy, this system enables precise detection of single-nucleotide polymorphisms. We further developed TOPIC (TnpB-based One-Pot nucleIC acid detection), a one-pot detection platform coupling enSfaTnpB with recombinase-aided amplification (RAA) or loop-mediated isothermal amplification that enables ultrasensitive detection of human papillomavirus (HPV) subtypes 16 and 18 (∼4 copies/μl) and African swine fever virus DNA (∼3 copies/μl). Finally, RAA-TOPIC accurately detected and genotyped 14 high-risk HPV subtypes with high-fidelity subtype discrimination, showing complete concordance with quantitative real-time PCR-based clinical diagnostics. These findings establish TOPIC as a compact, programmable, and scalable molecular detection tool with broad potential for precision diagnostics and point-of-care testing, particularly in resource-limited settings.},
}
@article {pmid41505207,
year = {2026},
author = {Gao, JL and Li, Z and Calderon-Perez, R and Pavek, A and Kim, L and McDermott, DH and Murphy, PM},
title = {Gene therapy via CRISPR/Cas9-mediated Cxcr4 disease allele inactivation reverses leukopenia in WHIM mice.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {5},
pages = {},
pmid = {41505207},
issn = {1558-8238},
mesh = {Animals ; *Receptors, CXCR4/genetics ; Mice ; *CRISPR-Cas Systems ; *Genetic Therapy ; *Leukopenia/therapy/genetics/pathology ; *Warts/therapy/genetics/pathology ; *Alleles ; *Immunologic Deficiency Syndromes/therapy/genetics/pathology ; Primary Immunodeficiency Diseases/therapy/genetics ; Hematopoietic Stem Cell Transplantation ; Humans ; Gene Editing ; },
abstract = {Warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is an immunodeficiency caused by autosomal dominant hyperfunctional mutations in chemokine receptor CXCR4 that promote panleukopenia due to BM retention. We previously reported a preclinical gene therapy protocol involving allele-nonspecific Cxcr4 CRISPR/Cas9 inactivation, leveraging the known in vivo dominance of Cxcr4+/o (+, WT; o, inactivated) hematopoietic stem cells (HSCs) for autologous BM engraftment and leukocyte reconstitution over HSCs with other Cxcr4 genotypes. Here, we show that without BM conditioning, this approach is not able to correct leukopenia in WHIM mice. We therefore modified the protocol by adding conditioning with a nongenotoxic CD117-targeted immunotoxin, CD117-antibody-saporin-conjugate. With this change, donor-derived blood cells rapidly reached ~95% chimerism after transplantation, which was stable without adverse events. Mice receiving edited HSCs showed rapid normalization of absolute myeloid cell counts, the key blood subset responsible for WHIM syndrome. In competitive transplants using equal numbers of edited and unedited donor HSCs, over 80% of blood cells originated from the edited population, predominantly with the Cxcr4+/o genotype. These results provide proof of principle that CRISPR/Cas9-mediated inactivation of the Cxcr4 disease allele, combined with nongenotoxic HSC-targeted conditioning, may offer a safe and effective gene therapy strategy generalizable to all WHIM-causing mutations.},
}
@article {pmid41505468,
year = {2026},
author = {Ma, J and Zhang, Y and Li, Z and Liu, L and A, J and Liang, R and Cao, C and Zhou, J and Cheng, P and Li, Y and Li, Z and Ma, L and Jiang, L and A, X},
title = {CRISPR/Cas12a-RCA enables ultrasensitive detection of circulating free DNA for noninvasive diagnosis of echinococcosis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {1},
pages = {e0013069},
pmid = {41505468},
issn = {1935-2735},
mesh = {Humans ; *CRISPR-Cas Systems ; *Echinococcosis/diagnosis/blood/parasitology ; *Echinococcus/genetics/isolation &amp; purification ; *Cell-Free Nucleic Acids/blood/genetics ; Sensitivity and Specificity ; *DNA, Helminth/blood/genetics ; *Molecular Diagnostic Techniques/methods ; Animals ; Male ; Female ; Limit of Detection ; Middle Aged ; High-Throughput Nucleotide Sequencing ; Adult ; },
abstract = {OBJECTIVE: To develop a novel non-invasive CRISPR/Cas12a-RCA assay for the primary screening of human echinococcosis via detection of circulating Echinococcus cell-free DNA (cfDNA) in peripheral blood.<br><br>METHODS: Plasma cfDNA from 20 AE patients was analyzed via high-throughput sequencing to identify conserved repetitive Echinococcus fragments.A one-pot RCA system coupled with CRISPR/Cas12a was optimized for Echinococcus-cfDNA detection. The limit of detection (LOD) was determined using serially diluted synthetic standards, while specificity was validated through mismatch probes and cross-reactivity testing. Clinical validation included 50 AE cases, 22 cystic echinococcosis (CE) cases, 43 non-Echinococcus hepatic disease (HD) cases, and 53 healthy controls (CON).<br><br>RESULTS: A conserved repetitive 28S rDNA fragment (pan-Echinococcus-28S) was identified as a biomarker. The CRISPR/Cas12a-RCA assay achieved amplification within 30 minutes at 37 &#xb0;C, with a linear range of 1 aM to 100 pM and an LOD of 1.41 aM. Visual detection limits were 10 aM (UV light) and 1 aM (blue light). The assay demonstrated high sensitivity (87.5%) and specificity (96.9%, AUC&#x2009;=&#x2009;0.933) in distinguishing Echinococcus infection (AE/CE) from HD and CON.<br><br>CONCLUSION: The optimized one-pot CRISPR/Cas12a-RCA system enables rapid and ultrasensitive detection of pan-Echinococcus cfDNA, providing a cost-effective and highly accurate solution for the primary screening of echinococcosis.},
}
@article {pmid41506042,
year = {2026},
author = {Jiang, H and Yang, J and Qian, C and Li, A and Liu, Y and Zhang, F and Deng, Y and Duan, J and Lv, X},
title = {Logic-gated CRISPR-Cas12a assay with engineered signal amplification for sensitive multiplexed detection of HCC miRNAs.},
journal = {Biosensors &amp; bioelectronics},
volume = {297},
number = {},
pages = {118376},
doi = {10.1016/j.bios.2026.118376},
pmid = {41506042},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification ; Humans ; *Liver Neoplasms/genetics/diagnosis/blood ; *Carcinoma, Hepatocellular/genetics/diagnosis/blood ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Biomarkers, Tumor/genetics ; },
abstract = {Rapid, sensitive, and accurate multi-target analysis is of great significance in biomedical detection. Traditional methods, however, often function as a mere "one-pot" collection of individual assays, ignoring the intrinsic relationships among biomarkers. To address this, we established a novel biosensing platform that integrates circle-to-circle AND logic gate circuit with an engineered CRISPR-Cas system for the early screening of hepatocellular carcinoma. In this design, the logic gate processes multiple miRNA inputs (e.g., miRNA 122 and miRNA 223), and only upon co-recognition, generates a unified DNA output. This output then directly activates a CRISPR-Cas12a system, which has been enhanced by a multi-legged crRNA assembled on a DNA tetrahedra and a cube-based framework probe to enhance the trans-cleavage activity and reaction kinetics. This integration converts complex multi-target recognition into a single, amplified detection signal, minimizing systemic interference. Under optimal conditions, the method achieved detection limits as low as 78.88 fM for miRNA 122 and 65.26 fM for miRNA 223, with serum recovery rates of 89.66 %-108.08 %. Clinical validation using 36 samples showed that excellent correlation with RT-qPCR (all R[2] > 0.98) and areas under the ROC curves of 0.8514 and 0.9244, effectively distinguishing liver cancer patients from healthy individuals. Combining high sensitivity, specificity, and clinical applicability, this strategy provides a universal platform for logic-operated multiple biomarkers analysis. Looking forward, integration with microfluidic systems could enable automated, high-throughput testing, further enhancing its utility in point-of-care diagnostics. This approach holds great promise not only for early hepatocellular carcinoma screening but also, with adaptation of the input logic, for the detection of a broad spectrum of cancers and other diseases.},
}
@article {pmid41506049,
year = {2026},
author = {Yaşar, S and Gehrke, F and Capdeville, N and Puchta, H},
title = {Recent progress in plant genome engineering: from large insertions to chromosome number changes.},
journal = {Current opinion in biotechnology},
volume = {97},
number = {},
pages = {103426},
doi = {10.1016/j.copbio.2025.103426},
pmid = {41506049},
issn = {1879-0429},
mesh = {*Genome, Plant/genetics ; *Genetic Engineering/methods ; *Chromosomes, Plant/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing ; Plants/genetics ; },
abstract = {The adaptation of the CRISPR/Cas system as a biotechnological tool has enabled a wide spectrum of targeted genome modifications. Whereas earlier approaches focused on small sequence changes, recent years have seen a shift toward larger-scale alterations. Advances in homology-directed gene targeting now enable efficient, scar-free kilobase insertions, while combining nuclease-deficient Cas effectors with recombinases or transposases allows the integration of much larger sequences. Prime editing further expands this scope, enabling inversions, replacements, and deletions spanning hundreds of kilobases to several megabases. More recently, genome engineering has reached a new stage with chromosome fission and fusion, demonstrating the feasibility of controlled karyotype restructuring. Together, these advances open new opportunities for crop improvement, from establishing reproductive barriers and mimicking evolutionary processes to trait stacking on Plant Artificial Chromosomes.},
}
@article {pmid41506374,
year = {2026},
author = {Marschhofer, M and Chen, S and Molbay, M and Winkeljann, B and Villano, E and Giancaspro, C and Kourou, A and Berninghausen, O and Rieder, S and Ungewickell, C and Beckmann, R and Popper, B and Torres, AM and Vidal, A and Merkel, OM and Carneiro, SP},
title = {Optimized lipid nanoparticles for pulmonary delivery of CRISPR/Cas9 targeting KRAS G12S in lung cancer.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114607},
doi = {10.1016/j.jconrel.2026.114607},
pmid = {41506374},
issn = {1873-4995},
mesh = {Animals ; *Lung Neoplasms/genetics/therapy ; *CRISPR-Cas Systems ; Humans ; *Nanoparticles/administration &amp; dosage/chemistry ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Lipids/chemistry/administration &amp; dosage ; Gene Editing ; A549 Cells ; *Carcinoma, Non-Small-Cell Lung/genetics/therapy ; Mice ; *CRISPR-Associated Protein 9/genetics ; Mice, Nude ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/genetics ; Female ; RNA, Messenger/administration &amp; dosage/genetics ; Apoptosis ; Cell Line, Tumor ; Liposomes ; },
abstract = {KRAS G12S mutations in non-small cell lung cancer (NSCLC) remain refractory to current targeted therapies, with few clinical options and frequent resistance. While CRISPR/Cas9 enables mutation-specific gene disruption, its pulmonary application is limited by systemic clearance, hepatic tropism, and airway mucus barriers. Here, we present lipid nanoparticles (LNPs) specifically engineered for pulmonary delivery of Cas9 mRNA and KRAS G12S-targeting sgRNA, optimized through mRNA surrogate screening and orthogonal mixture design to guide lipid composition and Cas9:sgRNA weight-to-weight ratios. Two lead LNP formulations, A6 3:1 and A8 1:1, exhibited robust critical quality attributes, including particle sizes below 120 nm, low polydispersity, near-neutral zeta potential, and over 80 % encapsulation efficiency. Cryo-TEM revealed distinct morphologies correlated with enhanced transfection. In vitro, A8 1:1 achieved up to 90 % on-target gene editing in A549 cells and a 3.6-fold increase in apoptosis, while A6 3:1 induced a 3.7-fold apoptotic response. Both formulations efficiently traversed airway mucus in air-liquid interface cultures and preserved over 80 % cell viability across doses. In vivo, repeated pulmonary administration was well tolerated, with no signs of systemic toxicity or cytokine elevation in healthy or tumor-bearing mice. In an orthotopic A549-luc lung tumor model, intratracheal delivery of A6 3:1 and A8 1:1 modestly suppressed tumor growth, with histological evidence of tumor cell apoptosis for A8 1:1. Quantification confirmed a statistically significant increase of apoptosis in the A8 1:1 group, consistent with effective KRAS disruption in vivo. Overall, lead LNPs, particularly A8 1:1, enabled efficient and localized RNA-based gene editing that induced downstream apoptotic signaling, demonstrating a preliminary, yet promising, proof-of-concept for CRISPR/Cas9 therapy in NSCLC.},
}
@article {pmid41506441,
year = {2026},
author = {Mokhles, F and Moosavi, MA and Gutierrez-Uzquiza, A and Velasco, G and Li, M and Cordani, M},
title = {Unraveling stress-adaptation pathways in cancer: Functional dissection through CRISPR-based genetic screens.},
journal = {Cancer letters},
volume = {644},
number = {},
pages = {218246},
doi = {10.1016/j.canlet.2026.218246},
pmid = {41506441},
issn = {1872-7980},
mesh = {Humans ; *Neoplasms/genetics/pathology/metabolism ; *CRISPR-Cas Systems ; Animals ; Endoplasmic Reticulum Stress/genetics ; *Genetic Testing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Tumor Microenvironment ; Unfolded Protein Response/genetics ; *Stress, Physiological ; Adaptation, Physiological/genetics ; },
abstract = {Cancer cells face a hostile microenvironment characterized by hypoxia, nutrient deprivation, endoplasmic reticulum (ER) stress, and oxidative imbalance. To cope with these challenges, they activate an interconnected network of adaptive pathways including autophagy, the unfolded protein response, metabolic reprogramming, and the integrated stress response., which promote cell survival, therapy resistance, immune evasion, and metastasis. CRISPR-based functional genomics has emerged as a powerful strategy to systematically dissect these stress-adaptive networks, enabling the identification of key regulators and vulnerabilities across diverse contexts. In this review, we first summarize tumor progression in major stress conditions and then highlight how CRISPR screening strategies ranging from genome-wide loss-of-function studies to single-cell and combinatorial platforms, are unraveling critical stress regulators. We further discuss emerging tools, model systems, and translational perspectives, underscoring how the integration of CRISPR technologies with multi-omics, artificial intelligence, and advanced preclinical models is reshaping our understanding of cancer stress biology and guiding the development of novel therapeutic strategies. Finally, we addressed how these novel dissection technologies influence translational opportunities, specifically in the context of combining stress-pathway modulators with immunotherapy and targeted therapy drugs.},
}
@article {pmid41506457,
year = {2026},
author = {Jiménez Lancho, V and Leitner, K and Agarwal, K and Krishnakumar, A and Khetan, A and Borth, N and Marx, N},
title = {CRISPR-based precise methylation of specific FUT8 promoter regions allows isolation of CHO cells with a fine-tuned glycoprofile.},
journal = {Journal of biotechnology},
volume = {410},
number = {},
pages = {341-352},
doi = {10.1016/j.jbiotec.2026.01.001},
pmid = {41506457},
issn = {1873-4863},
mesh = {CHO Cells ; Cricetulus ; Animals ; *DNA Methylation/genetics ; *Promoter Regions, Genetic/genetics ; *Fucosyltransferases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Glycosylation ; Gene Editing/methods ; },
abstract = {A major advantage of producing therapeutic proteins in mammalian cells is their ability to tailor proteins with human-like posttranslational modifications such as glycosylation, which ultimately defines aspects like stability, protein folding or immunogenicity. However, producing therapeutic proteins with a consistent and reproducible glycoprofile remains a major challenge for the biopharmaceutical industry, especially with biosimilar production. While the enzymes responsible for glycosylation of proteins have been the subject of various cell engineering approaches, tuning their gene expression to precise levels is still difficult to achieve. While CRISPR/Cas9 enabled the genetic engineering of cells to drastically overexpress or remove a target gene, CRISPR/dCas9-based epigenetic editing by targeted DNA methylation promises to stably change the expression pattern of target genes after transient transfection of the CRISPR-tool. Application of targeted DNA methylation so far has mostly been used to completely silence gene expression by fully methylating the corresponding promoter regions. Here, we aim to tune expression of the associated gene by DNA methylation of confined promoter regions and to apply this technique as a new glycoengineering approach. By coupling CRISPR-based targeted DNA methylation with lectin-FACS assisted sorting we obtained CHO cell lines with a fine-tuned phenotype. First, dCas9-DNMT3A3L in combination with one single gRNA is targeted to the FUT8 promoter to induce confined DNA methylation, resulting in a phenotypically diversified population. Next, a window sorting strategy based on lectin-stained cells using five different sorting gates spanning from low to high FUT8 expression was applied to isolate single clones with a defined phenotype. Isolated clones were phenotypically assessed and re-sorted to obtain a homogenous expression profile. The resulting clonal cell lines showed either tuned or knock-down phenotypes with varying gene expression levels. Two out of seven clones that showed tuned FUT8 gene expression were phenotypically stable over 60 days. Gene expression levels, on the other hand, showed a steady decline over time that in part, however, can be explained by the general variation of FUT8 expression in different growth phases. Importantly, glycan analysis of recombinant EpoFc produced in the tuned clonal cell lines showed ranges of 35-70 % fucosylation, demonstrating that isolated clones can produce recombinant proteins with a distinct glycosylation profile. To understand why some clones showed tuned FUT8 gene expression levels while others were completely knocked-down, we analyzed the DNA methylation status of their respective FUT8 promoter. Critical areas within the FUT8 promoter were identified, with some associated with general repression and others with the tuning of FUT8 gene expression when affected by DNA methylation. Additionally, a combination of histone marks associated with active and repressed promoters was found to potentially define clones with a fine-tuned expression. Combined, the data demonstrates that using targeted DNA methylation in a manner confined to specific promoter regions opens new engineering strategies to fine-tune gene expression in mammalian cells.},
}
@article {pmid41507205,
year = {2026},
author = {Wan, H and Kong, D and Yan, T and Zhou, Y and Liu, M and Ma, X and Zhao, T and Zhou, W and Liu, X and Yin, J and Guan, N and Ye, H},
title = {A compact and inducible dCas12f-based CRISPRa platform for programmable in vivo gene activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1447},
pmid = {41507205},
issn = {2041-1723},
support = {25J22800100//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; 32250010//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Dependovirus/genetics ; Mice ; *CRISPR-Cas Systems/genetics ; Humans ; *Transcriptional Activation/genetics ; HEK293 Cells ; *Gene Editing/methods ; Genetic Therapy/methods ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Precise activation of endogenous genes is a powerful strategy for functional genomics and therapeutic development, but current CRISPR-based transcriptional activation (CRISPRa) systems are limited by the large size of Cas proteins for adeno-associated virus (AAV) delivery. Here, we present a high-efficiency dCas12f-based transcriptional activation system (HEAL), which recruits transactivators through MS2 coat protein binding to MS2 aptamers embedded within the sgRNA scaffold. Engineered to enhance DNA binding, nuclear localization, and transactivator recruitment, HEAL induces over 100,000-fold activation of endogenous genes and outperforms existing CRISPRa systems in vitro and in vivo. We further develop red-light-inducible OptoHEAL and small-molecule-inducible ChemHEAL for remote and precise transcriptional control. AAV-delivered HEAL targeting interleukin 10 alleviates acute kidney injury in mice, while ChemHEAL-mediated activation of thymic stromal lymphopoietin reduces body weight in obese mice. HEAL provides a modular, compact, and controllable platform for endogenous gene activation with strong potential for fundamental research and gene therapy.},
}
@article {pmid41507381,
year = {2026},
author = {Gonzales, F and Schneider, C and Alexe, G and Lin, S and Khalid, D and Alvarez, M and Basanthakumar, A and Ellegast, JM and Merickel, L and Salhotra, S and Taillon, A and Giaimo, M and Wunderlich, M and Ansari, M and Perry, JA and Degar, B and Pikman, Y and Stegmaier, K},
title = {Identifying targeted therapies for CBFA2T3::GLIS2 acute myeloid leukemia.},
journal = {Leukemia},
volume = {40},
number = {2},
pages = {383-396},
pmid = {41507381},
issn = {1476-5551},
support = {R00 CA263161/CA/NCI NIH HHS/United States ; R35 CA283977/CA/NCI NIH HHS/United States ; R50 CA211404/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/drug therapy/genetics/pathology/metabolism ; Animals ; Mice ; CRISPR-Cas Systems ; Janus Kinase 2/antagonists &amp; inhibitors/genetics ; Nitriles ; *Molecular Targeted Therapy ; Pyrazoles/pharmacology ; Protein Kinase Inhibitors/pharmacology ; Xenograft Model Antitumor Assays ; Cell Proliferation ; Pyrimidines ; Cell Line, Tumor ; Apoptosis ; Drug Resistance, Neoplasm ; },
abstract = {CBFA2T3::GLIS2-positive pediatric acute myeloid leukemia (AML) remains one of the worst prognostic AML subgroups. To uncover innovative targeted therapy approaches in this disease subtype we performed genome-scale CRISPR-Cas9 screening that highlighted a strong, selective dependency on JAK2 compared to other types of cancer. Using a doxycycline-inducible JAK2 knockout (KO) system, we validated JAK2 dependency in CBFA2T3::GLIS2 cell lines, observing impaired proliferation in vitro and in vivo and apoptosis induction in vitro. Both type I (ruxolitinib) and type II (CHZ868) JAK2 inhibitors showed selective in vitro activity in CBFA2T3::GLIS2-positive AML models. To identify resistance and sensitizer mechanisms to JAK2 inhibitors, we used CRISPR-Cas9 ruxolitinib anchor screening in CBFA2T3::GLIS2 AML. sgRNAs targeting negative regulators of the MAPK pathway were enriched in the ruxolitinib-treated cells. Similarly, CBFA2T3::GLIS2 AML sublines grown to resistance under chronic ruxolitinib treatment expressed pathogenic NRAS mutations. Both approaches converged on MAPK pathway activation as a resistance mechanism to ruxolitinib treatment. Combining ruxolitinib with MEK inhibitors showed a synergistic effect in cell lines and patient-derived xenograft (PDX) cells expressing the fusion and in vivo activity in a CBFA2T3::GLIS2 AML PDX, suggesting a potential approach to target this signaling circuitry in this poor outcome AML subtype.},
}
@article {pmid41508115,
year = {2026},
author = {Xiao, Y and Li, X and Jiang, L and Zhao, Y and Wang, L and Feng, Y},
title = {Construction of Escherichia coli L-isoleucine cell factories based on propionate pathway.},
journal = {Journal of biological engineering},
volume = {20},
number = {1},
pages = {28},
pmid = {41508115},
issn = {1754-1611},
abstract = {UNLABELLED: Isoleucine, an essential branched-chain amino acid with broad applications in food, pharmaceuticals, and feed, is predominantly produced via the microbial threonine pathway, which suffers from catalytic complexity and regulatory inefficiency. The propionate pathway offers a streamlined alternative but remains unexplored for L-isoleucine biosynthesis. Here, we engineered Escherichia coli to establish the first propionate pathway-based L-isoleucine cell factory. Critical enzymes—propionyl-CoA synthase (PCS), propionyl-CoA transferase (PCT), and α-ketobutyrate synthase (OBS)—were identified for converting propionate to α-ketobutyrate. Key genes (prpE from Salmonella, pctcP from Gibberella, pctcN from Clostridium propionicum, and nifJ from Moorella thermoacetica) were integrated with the propionate transporter (prpP) and carbonic anhydrase (can) to enhance substrate utilization. ILE-5a, derived from E. coli BW25113 with deletions in brnQ, livJ, and livK, and containing specific insertions, was further modified to create ILE-5b with an additional deletion in yjiP. Plasmid-based expression in these strains, ILE-5a and ILE-5b, yielded top producers ILE-5a-P10 and ILE-5b-P10, which achieved L-isoleucine titers of 304 mg/L and 235 mg/L, respectively, in shake-flask fermentation using glucose and propionate as carbon sources. To stabilize production, the optimal gene set (pctN, nifJ, prpP, can) was genomically integrated via a transposon-encoded CRISPR-Cas system, generating mutants ILE-5a-P11 and ILE-5b-P11. Response surface methodology-optimized medium and 3-L fed-batch fermentation further elevated titers to 1.13 g/L (ILE-5a-P11) and 11.33 g/L (ILE-5b-P11). This study pioneers the propionate pathway for efficient L-isoleucine production, demonstrating its industrial potential through systematic metabolic engineering and process optimization.To our knowledge, this is the first study to design and demonstrate an E. coli platform for isoleucine production that simultaneously leverages the glutamate and propionate precursors.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13036-025-00609-6.},
}
@article {pmid41508607,
year = {2026},
author = {Wang, F and Lu, S and Zhu, C and Yang, L},
title = {PCdb: A comprehensive plant genome-editing database integrating sgRNA efficiency, off-target predictions, and epigenomic landscapes.},
journal = {Plant communications},
volume = {7},
number = {4},
pages = {101708},
pmid = {41508607},
issn = {2590-3462},
mesh = {*Genome, Plant/genetics ; *Gene Editing ; Epigenomics ; *Databases, Genetic ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; *Plants/genetics ; },
abstract = {CRISPR-Cas-based genome editing has transformed plant biotechnology by enabling precise genomic modifications for crop improvement and functional genomics. The success of these applications hinges on the design of single guide RNAs (sgRNAs) that maximize on-target efficiency while minimizing off-target effects. However, existing resources for sgRNA design and performance evaluation in plants remain fragmented and lack integration with genomic and epigenomic contexts that influence both editing efficacy and specificity. Here, we present PCdb (Plant CRISPR Database; https://gmo.sjtu.edu.cn/pcdb), a comprehensive plant-focused database that integrates experimentally validated sgRNAs, annotated genomic contexts, genome-wide off-target predictions, and multilayer epigenomic annotations. PCdb encompasses 6172 manually curated editing records from 2132 publications, covering 4320 unique sgRNAs and 6 117 424 predicted off-target sites across nine major plant species. Notably, PCdb contextualizes potential editing outcomes-both on target and off target-within the chromatin landscape by incorporating DNA methylation profiles, chromatin accessibility data, and histone modification patterns. The database features an intuitive web interface that supports flexible queries, interactive visualization tools, and comprehensive analytical modules for sgRNA efficiency assessment and off-target analysis. A case study reanalysis of Oryza sativa yield-related genes demonstrates PCdb's ability to generate a detailed performance profile by evaluating both on-target characteristics and off-target risks within their native epigenomic context. Systematic analysis of the database further identifies key sequence and chromatin features that influence editing outcomes, providing novel insights to improve gene-editing efficacy and specificity.},
}
@article {pmid41508907,
year = {2026},
author = {Liu, CF and Leon, S and Herrig, I and Wessely, O and Tang, WHW},
title = {Generation of Mice Harboring Bicc1 Conditional Null Alleles.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {1},
pages = {e70038},
pmid = {41508907},
issn = {1526-968X},
support = {R01 DK080745/DK/NIDDK NIH HHS/United States ; MF-2203-02413//G. Harold and Leila Y. Mathers Charitable Foundation/ ; R01DK080745/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; Alleles ; Mice, Knockout ; CRISPR-Cas Systems ; *Polycystic Kidney Diseases/genetics ; *RNA-Binding Proteins/genetics ; Gene Editing ; Exons ; Female ; },
abstract = {Bicaudal C1 (Bicc1) encodes an RNA-binding protein critical for many organ development and epithelial tissue homeostasis. Bicc1 null mutations have been shown to lead to the development of polycystic kidney disease (PKD) and death at an early prenatal stage. To elucidate the tissue-specific functions of Bicc1, we engineered two independent conditional knockout (cKO) mouse lines targeting distinct exonic regions of the gene. The first line was generated using a traditional embryonic stem (ES) cell-based approach, wherein loxP sites were inserted flanking exon 4 (E4), enabling Cre-mediated excision of a functionally essential coding region. The second line was created using CRISPR/Cas9 genome editing, introducing loxP sites around both exon 4 and exon 5 (E4-5) in a double-step zygote injection strategy. Both alleles were validated by PCR genotyping, sequencing, and functional recombination was confirmed via a tissue-specific Cre driver. These independent cKO models provide a robust platform for dissecting the role of Bicc1 in specific tissues and developmental stages, and offer new avenues for studying the mechanistic basis of PKD and other Bicc1-related pathologies.},
}
@article {pmid41510162,
year = {2026},
author = {Pan, J and Li, B and Wang, Y and Han, Y and Liu, G and Sun, SK},
title = {Physical stimuli-responsive CRISPR-Cas9 systems for spatiotemporally precise control of genome engineering.},
journal = {Theranostics},
volume = {16},
number = {6},
pages = {2984-3010},
pmid = {41510162},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; },
abstract = {The Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9 (CRISPR-Cas9) endonuclease system has revolutionized biology research by enabling precise, efficient, and versatile genome editing. However, achieving spatiotemporally controlled gene editing within specific organs, tissues, or cells remains a major challenge, as unregulated CRISPR-Cas9 activity can lead to severe off-target effects, hindering its clinical translation. To enhance the on-target precision and reduce the unwanted consequences of aberrant or premature CRISPR-Cas9 activation, various strategies have been developed to regulate its function at translational or post-translational stages using diverse external physicochemical stimuli. While chemical molecule-inducible CRISPR-Cas9 systems have demonstrated significant progress, most of them still suffer from inherent deficiencies, such as unsatisfactory spatiotemporal precision, irreversibility, pharmacokinetic dependence, internal disturbance, and safety concerns related to chemical inducers. By contrast, externally applied physical stimuli provide distinct advantages for triggering CRISPR-Cas9 activity, offering superior spatiotemporal precision, reversibility, and biocompatibility. These features significantly enhance the controllability, target specificity, and practical applicability of CRISPR-Cas9 systems across diverse biological settings. This review systematically explores recent advances in physical stimuli-responsive CRISPR-Cas9 platforms, detailing their design strategies, activation mechanisms, and proof-of-concept applications. Furthermore, we provide a comparative analysis of different stimulation strategies, highlighting their respective characteristics, current limitations, and future prospects. A discussion on the persistent bench-to-bedside gap is also included, aiming to guide future development toward clinically viable solutions.},
}
@article {pmid41510594,
year = {2026},
author = {Wei, J and Duan, Y and Xue, C and Zheng, L and Wei, Q and Wu, Z and Xin, H and Zeng, T and Deng, H and Fan, S and Xiong, W and Zeng, Z and Li, M and Zhou, M},
title = {Targeted demethylation of the BRD7 promoter based on CRISPR/dCas9 system inhibits the malignant progression of nasopharyngeal carcinoma.},
journal = {Clinical and translational medicine},
volume = {16},
number = {1},
pages = {e70583},
pmid = {41510594},
issn = {2001-1326},
support = {82473262//the National Natural Science Foundation of China/ ; 82403005//the National Natural Science Foundation of China/ ; 2023SK2008//the Human Provincial key Research and Development Program/ ; 2024ZZTS0160//the Free Exploration Program of Central South University/ ; 20256508//the scientific research plan project of Hunan Provincial Health Commission/ ; 111-2-12//the program of Introducing Talents of Discipline to Universities/ ; },
mesh = {Humans ; *Nasopharyngeal Carcinoma/genetics/pathology ; Promoter Regions, Genetic/genetics ; Mice ; Animals ; *Nasopharyngeal Neoplasms/genetics/pathology ; *Chromosomal Proteins, Non-Histone/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Disease Progression ; Cell Line, Tumor ; DNA Methylation/genetics ; Cell Proliferation/genetics ; Female ; Bromodomain Containing Proteins ; },
abstract = {BACKGROUND: BRD7 has been confirmed to be lowly expressed in nasopharyngeal carcinoma (NPC) tissues and exerts tumour suppressive roles. However, the molecular mechanism of the downregulation of BRD7 expression and whether the strategy of activating BRD7 expression plays anti-tumour effects still needs to be clarified.<br><br>METHODS: Methylation-specific polymerase chain reaction (PCR) was used to identify the methylation levels of BRD7 promoter. In vitro experiments were used to evaluate the effects of BRD7-targeted demethylation system on the malignant progression of NPC cells. Chromatin immunoprecipitation (ChIP)-qPCR experiment was employed to examine the regulatory mechanisms underlying the demethylation system. Xenograft tumour models were used to assess impact of this demethylation system on tumour growth in vivo and the anti-tumour effects of the lentivirus-mediated demethylation system in NPC.<br><br>RESULTS: There was hypermethylation modification in BRD7 promoter, which was negatively correlated with BRD7 expression. Next, we constructed a LentiCRISPRv2/dCas9-TET1CD-sgRNAs system targeting specific methylation sites of BRD7 promoter based on five sgRNAs, and confirmed that all five sgRNA-guided CRISPR/dCas9 systems could activate BRD7 and inhibit cell proliferation to varying degrees, among which sgRNA2&amp;sgRNA5 were the most significant. Further, we constructed NPC cell lines stably transfected with LentiCRISPRv2/dCas9-TET1CD-sgRNA2&amp;5, and confirmed that both sgRNA2&amp;sgRNA5 could promote the transcriptional activation by reducing its methylation, and inhibit the cell proliferation, migration, invasion and tumour growth in vivo of NPC, and the combination of them has a more significant demethylation, transcriptional activation and anti-tumour effect. In addition, BRD7 had hypermethylation modification in its promoter and decreased expression in NPC tissues, and both of them were negatively correlated, making it a potential diagnostic marker for NPC diagnosis.<br><br>CONCLUSIONS: The hypermethylation modification of BRD7 is an important mechanism leading to the inactivation of BRD7, and targeting demethylation of BRD7 inhibits the malignant progression of NPC, which might be a promising targeted therapeutic approach for treating NPC.},
}
@article {pmid41510755,
year = {2026},
author = {Wang, L and Su, L and Gu, W and Nie, S and Luo, J and Li, Y and Pan, A and Li, Y},
title = {A photonic crystal sensing array based on a tandem CRISPR/Cas13a system for ultra-sensitive and high-throughput detection of the CVA6 virus.},
journal = {The Analyst},
volume = {151},
number = {3},
pages = {903-912},
doi = {10.1039/d5an01178g},
pmid = {41510755},
issn = {1364-5528},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Photons ; *Biosensing Techniques/methods ; *RNA, Viral/genetics/analysis ; Fluorescent Dyes/chemistry ; High-Throughput Screening Assays/methods ; },
abstract = {Coxsackievirus A6 (CVA6) has emerged as a major cause of hand-foot-mouth disease (HFMD), yet no standardized detection method for it is currently available. Developing a simple, sensitive, and specific CVA6 test is crucial for HFMD control and safeguarding the health of at-risk children. Herein, a photonic crystal (PC) sensing array based on a tandem CRISPR/Cas13a system has been proposed for highly specific and ultra-sensitive analysis of CVA6 RNA, without the need for reverse transcription and amplification procedures. In this strategy, two crRNAs targeting CVA 6 RNA were designed and screened, and the fluorescence signal of the tandem CRISPR/Cas13a system was found to be up to 4.2 times higher than that of the non-tandem CRISPR system. The PC array with periodic nanostructures was prepared through self-deposition and further enhanced the fluorescent signal output from the tandem CRISPR system, owing to the match of the emission wavelength of the fluorescent dyes and the photonic band gap (PBG) of the PC. Benefitting from the synergistic effect of the tandem CRISPR system and PC array, as well as the high trans-cleavage activity of Cas13a protein, this engineered sensing array enables ultra-sensitive detection with a limit of detection (LOD) as low as 24.9 fM for CVA6. Meanwhile, this sensing strategy also achieved high-throughput and rapid analysis with a detection frequency of about 96 samples every 3.4 minutes. Therefore, the proposed strategy offers a simple workflow without reverse transcription or amplification, along with high sensitivity and high throughput, demonstrating strong potential for applications in biometrics and clinical diagnostics.},
}
@article {pmid41510784,
year = {2026},
author = {Hejlesen, R and Bakkeren, C and Damsgaard, C and Laursen, LS and Kjær-Sørensen, K and Corti, P and Malte, H and Oxvig, C and Fago, A},
title = {Myoglobin Affects Tissue-Specific Transcriptome, Heart Regeneration and Whole Animal Metabolic Rates.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {1},
pages = {e71424},
doi = {10.1096/fj.202503482RR},
pmid = {41510784},
issn = {1530-6860},
support = {NNF19OC0057938//Novo Nordisk Fonden (NNF)/ ; 3103-0011B//Danmarks Frie Forskningsfond (DFF)/ ; DMMTF24081545//Company of Biologists (Co_Biologists)/ ; //Graduate School Natural Science Aarhus University/ ; },
mesh = {Animals ; Zebrafish/genetics/metabolism ; *Myoglobin/genetics/metabolism ; *Regeneration/physiology ; *Transcriptome ; *Heart/physiology ; Muscle, Skeletal/metabolism ; Myocytes, Cardiac/metabolism ; *Myocardium/metabolism ; *Zebrafish Proteins/genetics/metabolism ; },
abstract = {Myoglobin (Mb) is a small haem-containing protein traditionally associated with oxygen carrier functions in cardiac and skeletal muscle. However, studies using Mb knockout mice have yielded conflicting results regarding its functional roles in vivo. Here, we used a CRISPR-Cas-generated zebrafish Mb knockout model to investigate the consequences of Mb loss across skeletal muscle types, transcriptomics profiles, and whole-animal metabolic rates under both resting and maximal exercise conditions. Mb deficiency did not alter skeletal muscle fiber composition or overall mitochondrial respiratory capacity but induced multiple tissue-specific transcriptomic changes, including downregulation of gene sets involved in respiration and differentiation pathways in the heart, while upregulating those associated with respiration and glycogen metabolism in the skeletal muscle. During cardiac regeneration following ventricle amputation in wild-type zebrafish, Mb expression was transiently suppressed, consistent with a role in maintaining the cardiomyocytes in a differentiated state. Physiologically, Mb knockout zebrafish displayed a reduced standard metabolic rate at rest, enhanced hypoxia tolerance (i.e., a lower critical oxygen tension), and increased maximal swimming speed, while maintaining unchanged maximal metabolic rate and aerobic scope relative to wild-type counterparts. Collectively, these findings show that loss of Mb in zebrafish elicits coordinated tissue-specific transcriptional changes, potentially facilitates cardiac regeneration, lowers standard metabolic rate, and enhances maximal swimming speed and hypoxia tolerance, thereby providing new insights into the multifaceted in vivo functions of Mb.},
}
@article {pmid41511319,
year = {2025},
author = {Xu, J and Zhang, J and Yang, D},
title = {Editorial: CRISPR-Based Genome Editing in Translational Research-2nd Edition.},
journal = {Cells},
volume = {15},
number = {1},
pages = {},
pmid = {41511319},
issn = {2073-4409},
support = {R42 GM122181/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Translational Research, Biomedical ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Genome editing technologies represented by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) have transformed biomedical research and therapeutic development [...].},
}
@article {pmid41511442,
year = {2026},
author = {Li, Y and Li, X and Chen, Y and Wang, Y and Zuo, Z},
title = {Differential Allosteric Modulation of Cas9 Specificity.},
journal = {Journal of chemical theory and computation},
volume = {22},
number = {2},
pages = {806-817},
doi = {10.1021/acs.jctc.5c01919},
pmid = {41511442},
issn = {1549-9626},
mesh = {Allosteric Regulation ; Molecular Dynamics Simulation ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Mutation ; },
abstract = {Both RNA- and protein-based strategies have been developed to mitigate off-target cleavage by CRISPR-Cas9, yielding noncanonical guide RNAs (gRNAs) and Cas9 variants with enhanced gene-editing precision. However, the molecular mechanisms by which such PAM-distal alterations─remote from the nuclease centers─modulate Cas9 activity and specificity remain incompletely understood. Here, we performed near-millisecond all-atom molecular dynamics simulations to elucidate how diverse PAM-distal perturbations─including gRNA truncation, base mismatching, and evolved mutations─reshape the conformational dynamics and allosteric regulation of Cas9. Despite their distinct origins, all perturbations ultimately modulate Cas9 function by altering HNH dynamics that impede the transition from the checkpoint to the catalytically active state, yet they do so through distinct allosteric routes. The 16-nt gRNA induces a pronounced REC3 reorientation toward the L2 linker and HNH domain, while PAM-distal mismatches with the 18-nt gRNA promote engagement of the unwound target DNA strand with L2─both effectively restraining HNH rotation. In contrast, evolved mutations remodel the global motional modes so that REC2 swivels inward, constraining the HNH flexibility. These perturbations delineate multiple structural paths converging on a shared allosteric outcome─HNH immobilization and catalytic suppression─thereby unifying RNA-, DNA-, and protein-level effects within a single dynamic framework linking distal structural perturbations to activity control. This work provides mechanistic insight into the regulation of Cas9 fidelity and offers principles for the design of next-generation genome editors.},
}
@article {pmid41512012,
year = {2026},
author = {Chaudhari, VR and Lin, MT and Hines, KM and Hanson, MR},
title = {Rewinding evolution in planta: A Rubisco-null platform validates high-performance ancestral enzymes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {3},
pages = {e2523360123},
pmid = {41512012},
issn = {1091-6490},
support = {DE-SC0020142//DOE Office of Basic Energy Sciences, Chemical Sciences, Geosciences, and Biosciences Division/ ; MCB-2131582//NSF (NSF)/ ; },
mesh = {*Ribulose-Bisphosphate Carboxylase/genetics/metabolism ; *Nicotiana/genetics/enzymology ; Plants, Genetically Modified/genetics ; Photosynthesis/genetics ; CRISPR-Cas Systems ; Chloroplasts/genetics ; Evolution, Molecular ; Kinetics ; Gene Knockout Techniques ; },
abstract = {Improving the photosynthetic enzyme Rubisco is a key target for enhancing C3 crop productivity, but progress has been hampered by the difficulty of evaluating engineered variants in planta without interference from the native enzyme. Here, we report the creation of a Rubisco-null Nicotiana tabacum platform by using CRISPR-Cas9 to knock out all 11 nuclear-encoded small subunit (rbcS) genes. Knockout was achieved in a line expressing cyanobacterial Rubisco from the plastid genome, allowing the recovery of viable plants. We then developed a chloroplast expression system for coexpressing both large and small subunits from the plastid genome. We expressed two resurrected ancestral Rubiscos from the Solanaceae family. The resulting transgenic plants were phenotypically normal and accumulated Rubisco to wild-type levels. Importantly, kinetic analyses of the purified ancestral enzymes revealed they possessed a 16 to 20% higher catalytic efficiency (kcat,air/Kc,air) under ambient conditions, driven by a significantly faster turnover rate (kcat,air). We have demonstrated that our system allows robust in vivo assessment of novel Rubiscos and that ancestral reconstruction is a powerful strategy for identifying superior enzymes to improve photosynthesis in C3 crops.},
}
@article {pmid41512333,
year = {2026},
author = {Zhao, J and Sui, Z and Chen, B and Wu, R and Xu, J and Dong, H},
title = {Customizable NAND Logic-Gate Biosensing System Enabled by an Engineered Methylation-CRISPR/Cas12a Consensus Sequence for Ultrasensitive DNA Methyltransferase Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2368-2378},
doi = {10.1021/acs.analchem.5c06772},
pmid = {41512333},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Site-Specific DNA-Methyltransferase (Adenine-Specific)/metabolism/analysis ; Limit of Detection ; Humans ; Logic ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {DNA methyltransferases (MTases) play crucial roles in epigenetic regulation, and their abnormal activity is closely associated with various human diseases. Here, we report a customizable NAND logic-gate biosensing platform for highly sensitive and intelligent detection of DNA adenine methyltransferase (Dam MTase). An engineered methylation-CRISPR/Cas12a consensus sequence (MCCS, 5'-TTTGATC-3') was rationally designed to integrate the Cas12a PAM site, Dam methylation site, and DpnI recognition sequence into a unified functional motif. Coupled with a primer-triggered hybridization chain reaction (HCR), multiple tandem MCCS units were generated to amplify the fluorescence signal output. In this logic circuit, Dam, SAM, and DpnI serve as three biochemical inputs, and their combined presence ("1,1,1") yields a low-fluorescence "OFF" output according to the NAND logic rule. The system exhibited a broad linear detection range with an ultralow detection limit of 0.00032 U mL[-1], outstanding selectivity toward nontarget MTases, and satisfactory recoveries (98.16-100.03%) in human serum samples. Furthermore, it enabled quantitative evaluation of Dam inhibitors, revealing IC50 values of 1.75 μM for 5-fluorouracil and 11.9 μM for penicillin G. This strategy provides a universal molecular computation-driven biosensing framework for enzyme activity analysis and inhibitor screening in complex biological systems.},
}
@article {pmid41512493,
year = {2026},
author = {Ahmad, R and Gastoldi, G and Wong, ST and Baeza Garcia, A and Caljon, G},
title = {The EamA metabolite transporter does not affect antileishmanial drug susceptibility.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {30},
number = {},
pages = {100632},
pmid = {41512493},
issn = {2211-3207},
mesh = {*Leishmania infantum/drug effects/genetics/metabolism ; *Antiprotozoal Agents/pharmacology ; *Protozoan Proteins/genetics/metabolism ; Drug Resistance/genetics ; Animals ; CRISPR-Cas Systems ; *Membrane Transport Proteins/genetics/metabolism ; Macrophages/parasitology ; Gene Editing ; Mice ; Humans ; Parasitic Sensitivity Tests ; Leishmaniasis, Visceral/parasitology/drug therapy ; },
abstract = {Leishmaniasis is a major neglected tropical disease, exists in 98 countries and constitutes a global public health threat. As chemotherapy is confronted with drug resistance and treatment failure, understanding the underlying mechanisms and continued drug discovery efforts are needed in the fight against leishmaniasis. A previous cosmid-based overexpression study suggested a role for EamA (LINF_020008400), annotated as a putative drug-metabolite transporter, in resistance to novel antileishmanial oxaboroles. To assess fitness cost and drug susceptibility, gene deficient Leishmania infantum lines were generated using CRISPR-Cas9 gene editing and overexpression from the ssu locus was achieved using the pLEXSY system. While in vitro parasite growth and survival were unchanged compared to control lines, the intracellular burden of the null mutant was lower. In vitro exposure to current antileishmanial drugs and several novel leads revealed an unchanged drug sensitivity profile in extracellular and intramacrophage assays. Similarly, the overexpression lines showed a significantly lower infection rate, but their drug susceptibility profiles showed no significant differences from the control. Collectively, these data suggest that -under the tested conditions- LINF_020008400 is not essential for parasite fitness, host cell infectivity and survival following exposure to antiparasitic drugs.},
}
@article {pmid41512567,
year = {2026},
author = {Sushmita, and Srivastava, A and Jain, G and Singh, M and Verma, PC},
title = {Targeted disruption of a cell wall-modifying gene α-Mannosidase using CRISPR-Cas9 enhances post-harvest shelf life in tomato through ABA accumulation.},
journal = {Plant physiology and biochemistry : PPB},
volume = {231},
number = {},
pages = {111017},
doi = {10.1016/j.plaphy.2026.111017},
pmid = {41512567},
issn = {1873-2690},
mesh = {*Solanum lycopersicum/genetics/metabolism/enzymology ; *Abscisic Acid/metabolism ; *Cell Wall/metabolism/genetics ; *CRISPR-Cas Systems ; *alpha-Mannosidase/genetics/metabolism ; Plant Proteins/genetics/metabolism ; Fruit/genetics/metabolism ; Gene Expression Regulation, Plant ; Reactive Oxygen Species/metabolism ; },
abstract = {Tomato ripening is a complex process regulated by transcription factors, hormones, and physiological changes. While this intricate regulation ensures desirable traits such as color, texture, and flavor, it is often accompanied by rapid post-harvest deterioration and poor shelf life, leading to significant economic losses and limiting market potential. Various cell wall-modifying enzymes, including N-glycan-processing enzyme α-Mannosidase, play crucial roles in the softening and senescence of tomato fruits. Our study shows that α-Mannosidase knockout through CRISPR/Cas9 results in fruits with enhanced firmness, longer shelf life, and improved moisture retention. Additionally, the expression of SlRIN (Ripening Inhibitor), a key regulator of ripening and several downstream genes, including those involved in cell wall degradation, ethylene biosynthesis, and signaling, was downregulated. Interestingly, in the later stages of storage, they also exhibited higher accumulation of abscisic acid (ABA) and lower accumulation of reactive oxygen species, along with better antioxidant capacity, compared to the control fruits, which may confer delayed softening and increased shelf life. Our findings highlight α-Mannosidase as a ripening-specific regulator and a promising genetic target for extending tomato shelf life, offering a sustainable strategy to minimize post-harvest losses without compromising plant development.},
}
@article {pmid41513195,
year = {2026},
author = {Luo, Q and Huang, Y and Zheng, H and Ke, W and Zheng, H and Sun, Q and Wang, M and Weng, Z},
title = {CRISPR-engineered zebrafish expression system for human type III collagen: Therapeutic efficacy in wound healing.},
journal = {International journal of biological macromolecules},
volume = {340},
number = {Pt 1},
pages = {150161},
doi = {10.1016/j.ijbiomac.2026.150161},
pmid = {41513195},
issn = {1879-0003},
mesh = {Animals ; *Zebrafish/genetics ; Humans ; *Wound Healing/drug effects/genetics ; Animals, Genetically Modified ; *CRISPR-Cas Systems/genetics ; *Collagen Type III/genetics/pharmacology ; Mice ; Recombinant Proteins/pharmacology/genetics ; Cell Proliferation ; },
abstract = {Human type III collagen (Col III) is a critical component for skin tissue repair and anti-aging, yet its heterologous expression often faces challenges such as incomplete structure and poor thermostability. Here, we established a transgenic zebrafish expression system via CRISPR/Cas9 technology, integrating the human Col3a1 gene into a non-functional region of zebrafish chromosome 4. The extraction yield of total zebrafish collagen (Col III-TC), a composite material comprising both recombinant human Col III and endogenous zebrafish collagens, was 45.76%. Structural analysis revealed intact fibrous architecture and a thermal shrinkage temperature of 71.3 °C, significantly superior to conventional systems. Functionally, Col III-TC exhibited remarkable free radical-scavenging capacity and suppressed LPS-induced inflammation in 3T3-L1 cells (downregulating Tnfα, Il1b, and Il6, while upregulating Il10), alongside promoting fibroblast proliferation. In a murine acute wound model, Col III-TC-based dressings achieved outstanding healing efficacy (>95% closure within 15 days), with histological analysis showing improved neoskin thickness and collagen deposition. The Col3a1 transgenic zebrafish system developed in this study not only provides a novel strategy for heterologous expression of fully functional human proteins, but also highlights the broad application potential of its high-yield collagen in biomedical fields, particularly in wound healing and anti-aging therapies.},
}
@article {pmid41513250,
year = {2026},
author = {Zhu, Y and Luo, S and Cui, X and Wu, J and Cheng, W and Wang, X and Zhang, Q and Tan, G and Yang, H and Zheng, Y and Peng, W},
title = {Converting an Untransformable Vibrio parahaemolyticus Isolate into a Fast Genetic Engineering Platform.},
journal = {ACS synthetic biology},
volume = {15},
number = {2},
pages = {740-747},
doi = {10.1021/acssynbio.5c00771},
pmid = {41513250},
issn = {2161-5063},
mesh = {*Vibrio parahaemolyticus/genetics ; Plasmids/genetics ; CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; Bacterial Proteins/genetics/metabolism ; Deoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; },
abstract = {The pathogenic bacterium Vibrio parahaemolyticus represents a substantial economic and public health concern; however, elucidating its virulence mechanisms has been significantly impeded by its inherent resistant to genetic manipulation, primarily attributed to sophisticated immune defense systems including restriction-modification (R-M) modules, CRISPR-Cas systems, standalone DNases, and DdmDE systems. Paradoxically, while genetic modification is essential for overcoming these barriers, the very barriers themselves obstruct DNA introduction. Our investigation focused on the V. parahaemolyticus X1 strain, where initial plasmid transformation attempts proved unsuccessful. However, low-efficiency conjugation allowed knockout of defense genes, thereby silencing the host's defense mechanisms. Our findings revealed a standalone DNase, Vpn, as the predominant obstacle to foreign DNA entry in the X1 strain, while a DdmDE system executes elimination of invaded plasmids. Leveraging these insights, we created the V. parahaemolyticus X2 strain via sequential depletion of the Vpn nuclease and the DdmDE system. Capitalizing on the bacterium's exceptional growth rate, characterized by a generation time of approximately 10.5 min, we established a highly efficient molecular cloning platform capable of creating a new plasmid construct within a single day. This work not only presents a strategic framework for genetic manipulation of previously recalcitrant bacterial species but also underscores the potential of fast-growing marine bacteria as promising candidates for next-generation biotechnological applications.},
}
@article {pmid41516041,
year = {2025},
author = {Sahu, D and Ganguly, T and Mann, A and Gupta, Y and Nynatten, LRV and Fraser, DD},
title = {Emerging Technologies for Exploring the Cellular Mechanisms in Vascular Diseases.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516041},
issn = {1422-0067},
mesh = {Humans ; *Vascular Diseases/genetics/metabolism/pathology/diagnosis ; Animals ; Gene Editing/methods ; Artificial Intelligence ; CRISPR-Cas Systems ; Single-Cell Analysis/methods ; },
abstract = {Vascular diseases (VDs) and cardiovascular diseases (CVDs) are the leading causes of morbidity and mortality worldwide. Current diagnostic and therapeutic approaches are limited by insufficient resolution and a lack of mechanistic understanding at the cellular level. Traditional imaging and clinical assays do not fully capture the dynamic molecular and structural complexities underlying vascular pathology. Recent technological innovations, including single-cell and spatial transcriptomics, super-resolution and photoacoustic imaging, microfluidic organ-on-chip platforms, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene editing, and artificial intelligence (AI), have created new opportunities for investigating the cellular and molecular basis of VDs. These techniques enable high-resolution mapping of cellular heterogeneity and functional alterations, facilitating the integration of large-scale data for biomarker discovery, disease modeling, and therapeutic development. This review focuses on evaluating the translational readiness, limitations, and potential clinical applications of these emerging technologies. Understanding the cellular and molecular mechanisms of VDs is essential for developing targeted therapies and precise diagnostics. Integrating single-cell and multiomics approaches highlights disease-driving cell types and gene programs. Optogenetics and organ-on-chip platforms allow for controlled manipulation and physiologically relevant modeling, while AI enhances data integration, risk prediction, and clinical interpretability. Future efforts should prioritize multi-center, large-scale validation studies, harmonization of assay protocols, and integration with clinical datasets and human samples. Multi-omics approaches and computational modeling hold promise for unraveling disease complexity, while advances in regulatory science and digital simulation (such as digital twins) may further accelerate personalized medicine in vascular disease research and treatment.},
}
@article {pmid41516323,
year = {2025},
author = {Evseev, PV and Podoprigora, IV and Chaplin, AV and Khabadze, ZS and Malkov, AA and Kafarskaia, LI and Shagin, DA and Urban, YN and Borisova, OY and Efimov, BA},
title = {Bulleidia extructa PP_925: Genome Reduction, Minimalist Metabolism, and Evolutionary Insights into Firmicutes Diversification.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516323},
issn = {1422-0067},
mesh = {*Genome, Bacterial ; Phylogeny ; *Evolution, Molecular ; Humans ; *Bacillota/genetics/metabolism/classification ; },
abstract = {Bulleidia extructa strain PP_925, isolated from the periodontal pocket of a patient with periodontitis, is a Gram-positive Bacillota with an unusually compact genome of 1.38 Mb. Phylogenomic analyses place PP_925 within Erysipelotrichales and show close relatedness of Bulleidia to Solobacterium and Lactimicrobium, as well as the existence of previously undescribed related clades. The metabolic repertoire of PP_925 is strongly reduced: it retains glycolysis, the phosphotransacetylase-acetate kinase pathway, and arginine catabolism but lacks the tricarboxylic acid cycle and most de novo biosynthetic pathways for amino acids, nucleotides, fatty acids, cofactors, and vitamins, implying reliance on salvage and cross-feeding. Phylogenetic inference indicates independent peptidoglycan losses in multiple mycoplasma Erysipelotrichia-related lineages, while PP_925 has retained an ancestral Gram-positive cell wall despite extensive genomic reduction. The genome preserves systems crucial for host interaction and adaptability, including a horizontally acquired tad locus encoding type IV pili, a comG competence system, and several adherence-associated virulence factors. Defense mechanisms are diverse and include a CRISPR-Cas II-A system, a type II restriction-modification module adjacent to Gao_Qat-like genes, and the Wadjet system in a genome without prophages; CRISPR spacers indicate repeated encounters with Bacillota phages. Comparative genomics of PP_925 and related strains reveals a small core genome with lineage-specific adhesion and defense modules, indicating recent shared ancestry combined with adaptive flexibility under substantial genome reduction.},
}
@article {pmid41516361,
year = {2026},
author = {Deriglazova, IO and Shepelev, MV and Kruglova, NA and Georgiev, PG and Maksimenko, OG},
title = {The Chimeric Nuclease SpRYc Exhibits Highly Variable Performance Across Biological Systems.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516361},
issn = {1422-0067},
support = {075-15-2024-539//Ministry of Science and Education of the Russian Federation/ ; },
mesh = {Animals ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; HEK293 Cells ; Drosophila melanogaster/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {The CRISPR-Cas9 system has significantly advanced genome editing but remains constrained by its requirement for specific protospacer adjacent motifs (PAMs). To overcome this limitation, PAM-relaxed nucleases, including the novel near-PAMless chimeric SpRYc, have been developed. Here, we evaluated SpRYc editing activity across multiple experimental systems, including human HEK293 and CEM-R5 cells, as well as Drosophila melanogaster S2 cells and embryos. In HEK293 cells, SpRYc exhibited broad PAM compatibility, enabling editing at non-canonical PAMs, albeit with reduced and variable efficiency at canonical NGG sites compared to SpCas9. This context dependency was more pronounced in CEM-R5 T cells, where SpRYc activity at endogenous CXCR4 and B2M loci was largely restricted to NGG PAMs. In contrast, unlike SpCas9, SpRYc displayed negligible genome-editing activity in Drosophila embryos in vivo. Notably, the transcriptional activator dSpRYc-VPR showed robust activity in Drosophila S2 cells at both canonical and non-canonical PAMs. Reduced chromatin occupancy of dSpRYc-VPR suggests a balance between expanded PAM recognition and DNA-binding stability, providing a mechanistic explanation for context-dependent performance of SpRYc. Overall, our results highlight that expanded targeting flexibility comes at the cost of variable efficiency, underscoring the need for extensive locus- and context-specific validation of PAM-relaxed genome-editing tools.},
}
@article {pmid41516375,
year = {2026},
author = {Firdaus, F and Yadav, V and Ramakrishnan, M and Wasi, A and Ganie, IB and Upadhyay, A and Shahzad, A and Ahmad, Z},
title = {2-Hydroxy-4-Methoxybenzaldehyde (2H4MB): Integrating Cell Culture, Metabolic Engineering, and Intelligent Genome Editing.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516375},
issn = {1422-0067},
support = {Y20240114//Young Foreign Talent Program/ ; },
mesh = {*Metabolic Engineering/methods ; *Gene Editing/methods ; *Benzaldehydes/metabolism/chemistry ; Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Biosynthetic Pathways ; },
abstract = {2-Hydroxy-4-Methoxybenzaldehyde (2H4MB) is a valuable aromatic compound with applications in flavour, fragrance, and pharmaceuticals. Because of its endangered status and root-specific accumulation, its production in native plants is restricted. In order to increase 2H4MB yield, this study emphasises recent developments in metabolic engineering, synthetic biology, in vitro culture methods, and AI-assisted route prediction. This review discussed about how CRISPR-based genome editing can be used to modify important biosynthetic genes and regulatory components, as well as how predictive machine learning techniques can be used to improve production conditions. Inadequate genetic resources, poorly understood biosynthetic pathways, and a dearth of reliable transformation systems are among the present constraints. The work highlights the importance of using integrative plant biotechnology techniques to fully realise the industrial and medicinal potential of this underutilised chemical.},
}
@article {pmid41516423,
year = {2026},
author = {Kongsomboonchoke, P and Ariyachet, C and Kaewsapsak, P and Sirichindakul, P and Tangkijvanich, P},
title = {Development of a Sensitive and Specific RPA-CRISPR/Cas12a Assay for Intrahepatic Quantification of HBV cccDNA.},
journal = {International journal of molecular sciences},
volume = {27},
number = {1},
pages = {},
pmid = {41516423},
issn = {1422-0067},
support = {//Second Century Fund (C2F)/ ; //Center of Excellence in Hepatitis and Liver Cancer/ ; },
mesh = {Humans ; *Hepatitis B virus/genetics ; *DNA, Circular/genetics/analysis ; *CRISPR-Cas Systems/genetics ; *DNA, Viral/genetics/analysis ; *Hepatitis B/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Liver/virology ; Sensitivity and Specificity ; Recombinases/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A-D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R[2] = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance.},
}
@article {pmid41517680,
year = {2026},
author = {Rehman, SU and Abbas, GH},
title = {CRISPR/CAS9-based gene editing in cancer therapy: A systematic review and meta-analysis on current status and future directions.},
journal = {Medicine},
volume = {105},
number = {2},
pages = {e47114},
pmid = {41517680},
issn = {1536-5964},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems ; *Neoplasms/therapy/genetics ; Humans ; *Genetic Therapy/methods/trends ; },
abstract = {BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has recently been discovered for gene editing and cancer therapy and its applications are expanding. This review and meta-analysis aim to assess the present and future of CRISPR/Cas9 based gene editing in cancer treatment and the way forward.<br><br>METHODS: The search was conducted in PubMed from 2015 to 2025 and 89 relevant studies were identified. The study design, CRISPR/Cas9 targets, delivery methods, therapeutic efficacy and limitations were extracted from the studies.<br><br>RESULTS: We reviewed the efficacy, challenges, and potential for translation of CRISPR/Cas9 in oncogene and tumor suppressor gene targeting and immune modulation. Several preclinical researches showed that CRISPR/Cas9 mediated disruption of oncogenes or restoration of tumor suppressor genes led to significant tumor regression. The evaluation was also extended to off target effects and integration with immunotherapy.<br><br>CONCLUSION: From the findings of this work, it can be concluded that CRISPR/Cas9 is a promising tool, but there are several limitations including off target effects, delivery systems and ethical issues that need to be solved in order to improve the clinical significance.},
}
@article {pmid41517973,
year = {2026},
author = {Jin, G and Li, H and Yu, H and Gu, Z and Cui, H and Chen, J and Li, X},
title = {Asp-2078-Gly mutation in ACCase confers quizalofop-p-ethyl resistance in Eleusine indica and establishment of a LAMP-CRISPR/Cas12a visual genotyping assay for the target mutation.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3374-3383},
doi = {10.1002/ps.70468},
pmid = {41517973},
issn = {1526-4998},
support = {//the Key Project in Soybean Bio breeding and Commercialization/ ; //the China Agriculture Research System/ ; 2022ZD04021//the Sci-Tech Innovation 2030 Agenda/ ; },
mesh = {*Herbicide Resistance/genetics ; *Acetyl-CoA Carboxylase/genetics/metabolism ; *Eleusine/genetics/drug effects/enzymology ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; *Herbicides/pharmacology ; Mutation ; *Genotyping Techniques/methods ; *Plant Proteins/genetics/metabolism ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Eleusine indica is a widespread, competitive weed causing yield losses in major crops. Repeated use of acetyl-CoA carboxylase (ACCase)-inhibiting herbicides in cotton fields has led to the evolution of resistant populations, posing a growing threat to cotton production in China. This study aimed to elucidate the target-site resistance mechanism of E. indica to quizalofop-p-ethyl and establish a rapid visual detection method based on the identified mutation.<br><br>RESULTS: The NJC-R population showed resistance to quizalofop-p-ethyl (resistance index&#x2009;=&#x2009;5.5). Gene sequencing revealed that an Asp-2078-Gly mutation in ACCase was one of the mechanisms underlying resistance. Loop-mediated isothermal amplification (LAMP) combined with the CRISPR/Cas12a system was developed to detect this mutation in E. indica. This method not only enabled genotype discrimination (wild-type, heterozygous, homozygous mutant), but also provided visual results within 70&#x2009;min, exhibiting superior performance compared with the derived cleaved amplified polymorphic sequences assay. In addition, this method eliminated false positives from nonspecific LAMP amplification, was ~100-fold more sensitive than a polymerase chain reaction, and the assay results were 100% concordant with Sanger sequencing for the 50 samples tested.<br><br>CONCLUSION: This study confirmed that the Asp-2078-Gly mutation confers quizalofop-p-ethyl resistance in E. indica from the cotton field in China, and LAMP-CRISPR/Cas12a was first applied for detecting ACCase target-site mutations in E. indica. Given its rapidity and high accuracy, this technique has the potential to be applied for resistance monitoring and to guide rational herbicide application. &#xa9; 2026 Society of Chemical Industry.},
}
@article {pmid41518077,
year = {2026},
author = {Liu, Y and Wang, H and Chen, L and Wu, X and Xu, Z and Huang, Q and Zhang, H and Cao, X and Liang, X and Zhong, X and Luo, C},
title = {CRISPR-Cas9-Generated TXNDC15 c.560delA Homozygous Mouse Model Exhibits Meckel-Gruber Syndrome Phenotype.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {1},
pages = {e70040},
pmid = {41518077},
issn = {1526-968X},
support = {SZSM202311005//Sanming Project of Medicine in Shenzen Municipality/ ; 2021A1515012495//Natural Science Foundation of Guangdong Province/ ; JCYJ20210324135009024//Shenzhen Science and Technology Innovation Commission/ ; },
mesh = {Animals ; Mice ; *Polycystic Kidney Diseases/genetics/pathology ; CRISPR-Cas Systems ; *Ciliary Motility Disorders/genetics/pathology ; *Encephalocele/genetics/pathology ; Disease Models, Animal ; Phenotype ; Homozygote ; *Retinitis Pigmentosa/genetics/pathology ; Frameshift Mutation ; },
abstract = {To determine whether TXNDC15 variation causes Meckel-Gruber syndrome (MKS), we assessed the pathogenicity of the frameshift variant c.560delA. A CRISPR-Cas9 generated mouse model carrying the equivalent Txndc15 c.512delA mutation was analyzed at embryonic day 15.5. Homozygous Txndc15[mt/mt] embryos displayed the complete MKS phenotype-fetal lethality, exencephaly, omphalocele, post-axial polydactyly, and polycystic kidneys-together with markedly reduced TXNDC15 protein in brain, liver, and kidney. These findings confirm TXNDC15 as a bona fide MKS disease gene.},
}
@article {pmid41518477,
year = {2026},
author = {Ghazaei, C},
title = {The role of bacteriophages and CRISPR-Cas in combating multidrug-resistant bacteria.},
journal = {Natural products and bioprospecting},
volume = {16},
number = {1},
pages = {14},
pmid = {41518477},
issn = {2192-2195},
abstract = {The alarming increase of multidrug-resistant (MDR) bacteria presents a serious global health crisis, reducing the effectivenessof traditional antibiotics and requiring alternative therapeutic strategies. Among the most promising innovations are bacteriophages-viruses that specifically infect bacteria-and CRISPR-Cas systems, molecular tools enabling precise genome editing. These technologies individually offer targeted antibacterial activity with minimal disturbance to the host microbiota. When combined, they forma synergistic platform capable of overcoming many limitations of conventional antibiotics, including broad-spectrum activity, resistance development, and limited adaptability. This review examinesmechanisms of bacterial resistance, the biological foundation of bacteriophages and CRISPR-Cas systems, and their application in fighting MDR pathogens. However, significant challenges remain, including delivery barriers, off-target effects, regulatory uncertainty, and public acceptance of gene-editing tools. Antimicrobial resistance now tanks among the top threats to global health, with an estimated burden exceeding one million deaths annually, surpassing many other infectious diseases. The article concludes with a discussion of the clinical prospects of phage-CRISPR therapies and highlights key areas for future research. By merging the specificity of phages with the programmable strength of CRISPR, these biotechnological advances provide a powerful and approach to address the growing threat of antibiotic resistance.},
}
@article {pmid41518500,
year = {2026},
author = {Álvarez, E and Franco-Zorrilla, JM},
title = {Targeted DNA Affinity Purification for Quantifying DNA-Binding Specificities of Transcription Factors.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2985},
number = {},
pages = {89-104},
pmid = {41518500},
issn = {1940-6029},
mesh = {*Transcription Factors/metabolism/genetics ; Binding Sites ; *DNA, Plant/metabolism/genetics ; High-Throughput Nucleotide Sequencing/methods ; Protein Binding ; *Chromatography, Affinity/methods ; Gene Expression Regulation, Plant ; *DNA/metabolism ; Arabidopsis/genetics/metabolism ; },
abstract = {Plant growth relies on flexible gene regulation to adapt to environmental changes. This process is ultimately controlled by transcription factors (TFs), which bind to specific DNA motifs, known as TF-binding sites (TFBS), located in the gene regulatory regions to regulate their expression. These interactions play crucial roles in plant development and responses to environmental cues, as well as in plant evolution and domestication, making both cis- (i.e., TFBS) and trans-regulatory factors as potential molecular targets in plant breeding for traits such as yield, quality, and stress resilience. These biotechnological approaches require precise knowledge of the target gene sets and TFBS specifically recognized by TFs. Recent advances in high-throughput sequencing techniques have enabled precise identification of TF target genes, especially thanks to methodologies that combine the main features of both in vitro and in vivo approaches. However, small scale and targeted approaches are still needed to evaluate the relative contribution of specific nucleotide positions in TF recognition. In this chapter, we describe a modified version of DNA Affinity Purification sequencing (DAP-seq) that replaces genomic DNA with a PCR-generated library of TFBS variants. This approach, termed targeted-DAP, allows a flexible and quantitative analysis of TF-binding using next-generation sequencing. Additionally, expressing TFs in Escherichia coli provides an economical source of proteins, enabling scalable and cost-effective analysis of DNA-binding specificities. We showed the benefits of this technique to demonstrate the contribution of the genomic context around the TFBS for specific recognition of a bHLH TF. Development of targeted DAP-seq would be of interest for the evaluation of nucleotide variation-either allelic or generated by CRISPR/Cas-within TFBS in TF recognition with predictable consequences on plant phenotypes.},
}
@article {pmid41519779,
year = {2026},
author = {Lim, MYT and Tan, C and Subhramanyam, CS and Teo, SJ and DeFalco, L and Pasaribu, SK and Koh, CH and Rayamajhi, D and Chi, J and Li, S and Wee, KB and Roy, S and Huber, RG and Aw, SS},
title = {A programmable ribozyme for RNA signal transduction.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1428},
pmid = {41519779},
issn = {2041-1723},
support = {H20C6a0034//Agency for Science, Technology and Research (A*STAR)/ ; UIBR//Agency for Science, Technology and Research (A*STAR)/ ; Cell and Gene Therapy Flagship Grant C253623010//Agency for Science, Technology and Research (A*STAR)/ ; IAF-PP H20C6a0034//Agency for Science, Technology and Research (A*STAR)/ ; OFYIRG16may045//MOH | National Medical Research Council (NMRC)/ ; Core funding//A*STAR | Institute of Molecular and Cell Biology (Institute of Molecular and Cell Biology - A STAR)/ ; },
mesh = {*RNA, Catalytic/metabolism/genetics/chemistry ; Humans ; Animals ; Zebrafish/genetics/embryology ; Aptamers, Nucleotide/metabolism/genetics ; CRISPR-Cas Systems/genetics ; *Signal Transduction/genetics ; Gene Editing/methods ; HEK293 Cells ; MicroRNAs/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA/metabolism/genetics ; },
abstract = {RNA detection applications can be augmented if a sensed RNA can be directly functionally transduced. However, there is no generalisable approach that allows an RNA trigger itself to directly activate diverse non-coding RNA effectors. Here, we report engineering of a programmable, RNA trigger-activated, dual-site self-cleaving ribozyme with modular sensing domain and cleavage product. This platform, UNlocked by Activating RNA (UNBAR), is entirely encoded within one RNA strand. The ribozyme can be designed to be almost completely inactive in absence of trigger, and to exhibit single-nucleotide trigger specificity. UNBAR ribozymes carry out cell-free sensing and protein-free amplification of microRNA and viral RNA sequences, and trigger-dependent release of ncRNA effectors sgRNA, shRNA and aptamer. We demonstrate RNA detection and functional transduction by a cleaved aptamer, whose fluorescence can be directly read out as a function of trigger RNA. We further engineer the ribozyme for function in cells, and demonstrate trigger-dependent regulation of CRISPR-Cas9 editing by sgRNA-embedded ribozymes in zebrafish embryos and human cells. UNBAR is a first-in-class modality with potential to be developed into a versatile platform for synthetic biology, diagnostics and gene regulation.},
}
@article {pmid41519853,
year = {2026},
author = {Michael Deans, PJ and Retallick-Townsley, KG and Li, A and Seah, C and Johnson, J and Garcia Gonzalez, J and Cao, E and Schrode, N and Yu, A and Cartwright, S and Voloudakis, G and Zhang, W and Wang, M and Fullard, JF and Girdhar, K and Stahl, E and Akbarian, S and Zhang, B and Roussos, P and O'Reilly, P and Huckins, LM and Brennand, KJ},
title = {Functional implications of polygenic risk for schizophrenia in human neurons.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1355},
pmid = {41519853},
issn = {2041-1723},
support = {R01ES033630//U.S. Department of Health &amp; Human Services | NIH | National Institute of Environmental Health Sciences (NIEHS)/ ; R56MH101454//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; T35 DK104689/DK/NIDDK NIH HHS/United States ; R01 ES033630/ES/NIEHS NIH HHS/United States ; R56 MH101454/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *Schizophrenia/genetics ; *Multifactorial Inheritance/genetics ; *Neurons/metabolism ; Induced Pluripotent Stem Cells/metabolism ; *Genetic Predisposition to Disease ; Genome-Wide Association Study ; CRISPR-Cas Systems ; Transcriptome ; Polymorphism, Single Nucleotide ; },
abstract = {Genome wide association studies of schizophrenia reveal a complex polygenic risk architecture comprised of hundreds of risk variants; most are common in the population, non-coding, and act by genetically regulating the expression of one or more gene targets ("eGenes"). It remains unclear how the myriad genetic variants that are predicted to confer individually small effects combine to yield substantial clinical impacts in aggregate. Here, we demonstrate that convergence (i.e., the shared downstream transcriptomic changes with a common direction of effect), resulting from one-at-a-time perturbation of schizophrenia eGenes, influences the outcome when eGenes are manipulated in combination. In total, we apply pooled and arrayed CRISPR approaches to target 21 schizophrenia eGenes in human induced pluripotent stem cell-derived glutamatergic neurons, finding that functionally similar eGenes yield stronger and more specific convergent effects. Points of convergence constrain additive relationships between polygenic risk loci: consistent with a liability threshold model, combinatorial perturbations of these same schizophrenia eGenes reveal that pathway-level convergence predicts when observed effects will fail to sum to levels predicted by an additive model. Targeting points of convergence as novel therapeutic targets may prove more efficacious than individually reversing the effects of multiple risk loci.},
}
@article {pmid41519897,
year = {2026},
author = {Boutin, J and Fayet, S and Marin, V and Bergès, C and Riandière, M and Toutain, J and Lamrissi-Garcia, I and Thibault, C and Cappellen, D and Dabernat, S and Poulet, A and Francillette, M and Droin, N and Debeissat, C and Brunet de la Grange, P and Moreau-Gaudry, F and Bedel, A},
title = {Single-cell multiplex approaches deeply map ON-target CRISPR-genotoxicity and reveal its mitigation by palbociclib and long-term engraftment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1429},
pmid = {41519897},
issn = {2041-1723},
mesh = {*Piperazines/pharmacology ; Animals ; *Pyridines/pharmacology ; Humans ; *Single-Cell Analysis/methods ; Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; Polymorphism, Single Nucleotide ; Hematopoietic Stem Cells/drug effects/metabolism ; Loss of Heterozygosity ; Female ; },
abstract = {Genome editing by CRISPR-Cas9-nuclease is promising for gene therapy. However, safety concerns remain. Monitoring ON-target genotoxicity is essential, especially to assay megabasic rearrangements at the targeted locus. Here, we developed a combined single-cell resolution approach with DNA sequencing focused on single nucleotide polymorphism (scSNP-DNAseq), micronuclei and LOH cytometry-reporter assays. This sensitive multiplexed strategy enables the sensitive monitoring of CRISPR-mediated genotoxicity in primary cells. Using this approach, we detect, map and characterize various types of induced-losses of heterozygosity and assess editing-associated chromosomal instability. Importantly, palbociclib prevents the appearance of such genomic rearrangements in hematopoietic stem cells without impairing cell fate or graft capability. Conversely, short-term risk is significantly increased with DNA-PKcs inhibitor AZD7648. Fortunately, targeting HBG1/2p, scSNP-DNA-seq reveals that ON-target genotoxic events are no longer detectable after long-term xenografts. This work demonstrates that scSNP-DNA-seq should be routinely implemented to monitor chromosomal rearrangements before and after CRISPR-edited cell infusions.},
}
@article {pmid41520043,
year = {2026},
author = {Senger, J and Keutgen, M and Roth, N and Seitl, I and Fischer, L},
title = {Toward food-grade production of the Bacteroides helcogenes protein-glutamine glutaminase with an optimized Bacillus subtilis strain.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {8},
pmid = {41520043},
issn = {1432-0614},
mesh = {*Bacillus subtilis/genetics/metabolism ; *Glutaminase/genetics/biosynthesis/metabolism ; *Bacteroides/enzymology/genetics ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Bioreactors/microbiology ; },
abstract = {Protein-glutamine glutaminases (PGs; EC 3.5.1.44) have gained attention in the food industry due to their application in plant protein products. The recently discovered PG from Bacteroides helcogenes (PGB) has especially been shown to provide promising characteristics for improving the techno-functional properties of plant proteins. A prerequisite for food enzymes, such as the PG, is their production with an expression host that meets food safety and yield requirements. The antibiotic-free and secretory production of the PGB was targeted in this study using the undomesticated Bacillus subtilis 007. The CRISPR/Cas9-mediated approach enabled specific genomic PGB integrations, while simultaneously deleting unwanted B. subtilis traits. Firstly, the PGB expression cassette was integrated into the sigF gene, leading to an asporogenic strain and extracellular activity of 4.1 µkat/Lculture in bioreactor cultivations. However, excessive foaming hampered the production process tremendously. Consequently, a second PGB copy was integrated into the sfp locus, which is involved in the production of lipopeptides, such as surfactin. As a result, the PGB activity was increased to 5.4 µkat/Lculture, and foaming during cultivation was reduced significantly. The introduction of a third PGB copy for preventing cell motility did not increase production; however, the integration into the well-established amyE locus improved the PGB yield during reactor cultivations. A final extracellular activity of 9.5 µkat/Lculture was reached. The multiple genomic integrations of the PGB gene enabled the efficient PGB secretion in an optimized B. subtilis host without the need for antibiotics. KEY POINTS: • Site-specific PGB integration enabled by genome sequencing of B. subtilis 007. • Antibiotic-free and secretory PGB production with an optimized B. subtilis host. • Increased PGB production reaching 9.5 µkat/Lculture.},
}
@article {pmid41520927,
year = {2026},
author = {Nishizawa, H and Daimon, T},
title = {DIL-CRISPR: a practical approach to mitigate G0 mosaic lethality in insect gene editing.},
journal = {Insect biochemistry and molecular biology},
volume = {188},
number = {},
pages = {104492},
doi = {10.1016/j.ibmb.2026.104492},
pmid = {41520927},
issn = {1879-0240},
mesh = {Animals ; *Gene Editing/methods ; *Spodoptera/genetics/growth &amp; development ; *CRISPR-Cas Systems ; *Mosaicism ; Larva/genetics/growth &amp; development ; Genes, Lethal ; },
abstract = {Genome editing in insects is typically conducted by injecting genome editing reagents into early embryos, producing generation zero (G0) individuals that develop as genetic mosaics. Targeting genes whose disruption induces mosaic lethality is therefore challenging, since most or all G0 individuals frequently fail to survive to adulthood, preventing germline transmission of edited alleles. Here, we present a straightforward and practical approach, DIL-CRISPR, to mitigate G0 mosaic lethality by systematically diluting the CRISPR/Cas9 injection mix. Using the tobacco cutworm Spodoptera litura and the juvenile hormone receptor gene Met1 as a benchmark, we demonstrate that dilution of the injection mix lessens the severity of larval-pupal mosaic phenotypes and increases G0 survival in a dose-dependent manner. Amplicon sequencing further showed that somatic mutation frequencies decline with dilution, while germline mutation rates remain sufficient to establish mutant lines. Notably, we detected a substantial discrepancy between somatic and germline editing efficiencies, likely reflecting selective loss of highly edited, lethal mosaics before they reach adulthood. We conclude that DIL-CRISPR therefore offers a reliable means to balance G0 survival with germline editing, converting an empirically used dilution practice into a generalizable strategy. Overall, this approach provides a practical solution for generating mutant lines of mosaic lethal genes and is broadly applicable across diverse insect species, facilitating functional genetic studies in non-model insects.},
}
@article {pmid41521358,
year = {2026},
author = {S Marques, B and Vitorino, C and V Ventura, F},
title = {CRISPR Applications in HIV Management - Prevention, Diagnosis, Monitoring and Treatment.},
journal = {Current HIV/AIDS reports},
volume = {23},
number = {1},
pages = {1},
pmid = {41521358},
issn = {1548-3576},
mesh = {Humans ; *HIV Infections/diagnosis/prevention &amp; control/therapy ; *CRISPR-Cas Systems ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Despite global efforts to combat the human immunodeficiency virus (HIV) epidemic, acquired immunodeficiency syndrome (AIDS) still claims one life every minute, underscoring the persistent need for improved control strategies. Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated protein (Cas) technologies have emerged as promising tools that may transform HIV management. The objective of this review is to summarise recent advancements in CRISPR/Cas-based approaches for HIV prevention, diagnosis, monitoring, and treatment, and to evaluate their potential and current challenges. A systematic literature search was conducted to identify relevant CRISPR/Cas applications in HIV infection. In prevention, CRISPR/Cas strategies aim to hinder viral integration and enhance host immune response, although substantial development is required before clinical translation. In diagnosis, CRISPR/Cas methods show high specificity and sensitivity, yet their reliance on specialised equipment and expertise limits their accessibility. In HIV monitoring, CRISPR/Cas-based methods have not yet demonstrated superiority over the quantitative PCR. In treatment, two ongoing clinical trials - one targeting a viral co-receptor on hematopoietic stem cells (HSCs) and the other excising proviral DNA - illustrate the potential of CRISPR/Cas-mediated cures, despite challenges such as low editing efficiency and off-target effects. Overall, CRISPR/Cas technologies hold considerable promise for advancing HIV management, but issues of accessibility, affordability, and scalability must be addressed to ensure global impact.},
}
@article {pmid41521468,
year = {2026},
author = {Hsieh, JA and Wu, FH and Yang, DX and Wu, AE and Liu, CA and Chen, CH and Lin, SZ and Lin, YJ and Lin, CS},
title = {Protoplast-Based Functional Genomics and Genome Editing: Progress, Challenges and Applications.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {4},
pages = {2183-2199},
pmid = {41521468},
issn = {1365-3040},
support = {//Academia Sinica/ ; //Buddhist Tzu Chi Medical Foundation/ ; NSTC 113-2313-B-001-006-//National Science and Technology Council, Taiwan/ ; NSTC 114-2628-B-002-008-//National Science and Technology Council, Taiwan/ ; NSTC 114-2314-B-303-006-//National Science and Technology Council, Taiwan/ ; },
mesh = {*Protoplasts/physiology/metabolism ; *Gene Editing/methods ; *Genomics/methods ; *Genome, Plant ; Crops, Agricultural/genetics ; CRISPR-Cas Systems ; },
abstract = {Protoplast-based systems provide a powerful and versatile platform for exploring how plants sense and respond to their environment. By enabling the direct delivery of proteins, DNA, and RNA into plant cells after cell wall removal, this approach facilitates precise molecular dissection of signaling, stress adaptation, and gene regulation across both model species and economically important crops. In this review, we analyzed 1050 published articles and categorizing them by delivery methods, research focus, plant species, and tissue types. We further highlight recent advances, including the application of single-cell transcriptomics, which provides unprecedented resolution for dissecting cellular responses and offers deeper insights into the mechanisms underlying stress resilience. Importantly, protoplast regeneration is gaining renewed attention not only as a model system for studying cellular reprogramming but also as a practical platform for crop improvement. Applications of protoplast regeneration include protoplast fusion, which integrates nuclear and organellar DNA/genomes from divergent parents to accelerate breeding and enhance tolerance to both biotic and abiotic stresses. Another important application is CRISPR/Cas ribonucleoprotein (RNP)-based editing targeting stress-resilience-related genes. In asexually propagated or highly heterozygous perennial crops with limited sexual reproduction, protoplast-based RNP delivery offers a viable and regulation-compliant strategy. This approach may help address public concerns over transgenic technologies while enabling the rapid development of stress-tolerant cultivars.},
}
@article {pmid41521661,
year = {2026},
author = {Newman, A and Starrs, L and Burgio, G},
title = {Nuc domain electrostatics drive the trans cleavage activity of CRISPR-Cas12a.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41521661},
issn = {1362-4962},
support = {2018833//National Health and Medical Research Council/ ; 2027987//National Health and Medical Research Council/ ; //The Gordon and Gretel Bootes foundation/ ; //National Computing Infrastructure/ ; //Australian Government Research Training Program/ ; },
mesh = {Static Electricity ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; Catalytic Domain ; Gene Editing ; DNA Cleavage ; DNA/metabolism ; Protein Domains ; Kinetics ; Amino Acid Substitution ; Models, Molecular ; Mutation ; },
abstract = {The trans cleavage activity of Cas12a has been extensively used for the detection of biomolecules. Different Cas12a orthologues exhibit faster or slower trans cleavage kinetics, making some orthologues more suited for sensitive molecular detection. Ionic strength of reaction buffers and mutations that change the electrostatic environment near the RuvC active site have also been reported to strongly influence trans cleavage kinetics. Studying three commonly used Cas12a orthologues (FnCas12a, AsCas12a, and LbCas12a), we report that electrostatic interactions near the RuvC active site are critical for their trans cleavage activity. Alanine substitution of arginine and lysine residues in the Nuc domain can abolish trans cleavage while modestly reducing cis cleavage. Substitutions in the RuvC lid and substitutions to introduce positively charged residues in the Nuc could enhance both cis and trans cleavage. These Cas12a variants improved DNA detection and genome editing efficacy. Overall, this study provides a blueprint for rationally engineering the DNase activities of Cas12a.},
}
@article {pmid41522435,
year = {2025},
author = {Zalila-Kolsi, I},
title = {Engineered bacteria as living therapeutics: Next-generation precision tools for health, industry, environment, and agriculture.},
journal = {AIMS microbiology},
volume = {11},
number = {4},
pages = {946-962},
pmid = {41522435},
issn = {2471-1888},
abstract = {Synthetic biology has revolutionized precision medicine by enabling the development of engineered bacteria as living therapeutics, dynamic biological systems capable of sensing, responding to, and functioning within complex physiological environments. These microbial platforms offer unprecedented adaptability, allowing for real-time detection of disease signals and targeted therapeutic delivery. This review explores recent innovations in microbial engineering across medical, industrial, environmental, and agricultural domains. Key advances include CRISPR-Cas systems, synthetic gene circuits, and modular plasmid architectures that provide fine-tuned control over microbial behavior and therapeutic output. The integration of computational modeling and machine learning has further accelerated design, optimization, and scalability. Despite these breakthroughs, challenges persist in maintaining genetic stability, ensuring biosafety, and achieving reproducibility in clinical and industrial settings. Ethical and regulatory frameworks are evolving to address dual-use concerns, public perception, and global policy disparities. Looking forward, the convergence of synthetic biology with nanotechnology, materials science, and personalized medicine is paving the way for intelligent, responsive, and sustainable solutions to global health and environmental challenges. Engineered bacteria are poised to become transformative tools not only in disease treatment but also in diagnostics, biomanufacturing, pollution mitigation, and sustainable agriculture.},
}
@article {pmid41522810,
year = {2026},
author = {Heer, CD and Elia, JL and Menon, V and Johnson, SS and Arbelaez, SR and Friedman, S and Lopez-Giraldez, F and Sundaram, RK and Herzon, SB and Bindra, RS and Gueble, SE},
title = {Targeted CRISPR knockout screening identifies known and novel chemogenomic interactions between DNA damaging agents and DNA repair genes.},
journal = {NAR cancer},
volume = {8},
number = {1},
pages = {zcaf052},
pmid = {41522810},
issn = {2632-8674},
support = {DP5 OD036128/OD/NIH HHS/United States ; },
mesh = {Humans ; *DNA Repair/genetics/drug effects ; *DNA Damage/drug effects/genetics ; Cell Line, Tumor ; Gene Knockout Techniques ; CRISPR-Cas Systems ; *Glioma/genetics/drug therapy ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Genetic instability is a hallmark of cancer, often arising from mutations to DNA damage repair and response (DDR) genes. Classical genetic, biochemical, and structural approaches elucidated the foundational mechanisms of DDR pathways and provided a scientific understanding of their involvement in repair of lesions induced by broad classes of DNA-damaging agents (DDAs). However, given the chemical diversity of DDAs and resultant DNA lesions, along with the multitude of interconnected DDR factors, the chemogenomic landscape of DDA-DDR interactions remains incompletely mapped. To this end, we developed a DDR-targeted, CRISPR knockout screening approach and assessed relationships amongst 353 DNA repair genes and 15 DDAs in LN229 glioma cells. Within this dataset of 5295 DDR-related chemogenomic interactions, we identified many established interactions and discovered novel ones. For example, we observed a specific role of transcription-coupled nucleotide excision repair in the repair of adducts generated by monofunctional alkylating agents, a role for the Fanconi anemia pathway in addressing methyl lesions, overt differences in DSB repair following treatment with topoisomerase I versus II poisons, and repair dependencies associated with the imidazotetrazines temozolomide, mitozolomide, and KL-50. Future directions will continue to investigate the mechanisms of novel chemogenomic interactions that we have uncovered as well as work to identify chemogenomic interactions amenable to clinical translation.},
}
@article {pmid41523156,
year = {2025},
author = {Altpeter, F},
title = {Gene editing to enhance biotic stress tolerance in sugarcane.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1750169},
pmid = {41523156},
issn = {1664-462X},
}
@article {pmid41524400,
year = {2026},
author = {Clarke, JE and Faulkner, TR and Seipke, RF},
title = {A platform for CRISPRi-seq in Streptomyces albidoflavus.},
journal = {mBio},
volume = {17},
number = {2},
pages = {e0306525},
pmid = {41524400},
issn = {2150-7511},
support = {BB/T014962/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Streptomyces/genetics ; Operon ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; *CRISPR-Cas Systems ; Genomics/methods ; },
abstract = {UNLABELLED: Streptomyces produce a multitude of secondary metabolites, which have been exploited in drug discovery campaigns for more than three-quarters of a century. Our understanding of microbial physiology has been revolutionized by genome sequencing and large-scale functional studies. Technology for genome-wide investigations in Streptomyces species, however, has lagged behind that for other bacterial systems, hindering exploitation of unprecedented quantities of genomic data. Here, we develop a platform for en masse clustered regularly interspaced short palindromic repeats interference sequencing (CRISPRi-seq) for Streptomyces spp. By performing CRISPRi-seq with 2,160 unique sgRNAs targeting all operons (432 operons) encoding membrane transporters (629 genes) representing 1.1Mb of the 6.8Mb genome for S. albidoflavus, combined with hit validation, we discovered that only a small proportion (13 of 432 operons, 25 kb) contribute positively to fitness. Our work provides both a first-in-class platform for high-throughput functional genomics and a generalized blueprint for en masse screens in Streptomyces species.<br><br>IMPORTANCE: Streptomyces bacteria are prolific producers of clinically essential natural products, yet high-throughput tools to systematically interrogate their genomes remain underdeveloped. By establishing a robust CRISPRi-seq platform for en masse functional screening in Streptomyces albidoflavus, our work closes a critical technological gap in Streptomyces functional genomics. Our study not only identifies a small subset of transporter operons essential for fitness but also introduces a scalable, generalizable approach for dissecting gene function. This platform will accelerate systems-level understanding of an industrially and medically important genus.},
}
@article {pmid41524418,
year = {2026},
author = {Raftopoulou, O and Malmstrom, K and Pan, M and Barrangou, R},
title = {Enhanced editing of Bifidobacterium lactis using the endogenous Type I-G CRISPR-Cas system.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {2},
pages = {e0183925},
pmid = {41524418},
issn = {1098-5336},
support = {industry funding//IFF/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Plasmids/genetics ; *Bifidobacterium animalis/genetics ; Genome, Bacterial ; Probiotics ; },
abstract = {Diverse Bifidobacterium animalis subsp. lactis strains are widely used as commercial probiotics. While proof-of-concept studies have shown that some strains can be edited using several CRISPR-Cas approaches, this species remains difficult to engineer, hindering functional genomic studies to establish their molecular mode of action and enhance their probiotic functionalities. Here, we show that >95% of available B. lactis genomes harbor a conserved Type I-G CRISPR-Cas system, which we leverage to develop and validate a broadly applicable genome editing framework. We redesigned backbone plasmids with different replicons and antibiotic resistance markers and evaluated performance across six commercial strains for transformation efficiency. A vector carrying the pBC1 origin coupled with a chloramphenicol resistance marker improved transformation in most strains. Using synthetic CRISPR arrays with self-targeting spacers in combination with homologous editing templates, we tested multiple spacers and evaluated short (600 bp) versus long (1,000 bp) homology arms. To demonstrate applicability, we generated knockouts in three glycoside hydrolases within the Balac 1593-1601 cluster, readily cured editing plasmids in non-selective medium, and performed iterative genome editing. Growth phenotyping across carbohydrates confirmed that the GH36 α-galactosidase Balac 1601 knockout abolished melibiose and raffinose utilization, and that deletions within Balac 1596 and Balac 1593 carbohydrate hydrolases produced non-canonical phenotypes, suggestive of a modulatory role associated with shift in carbon use and compensation by other pathways. These results establish a practical toolkit for editing diverse B. lactis strains, unravel the genomics underlying probiotic attributes, and provide a blueprint for genome engineering in other non-model probiotic bacteria.IMPORTANCEBifidobacterium animalis subsp. lactis strains are prominent probiotics widely formulated in foods and dietary supplements, yet remain difficult to engineer, limiting efforts to connect genes to probiotic traits and to build strains with enhanced functions. Here, we harness the native Type I-G CRISPR Cas system to enable genome editing across commercial B. lactis strains by optimizing a compact plasmid backbone, testing multiple spacers to achieve efficient editing, and selecting homology arms of the appropriate length for recombination. With this framework, we generate knockouts at multiple, functionally distinct loci, demonstrating target-agnostic applicability, and we cure the CRISPR-editing vectors efficiently, enabling sequential edits. This toolkit enables systematic genotype-to-phenotype mapping in B. lactis and provides a practical framework for strain improvement in organisms of industrial relevance.},
}
@article {pmid41524478,
year = {2026},
author = {Kim, M and Kim, K and Lee, J and Lee, S and Choi, S and Park, SA and Jeong, E and Choi, SY and Park, HH and Park, TE and Kwon, T and Myung, K and Yoo, J and Cho, SW and Joo, J},
title = {FAST-CRISPR: Fusogenic Association and Secured Transfection of CRISPR/Cas9 Ribonucleoproteins Using Lipid-Silica Hybrid Nanoparticles for Therapeutic Genome Editing.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {15},
pages = {e11362},
pmid = {41524478},
issn = {1613-6829},
support = {RS-2024-00512120//Korean ARPA-H Project through the KHIDI/ ; 22A0102L1-11//Korean Fund for Regenerative Medicine (KFRM)/ ; RS-2024-00509412//National Research Foundation (NRF)/ ; RS-2023-00209822//National Research Foundation (NRF)/ ; RS-2023-00207746//National Research Foundation (NRF)/ ; IBS-R022-D1//Institute for Basic Science/ ; 1.250006.01//UNIST research fund/ ; RS-2024-00403508//Korea Basic Science Institute/ ; 1.250006.01//Ulsan National Institute of Science and Technology/ ; },
mesh = {*Nanoparticles/chemistry ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/metabolism/genetics ; *Silicon Dioxide/chemistry ; Humans ; *Lipids/chemistry ; Animals ; *Transfection/methods ; Mice ; Cell Line, Tumor ; },
abstract = {Clinical translation of CRISPR/Cas9 therapeutics is challenged by inefficient cytosolic delivery and toxicity issues associated with viral vectors and nanoparticle-based carriers. To overcome these concerns, herein we report a lipid-silica hybrid nanoparticle platform for fusogenic association and secured transfection of CRISPR/Cas9 (FAST-CRISPR), designed for rapid cytosolic delivery of CRISPR/Cas9 ribonucleoproteins, followed by efficient gene editing. Through direct fusion with the plasma membrane and bypassing conventional endocytic barriers, FAST-CRISPR nanoparticles displayed superior intracellular delivery efficacy. Optimizing lipid compositions, we discovered that a 1:1 weight mixture of cationic DOTAP and ionizable DODMA lipids, combined with tailored large-pore silica nanoparticles, enables enhanced loading capacity, rapid cytosolic dispersion, and significant nuclear transport of Cas9/gRNA complexes. FAST-CRISPR nanoparticles efficiently delivered multiplex genome-targeting ribonucleoproteins to induce targeted double-strand DNA breaks, triggering apoptosis in cancer cells and significantly suppressing tumor growth in a mouse xenograft model without systemic toxicity. Our findings demonstrate the therapeutic efficacy and translational potential of FAST-CRISPR nanoparticles as a safe and versatile non-viral delivery platform for precision genome editing.},
}
@article {pmid41524535,
year = {2026},
author = {Batra, SS and Cabrera, A and Spence, JP and Goell, J and Anand, SS and Hilton, IB and Song, YS},
title = {Predicting the effect of CRISPR-Cas9-based epigenome editing.},
journal = {eLife},
volume = {12},
number = {},
pages = {},
pmid = {41524535},
issn = {2050-084X},
support = {R35 GM134922/GM/NIGMS NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; R35-GM143532/GM/NIGMS NIH HHS/United States ; R35-GM134922/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; HEK293 Cells ; *Epigenome ; Protein Processing, Post-Translational ; Machine Learning ; Histones/metabolism ; K562 Cells ; *Epigenesis, Genetic ; *Epigenomics/methods ; Epigenome Editing ; },
abstract = {Epigenetic regulation orchestrates mammalian transcription, but functional links between them remain elusive. To tackle this problem, we use epigenomic and transcriptomic data from 13 ENCODE cell types to train machine learning models to predict gene expression from histone post-translational modifications (PTMs), achieving transcriptome-wide correlations of ∼0.70-0.79 for most cell types. Our models recapitulate known associations between histone PTMs and expression patterns, including predicting that acetylation of histone subunit H3 lysine residue 27 (H3K27ac) near the transcription start site (TSS) significantly increases expression levels. To validate this prediction experimentally and investigate how natural vs. engineered deposition of H3K27ac might differentially affect expression, we apply the synthetic dCas9-p300 histone acetyltransferase system to 8 genes in the HEK293T cell line and to 5 genes in the K562 cell line. Further, to facilitate model building, we perform MNase-seq to map genome-wide nucleosome occupancy levels in HEK293T. We observe that our models perform well in accurately ranking relative fold-changes among genes in response to the dCas9-p300 system; however, their ability to rank fold-changes within individual genes is noticeably diminished compared to predicting expression across cell types from their native epigenetic signatures. Our findings highlight the need for more comprehensive genome-scale epigenome editing datasets, better understanding of the actual modifications made by epigenome editing tools, and improved causal models that transfer better from endogenous cellular measurements to perturbation experiments. Together, these improvements would facilitate the ability to understand and predictably control the dynamic human epigenome with consequences for human health.},
}
@article {pmid41524770,
year = {2026},
author = {Basit, A and Zhu, J and Zheng, W},
title = {Assessing off-target effects in CRISPR/Cas9: challenges and strategies for precision DNA editing.},
journal = {Archives of microbiology},
volume = {208},
number = {2},
pages = {114},
pmid = {41524770},
issn = {1432-072X},
support = {YZ2024220//Work was partly supported by the 2024 Annual Special Funds for Municipal-School Cooperation Projects of Yangzhou City (YZ2024220) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD)./ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The emergence of CRISPR/Cas9 technology has transformed the landscape of gene editing, allowing for precise alterations in DNA that hold great promise for research and potential therapies. However, a significant concern is the occurrence of off-target effects, which can lead to unintended genetic modifications with potentially harmful consequences. This paper explores the nature of off-target effects in CRISPR/Cas9, discussing how they arise and their implications for the reliability of gene editing. We identify the challenges faced in detecting and predicting these off-target interactions, including limitations in current detection techniques and the complexities of cellular biology. We present strategies aimed at minimizing off-target effects, such as careful design of guide RNAs, the use of computational tools for prediction, and improved delivery methods. Through a review of case studies, we highlight successful cases where off-target activity has been significantly reduced, offering insights into best practices for enhancing the accuracy of CRISPR/Cas9 applications. Moreover, we provide a comparative overview of Cas9, Cas12, and Cas13 systems, emphasizing their distinct target specificities, mechanisms of action, and off-target profiles. This comparison offers a broader understanding of how alternative CRISPR effectors may be leveraged to improve genome and transcriptome editing precision. This study underscores the importance of continued research to address the challenges of off-target effects, ultimately supporting the development of safer and more effective gene editing methods for clinical use.},
}
@article {pmid41525963,
year = {2026},
author = {Habib, AH and Sain, ZM and Rafeeq, M and Karami, MM and Alsufyani, HA and Iqbal, J and Chaieb, K and Altayb, HN and Nadeem, MS and Al-Abbasi, FA and Kazmi, I},
title = {MicroRNA-CRISPR biosensors for cancer diagnostics.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {583},
number = {},
pages = {120837},
doi = {10.1016/j.cca.2026.120837},
pmid = {41525963},
issn = {1873-3492},
mesh = {Humans ; *Biosensing Techniques/methods ; *Neoplasms/diagnosis/genetics ; *MicroRNAs/genetics/blood/analysis ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/genetics ; },
abstract = {Circulating microRNAs (miRNAs) are promising minimally invasive biomarkers for cancer and cardiovascular disorders. However, their low sequence length, low abundance, high sequence homology (including iso-miRs), and strong matrix and preanalytical effects in biofluids require highly sensitive and robust analytical technologies. CRISPR-Cas systems, particularly Cas12a, Cas12b, Cas13a, and Cas9, offer programmable nucleic acid recognition with high mismatch discrimination combined with collateral nuclease activity, enabling versatile signal amplification through fluorescence, electrochemical, electrochemiluminescent (ECL), photoelectrochemical (PEC), colorimetric, and lateral-flow readouts. This review critically evaluates the latest advances in CRISPR-based miRNA biosensors, emphasizing their analytical performance and translational potential in clinical diagnostics across plasma/serum, saliva, whole blood, and extracellular vesicle samples. The detection limits are typically within the femtomolar to attomolar range. The requirements for clinical translation are equally influenced by factors such as sample preparation, inhibitor tolerance, miRNA panel multiplexing, quantitative readout, and reagent stability. We compared CRISPR-based workflows with RT-qPCR and digital PCR and provided a roadmap for standardization and quality control, as well as the minimal analytical and clinical validation standards required for adopting CRISPR technology in clinical chemistry laboratories.},
}
@article {pmid41526335,
year = {2026},
author = {Gao, J and Bader, A and Linder, MI and Cheng, J and Richter, M and da Costa, R and Zehrer, A and Mitt, K and Popper, B and Meissner, F and Wei, X and de Vega Gómez, E and Tatematsu, M and Rohlfs, M and Frenz-Wiessner, S and Kiziltug, M and Somekh, I and Yacobovich, J and Steinberg-Shemer, O and Somech, R and Soehnlein, O and Schmid, B and Klein, C and Walzog, B and Maier-Begandt, D},
title = {Mutations in VPS18 lead to a neutrophil maturation defect associated with disturbed vesicle homeostasis.},
journal = {Cell death &amp; disease},
volume = {17},
number = {1},
pages = {180},
pmid = {41526335},
issn = {2041-4889},
support = {CRC914 (project A02)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project C03)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC914 (project A13)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project A02)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; TRR332 (#449437943; project B01)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; CRC914 (projects A02 and Z03)//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Neutrophils/metabolism/pathology ; Animals ; Humans ; *Vesicular Transport Proteins/genetics/metabolism ; Zebrafish ; *Mutation/genetics ; *Homeostasis ; Cell Differentiation ; Induced Pluripotent Stem Cells/metabolism ; Autophagy ; CRISPR-Cas Systems ; Neutropenia/genetics/pathology ; Autophagosomes/metabolism ; Male ; },
abstract = {Neutrophils, the first cells to arrive at the site of inflammation, are rather short-lived cells and thus have to be constantly replenished. During neutrophil development, vesicle dynamics need to be fine-tuned and impaired vesicle trafficking has been linked to failure in neutrophil maturation. Here, we characterized the role of VPS18 as a central core component of CORVET &amp; HOPS tethering complexes for neutrophil development. Using CRISPR/Cas9-engineered Hoxb8 cells with heterozygous mutations in Vps18, we found that VPS18 deficiency interfered with neutrophil development due to tethering complex instability. As a result, vesicle dynamics were impaired with a strong increase in LC3B-II and p62 levels, indicating autophagosome accumulation and reduced autophagic flux. With transmission electron microscopy, we verified the increase in autophagosomes and also found irregularly shaped vesicular structures in Vps18 mutants. Subsequently, Vps18 mutant neutrophil progenitors underwent premature apoptosis. We described a novel patient with a heterozygous stop-gain mutation in VPS18 suffering from neutropenia and recurrent infections. To verify our findings in the human system, we used human induced pluripotent stem cells (iPSCs). Upon differentiation into neutrophils, loss of VPS18 resulted in an almost complete absence of iPSC-derived developing neutrophils. Heterozygous VPS18 mutant and patient mutation-harboring iPSCs were characterized by strongly reduced numbers of developing neutrophils. Zebrafish larvae with heterozygous mutations in vps18 were also characterized by significantly reduced neutrophil numbers. This study shows the pivotal impact of VPS18 for adequate vesicle dynamics during neutrophil development which might be relevant in the context of vesicle trafficking during granulopoiesis and congenital neutropenia.},
}
@article {pmid41526374,
year = {2026},
author = {Xie, N and Pan, Y and Tong, H and Lin, Y and Jiang, Y and Wang, Z and Wan, J and Zhang, W and Wang, X and Sun, X and Yan, S and Yin, P and Sun, Q and Qi, C and Tian, Y and Shen, L and Jiang, H and Liang, D and Tang, B and Li, S and Li, XJ and Liu, Q},
title = {Precise excision of expanded GGC repeats in NOTCH2NLC via CRISPR/Cas9 for treating neuronal intranuclear inclusion disease.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1683},
pmid = {41526374},
issn = {2041-1723},
support = {32071037//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Gene Editing/methods ; *Intranuclear Inclusion Bodies/genetics/pathology/metabolism ; *Neurodegenerative Diseases/genetics/therapy/pathology ; *Trinucleotide Repeat Expansion/genetics ; Disease Models, Animal ; Mice, Transgenic ; *Receptor, Notch2/genetics ; Cell Line ; Male ; Nerve Tissue Proteins ; Intercellular Signaling Peptides and Proteins ; },
abstract = {Neuronal intranuclear inclusion disease (NIID) is an adult-onset neurodegenerative disease caused by expanded GGC repeats in the 5' untranslated region of the human-specific NOTCH2NLC gene. The high sequence similarity between NOTCH2NLC and its paralogs poses a significant challenge for precise gene editing. Here, we develop a CRISPR/spCas9-based gene-editing strategy that precisely excises the expanded GGC repeats in NOTCH2NLC without detectable off-target effects on the highly homologous NOTCH2/NOTCH2NL family genes (<2% sequence divergence at this locus). The efficacy, specificity and safety of this approach are rigorously validated across multiple experimental models, including human cell lines, NIID iPSCs, and our previously established transgenic NIID mouse model. Our results demonstrate that precise excision of the expanded GGC repeats effectively alleviates NIID-related neuropathological, molecular and behavioral abnormalities. This study establishes the proof of concept for genome editing as a therapeutic strategy for NIID and other related repeat expansion disorders.},
}
@article {pmid41526382,
year = {2026},
author = {Van Vu, T and Thi Nguyen, N and Kim, J and Hoai Nguyen, T and Kim, JY},
title = {Development of an ultra-efficient prime editing system in tomato.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {95},
pmid = {41526382},
issn = {2041-1723},
support = {RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant ; },
abstract = {Prime editing (PE) enables precise genome modifications without donor DNA or double-strand breaks, but its application in dicot plants has faced challenges due to low efficiency, locus dependence, and poor heritability. Here, we develop an ultra-efficient prime editing (UtPE) system for dicots by integrating evolved PE6 variants (PE6c and PE6ec), an altered pegRNA (aepegRNA), an RNA chaperone, and a geminiviral replicon. UtPE significantly improves editing performance in tomatoes, with UtPEv1 excelling in simple edits (unstructured RTTs) and UtPEv3 effective for complex targets (structured RTTs or multiple nucleotide changes). Compared to a PE2max-based tool, UtPE increases desired editing efficiency by 3.39 to 8.89-fold, enables editing at previous inaccessible sites, achieves an average of 16.0% desired editing efficiency in calli, and produces high-frequency desired edits in up to 87.5% of T0 plants. Multiplexed editing at up to three loci and stable T1 inheritance are also achieved, resulting in traits such as jointless pedicels and glyphosate resistance, while minimizing off-target effects.},
}
@article {pmid41526501,
year = {2026},
author = {Garriga-Canut, M and Cannon, N and Benton, M and Zanon, A and Horsfield, ST and Scheurich, J and Remans, K and Lees, J and Paix, A and van Gestel, J},
title = {Unlocking CRISPR-Cas9 editing for widely diverse Dictyostelid species.},
journal = {Molecular systems biology},
volume = {22},
number = {4},
pages = {599-619},
pmid = {41526501},
issn = {1744-4292},
support = {101116560//EC | ERC | HORIZON EUROPE European Research Council (ERC)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Dictyostelium/genetics ; Gene Knockout Techniques ; Gene Knock-In Techniques ; Phylogeny ; },
abstract = {Dictyostelids are a species-rich clade of cellular slime molds that are widely found in soils and have been studied for over a century. Due to a lack of genome editing methods, most molecular research in Dictyostelids has focused on only a single species, Dictyostelium discoideum, which has severely limited broad-scale comparative analyses. Here, we introduce the first CRISPR-Cas9 editing approach that is cloning-free, selection-free, highly efficient, and effective across Dictyostelid species that diverged millions of years ago. Depending on the CRISPR-Cas9 target site, our editing approach generates knock-out efficiencies of up to 90% and knock-in efficiencies of up to 50% without a selective marker. We show that mutants can be isolated as soon as one day post-transfection, vastly outpacing existing methods for generating knock-outs, fusion proteins, and expression reporters. Leveraging single-cell sorting and fluorescent microscopy, we could readily apply our CRISPR-Cas9 editing approach to phylogenetically distant Dictyostelid species, including those that have never been genome edited before. Our methods therefore open the door to performing broad-scale genetic interrogations across the Dictyostelids.},
}
@article {pmid41526513,
year = {2026},
author = {Ngo, W and Wu, JLY and Wasko, KM and Doudna, JA},
title = {Targeted delivery of genome editors in vivo.},
journal = {Nature biotechnology},
volume = {44},
number = {1},
pages = {49-59},
pmid = {41526513},
issn = {1546-1696},
support = {/HHMI/Howard Hughes Medical Institute/United States ; R21 HL173710/HL/NHLBI NIH HHS/United States ; DE-AC52-07NA27344//DOE | LDRD | Lawrence Livermore National Laboratory (LLNL)/ ; },
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; *Drug Delivery Systems ; },
abstract = {Genome editing has revolutionized the treatment of genetic diseases, yet the difficulty of tissue-specific delivery currently limits applications of editing technology. In this Review, we discuss preclinical and clinical advances in delivering genome editors with both established and emerging delivery mechanisms. Targeted delivery promises to considerably expand the therapeutic applicability of genome editing, moving closer to the ideal of a precise 'magic bullet' that safely and effectively treats diverse genetic disorders.},
}
@article {pmid41526975,
year = {2026},
author = {Hu, Q and Zhang, R and Liu, J and Zhang, W and Liao, X and Guo, Y and Lu, Q and Yang, B and Zhang, T and Zhai, X and Luo, Q},
title = {A rapid and field-deployable RAA-CRISPR/Cas12a platform for detection of Mycoplasma gallisepticum in poultry.},
journal = {BMC veterinary research},
volume = {22},
number = {1},
pages = {117},
pmid = {41526975},
issn = {1746-6148},
support = {2025BEB053//the Hubei Province Technology Innovation Plan Project/ ; 2024BBA004//the Hubei Province Technology Innovation Plan Project/ ; NYWSWZX2025-3432027-04//the Major Special Project for the Development of Agricultural Microbial Industry in Hubei Province/ ; 2023HBSTX4-04//the Hubei Province Modern Agricultural Industry Technology System/ ; CARS-41//the China Agriculture Research System/ ; },
mesh = {Animals ; *Mycoplasma gallisepticum/isolation &amp; purification/genetics ; *Poultry Diseases/diagnosis/microbiology ; *Mycoplasma Infections/veterinary/diagnosis/microbiology ; *CRISPR-Cas Systems ; Chickens ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Recombinases ; Ducks ; Columbidae ; },
abstract = {BACKGROUND: Mycoplasma gallisepticum (MG) is a major pathogen that causes respiratory diseases 14in poultry, resulting in reduced production and severe economic losses. Current MG detection methods are time-consuming, labor-intensive, and expensive. Hence, the rapid and accurate detection of MG is critical for effective disease control. Therefore, this study aimed to develop a dual-mode diagnostic assay for sensitive and specific detection of MG by combining recombinase-aided amplification (RAA) with CRISPR/Cas12a technology. Conserved regions of the mgc2 gene were used for primer and CRISPR RNA design, and the reaction conditions were optimized to maximize detection efficiency.<br><br>RESULTS: The assay achieved a detection limit of 2 copies/&#xb5;L and demonstrated high specificity against seven other common avian pathogens. Detection was visualized within 1&#xa0;h using either fluorescence or lateral flow dipstick. Moreover, clinical validation of chicken samples showed complete concordance with quantitative real-time polymerase chain reaction results. Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for MG among chickens, ducks, and pigeons in Hubei Province.<br><br>CONCLUSIONS: This simple, rapid, field-deployable method is valuable for timely MG surveillance and effective disease management in poultry production.},
}
@article {pmid41527385,
year = {2026},
author = {Pang, Y and Duan, Y and Sun, Y and Zhou, T and Li, A and Ran, R and Hou, H and Liu, SM},
title = {One copy in one-pot for rapid and accurate SFTSV testing by LAC12b-2M.},
journal = {Clinical chemistry and laboratory medicine},
volume = {64},
number = {4},
pages = {957-967},
pmid = {41527385},
issn = {1437-4331},
mesh = {Humans ; *Phlebovirus/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; RNA, Viral/genetics/blood ; CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Real-Time Polymerase Chain Reaction ; },
abstract = {OBJECTIVES: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne pathogen that can cause a fatality rate as high as 12-50 %, posing a significant threat to public health. SFTSV is prevalent in mountainous and hilly regions with relatively poor medical conditions. Therefore, there is an urgent need to develop a new convenient, rapid and sensitive method for SFTSV detection in low-resource environments.<br><br>METHODS: We developed a one-pot and visualized method for SFTSV detection using loop-mediated isothermal amplification assisted by CRISPR/Cas12b with G478A/K396A double mutations (LAC12b-2M). The specificity, sensitivity, accuracy and limit of detection (LOD) of LAC12b-2M were evaluated using clinical reverse transcription-quantitative PCR (RT-qPCR) as the reference method, with gradient dilutions of strong positive SFTSV RNA samples and 215 clinical serum samples from two cohorts.<br><br>RESULTS: LAC12b-2M is sensitive to detect SFTSV with a LOD of 1 copy/&#x3bc;L at 61&#x202f;&#xb0;C within 30&#x202f;min. Compared to clinical RT-qPCR, LAC12b-2M demonstrated a sensitivity of 98.8&#x202f;% (82/83), a specificity of 100.0&#x202f;% (96/96), and an accuracy of 99.4&#x202f;% (178/179) in cohort 1 (n=179), and an accuracy of 100.0&#x202f;% in cohort 2 (n=36).<br><br>CONCLUSIONS: Our LAC12b-2M method holds promise for point-of-care SFTSV testing in different healthcare settings, particularly in low-resource region where SFTSV is prevalent.},
}
@article {pmid41527434,
year = {2026},
author = {Stewart, C and Liddle, TA and Tolla, E and Lewis, JE and Marshall, C and Evans, NP and Morgan, PJ and Ebling, FJP and Stevenson, TJ},
title = {Hypothalamic deiodinase type-3 establishes the period of circannual interval timing in mammals.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41527434},
issn = {2050-084X},
support = {/WT_/Wellcome Trust/United Kingdom ; Institutional Strategic Support Fund/WT_/Wellcome Trust/United Kingdom ; LT-RL-2019-06//Leverhulme Trust/ ; },
mesh = {Animals ; *Iodide Peroxidase/metabolism/genetics ; *Hypothalamus/enzymology/physiology ; Photoperiod ; Seasons ; Phodopus/physiology ; Male ; Cricetinae ; },
abstract = {Animals respond to environmental cues to time phenological events, but the intrinsic mechanism of circannual timing remains elusive. We used transcriptomic sequencing and frequent sampling of multiple hypothalamic nuclei in Djungarian hamsters to examine the neural and molecular architecture of circannual interval timing. Our study identified three distinct phases of transcript changes, with deiodinase type-3 (Dio3) expression activated during the early induction phase. Subsequent work demonstrated that targeted mutation of Dio3 using CRISPR-Cas resulted in a shorter period for circannual interval timing. Hamsters that are non-responsive to short photoperiods and fail to show any winter adaptations do not display changes in Dio3 expression and do not show any change in body mass or pelage. Our work demonstrates that changes in Dio3 induction are essential for setting the period of circannual interval timing.},
}
@article {pmid41527500,
year = {2026},
author = {Atceken, N and Kahya, A and Yigci, D and Tasoglu, S},
title = {CRISPR-on-Chip for Point-of-Care Diagnostics.},
journal = {ACS nano},
volume = {20},
number = {3},
pages = {2561-2577},
pmid = {41527500},
issn = {1936-086X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Point-of-Care Systems ; *Lab-On-A-Chip Devices ; Biosensing Techniques ; *Point-of-Care Testing ; Precision Medicine ; },
abstract = {CRISPR-based diagnostic platforms have gained significant momentum in recent years, enabling highly sensitive and specific detection of pathogens and diseases. Due to their practical benefits, these platforms have become widely adopted in point-of-care (PoC) applications. CRISPR-on-chip technology integrates CRISPR-Cas platforms with diverse microfluidic systems, allowing scalability and portable, real-time, and precise biomolecule detection. This approach enhances diagnostic accuracy, reduces processing times, and minimizes the need for complex laboratory infrastructures, unlike in conventional diagnostics. Using CRISPR-Cas enzymes in microfluidic systems, CRISPR-on-chip platforms offer key advantages such as single-molecule sensitivity, multiplex detection, and applicability. However, integration with microfluidics for PoC applications is still poorly understood, despite CRISPR-Cas being widely used. This study reviews recent developments in CRISPR-on-chip-based diagnostics and highlights its potential applications in infectious diseases, biosensors, and personalized medicine. Furthermore, challenges and future perspectives in achieving an ideal diagnostic solution are discussed.},
}
@article {pmid41528123,
year = {2025},
author = {Dong, Q and Luo, C},
title = {A recombinase polymerase amplification-coupled Cas12a for detection of Salmonella Typhi - a preliminary report.},
journal = {Folia histochemica et cytobiologica},
volume = {63},
number = {4},
pages = {185-192},
doi = {10.5603/fhc.108406},
pmid = {41528123},
issn = {1897-5631},
mesh = {*Salmonella typhi/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems/genetics ; *Typhoid Fever/diagnosis/microbiology ; *Recombinases/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Bacterial Proteins/genetics/metabolism ; DNA, Bacterial/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Associated Proteins ; },
abstract = {INTRODUCTION: . Typhoid fever, a disease resulting from an infection with Salmonella Typhi (S. Typhi) remains widespread in economically disadvantaged regions, where it continues to be a critical public health concern. As the symptoms and signs are non-specific, they are difficult to diagnose directly based on the clinical picture. Therefore, laboratory examinations are essential for diagnosis.<br><br>MATERIAL AND METHODS: . This research introduces a fast and equipment-independent approach for detecting S. Typhi by employing CRISPR/Cas12a-based technology. The optimized CRISPR/Cas12a system achieved a detection limit of 103 copies/&#x3bc;L of S. Typhi DNA per reaction, with the entire assay completed within 60 min.<br><br>RESULTS: . Four clinical isolates cultured from patients with typhoid fever were collected and evaluated using our CRISPR/Cas12a-based detection system. The assay results demonstrated that all four samples were accurately identified as positive.<br><br>CONCLUSIONS: . We showed that the developed CRISPR/Cas12a-based detection method provides a promising alternative for the on-site and simple detection of S. Typhi.},
}
@article {pmid41528700,
year = {2026},
author = {Wei, W and Yang, Y and Shih, J},
title = {Rapid and Sensitive Detection of Phytoplasma Diseases Using a CRISPR/Cas12a DETECTR Assay Combined with Isothermal Recombinase Polymerase Amplification.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3008},
number = {},
pages = {63-74},
pmid = {41528700},
issn = {1940-6029},
mesh = {*Phytoplasma/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Nucleic Acid Amplification Techniques/methods ; Recombinases/metabolism ; DNA, Bacterial/genetics ; CRISPR-Associated Proteins/genetics ; Endodeoxyribonucleases/genetics ; Bacterial Proteins/genetics ; },
abstract = {Our protocol outlines a DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay, which combines CRISPR/Cas12a technology with isothermal Recombinase Polymerase Amplification (RPA) for the rapid and specific detection of phytoplasma diseases in plants. This isothermal method utilizes RPA to amplify the target DNA fragment from the genomic DNA of phytoplasmas, followed by incubation with Cas12a nuclease and CRISPR RNAs (crRNAs) specifically designed to target unique phytoplasma DNA sequences. Upon initial cleavage of the amplified target DNA, Cas12a gains enzymatic activity to indiscriminately cleave single-stranded fluorescent oligonucleotide reporters, generating a fluorescent signal for highly sensitive detection of the pathogen. The protocol provides detailed instructions on: (i) sample collection and preparation; (ii) assay reaction setup, including RPA and Cas12a detection steps; (iii) reaction and detection conditions; and (iv) guidelines for accurately interpreting fluorescence data to detect phytoplasma DNA. This protocol is designed for researchers and agricultural professionals to effectively adopt and implement this advanced diagnostic technique.},
}
@article {pmid41529994,
year = {2026},
author = {Ebe, S and Nakamura, H and Matsuda, M and Terauchi, Y and Akada, R and Hoshida, H},
title = {Identification and overexpression of genes encoding sugar alcohol transporter and metabolic enzymes for accelerated utilization in the yeast Kluyveromyces marxianus.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {4},
pages = {221-229},
doi = {10.1016/j.jbiosc.2025.12.003},
pmid = {41529994},
issn = {1347-4421},
mesh = {*Kluyveromyces/genetics/metabolism/enzymology/growth &amp; development ; Mannitol/metabolism ; Sorbitol/metabolism ; CRISPR-Cas Systems ; *Fungal Proteins/genetics/metabolism ; L-Iditol 2-Dehydrogenase/genetics/metabolism ; },
abstract = {The yeast Kluyveromyces marxianus assimilates various sugars, including sorbitol and mannitol. However, the metabolic pathways for sugar utilization, including sugar transporters, remain to be elucidated. To identify these genes in this study, first 13 candidate transporter genes were disrupted using a newly developed non-homologous end joining (NHEJ)-mediated gene disruption method, combined with targeted digestion using the CRISPR-Cas9 system. While most disruptants exhibited no clear growth defects in various sugar media, a disruptant of the KmMLEV2025 gene (named KmSAT1) failed to grow in either sorbitol or mannitol media, suggesting that it encodes a sugar alcohol transporter. Next, we investigated the candidate dehydrogenase genes crucial for sugar alcohol metabolism, as they are converted to fructose by dehydrogenases. KmXyl2p, a known xylitol dehydrogenase, is a candidate sorbitol dehydrogenase. Disruption of KmXYL2 caused growth defects in sorbitol medium, but not in mannitol medium. We disrupted several genes to identify the mannitol dehydrogenase, revealing that the disruption of KmSOU2, annotated as a sorbose reductase, resulted in a growth defect in the mannitol medium. The identified genes were overexpressed for the efficient utilization of sugar alcohols. The strain overexpressing KmSAT1, but not the dehydrogenase genes, started growing immediately, whereas the wild-type strain exhibited a lag time of several days. Furthermore, the final cell optical densities in both the sorbitol and mannitol media were higher than those observed in the glucose medium. These results indicated that overexpression of a sugar alcohol transporter is a highly effective strategy for biotechnological applications.},
}
@article {pmid41530154,
year = {2026},
author = {Li, H and Liu, F and Li, J and Shi, C and Lin, Z and Qin, Y and Pan, R and Wu, X and Peng, Y and Xing, C and Wang, Y and Qu, Q and Li, G},
title = {Evolutionary dynamics of sex determination in Branchiostoma belcheri driven by repeated transposition of a single novel gene.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1616},
pmid = {41530154},
issn = {2041-1723},
support = {Start-up//Xiamen University (XMU)/ ; 32300346//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32200411//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32270439//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32061160471//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022J06004//Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)/ ; },
mesh = {Animals ; *Sex Determination Processes/genetics ; Female ; Male ; *Lancelets/genetics ; *Evolution, Molecular ; CRISPR-Cas Systems ; *DNA Transposable Elements/genetics ; Sex Chromosomes/genetics ; RNA, Long Noncoding/genetics ; },
abstract = {Sex determination systems display striking evolutionary flexibility, yet the mechanisms underlying their transitions remain poorly understood. Using newly generated genome assemblies, we investigated the evolving sex-determining system in the amphioxus Branchiostoma belcheri. We identified two female-specific sex-determining regions (SDRs) on chromosome 13, both derived from independent transpositions of the autosomal gene tesD, which shows testis-specific expression in amphioxus species. CRISPR/Cas9 knockout experiments in Branchiostoma floridae confirmed that tesD functions as a male-determination gene, with loss of function producing an all-female phenotype. In B. belcheri, the older SDR (tesDwa) inserted into the coding region of twai, while the younger SDR (tesDwb), flanked by active Zator-1 transposons, inserted into the 3' UTR of vps9c and later translocated to autosomes in ~10% of individuals. Transcriptomic analyses revealed that W-linked tesDwa and tesDwb produce antisense long non-coding RNAs that likely suppress tesD transcription in females, whereas autosomal tesDwb is not expressed and appears non-functional. The insertion sites and co-transcription with host genes suggest promoter hijacking. Together, these findings demonstrate that recurrent transpositions can generate new functional SDRs that coexist with older ones, driving dynamic turnover of sex determination in B. belcheri.},
}
@article {pmid41530266,
year = {2026},
author = {Freedman, BS and Bulte, JWM and Conklin, BR and Judge, LM and Dwinell, MR and Geurts, AM and Sitton, MJ and Mahajan, V and Kiani, S and Gersbach, CA and Ebrahimkhani, MR and Kelly, JJ and Ronald, JA and Morizane, R and Gupta, N and Shakeri-Zadeh, A and Vo, N and Saha, K and Saxena, S and Gamm, DM and Sinha, D and Tarantal, AF and Vandsburger, M and Matsubara, A and Fu, H and Tsai, SQ and , and , },
title = {Monitoring biological effects of somatic cell genome editing.},
journal = {Nature reviews. Genetics},
volume = {27},
number = {4},
pages = {323-342},
pmid = {41530266},
issn = {1471-0064},
support = {UH2 EB028904/EB/NIBIB NIH HHS/United States ; R01 NS119678/NS/NINDS NIH HHS/United States ; U01 EB028892/EB/NIBIB NIH HHS/United States ; R21 DK129909/DK/NIDDK NIH HHS/United States ; U01 AI176460/AI/NIAID NIH HHS/United States ; S10 RR025063/RR/NCRR NIH HHS/United States ; U19 NS132296/NS/NINDS NIH HHS/United States ; R35 GM119644/GM/NIGMS NIH HHS/United States ; S10 OD018102/OD/NIH HHS/United States ; DP2 DK133821/DK/NIDDK NIH HHS/United States ; U01 DK127587/DK/NIDDK NIH HHS/United States ; S10 OD016261/OD/NIH HHS/United States ; U01 EB028899/EB/NIBIB NIH HHS/United States ; UG3 NS111688/NS/NINDS NIH HHS/United States ; UH3 EB028904/EB/NIBIB NIH HHS/United States ; U01 HL145792/HL/NHLBI NIH HHS/United States ; S10 OD028713/OD/NIH HHS/United States ; R01 DK141567/DK/NIDDK NIH HHS/United States ; U01 DK127553/DK/NIDDK NIH HHS/United States ; U42 OD027094/OD/NIH HHS/United States ; UH3 EB028910/EB/NIBIB NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Genetic Therapy/methods ; },
abstract = {CRISPR-based genome editing therapeutics are entering the clinic, offering transformative potential but also presenting potential risks. Preclinical-to-clinical toolkits are needed to assess the safety and efficacy of these new therapies and accelerate progress. Emerging technologies to monitor the biological effects of genome editors cover a range of biological scales, from the direct measurement of editing outcomes in DNA, to human microphysiological systems, and non-invasive in vivo imaging. Measurements of on-target and off-target editing outcomes, including sequences unique to humans, provide essential benchmarks to understand functional responses. Microphysiological systems, including organoids and organs-on-chips, enable phenotypic evaluations of editing strategies in varied organ lineages and disease states. Non-invasive imaging modalities can track the biodistribution and activities of genome editors and edited cells in vivo. Collectively, these technologies provide complementary insights across different scales, from the single nucleotide to the whole organism, bridging preclinical therapeutics development with clinical trials.},
}
@article {pmid41531187,
year = {2026},
author = {Tamauchi, S and Yoshida, K and Xinyuan, W and Nakagawa, A and Yokoi, A and Yoshikawa, N and Niimi, K and Yamamoto, Y and Kajiyama, H},
title = {In-Tumor CRISPR-Cas9 Knockout Screening and Novel Therapy Development for Malignant Transformation of Ovarian Teratoma.},
journal = {Cancer science},
volume = {117},
number = {4},
pages = {983-995},
pmid = {41531187},
issn = {1349-7006},
support = {24K12578//Japan Society for the Promotion of Science/ ; 23ama221125h0001//Japan Agency for Medical Research and Development/ ; },
mesh = {Female ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; *Teratoma/genetics/pathology/drug therapy ; Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; *Cell Transformation, Neoplastic/genetics/drug effects ; Cell Proliferation/drug effects/genetics ; Apoptosis/drug effects ; Gene Knockout Techniques ; Cisplatin/pharmacology ; Oxidative Stress/drug effects ; },
abstract = {Malignant transformation of mature cystic teratoma (MTMCT) of the ovary is a rare but aggressive malignancy for which no standardized chemotherapy or effective targeted therapies currently exist. To identify therapeutic vulnerabilities in MTMCT, we performed a genome-wide CRISPR-Cas9 knockout screen using the MTMCT-derived NOSCC1 cell line. Two parallel selective pressures were applied: in vivo tumorigenicity in immunodeficient mice and cisplatin exposure in vitro. From this screen, 67 negatively selected genes were identified, among which SOD1 and NDUFB4 emerged as top candidates based on high basal expression levels and clinical relevance. Integration with spatial transcriptomic data from three independent MTMCT patient tumors further supported the prioritization of these targets. SOD1 was selected for further investigation due to the availability of known pharmacological inhibitors. Both siRNA-mediated knockdown and small-molecule inhibition of SOD1 using LCS-1 significantly suppressed MTMCT cell proliferation in vitro by inducing oxidative stress and impairing cell cycle progression. This antiproliferative effect was reversed by co-treatment with N-acetylcysteine, a reactive oxygen species scavenger. In vivo validation using patient-derived xenograft models demonstrated that oral administration of LCS-1 led to significant tumor growth suppression and increased expression of apoptotic and DNA damage markers, including cleaved caspase-3 and γH2AX. These findings establish SOD1 as a critical vulnerability in MTMCT and provide preclinical evidence supporting redox modulation as a therapeutic strategy for this highly chemoresistant and understudied ovarian cancer subtype. Our integrative approach combining functional genomics, spatial transcriptomics, and pharmacologic validation offers a framework for the discovery of novel targets in rare gynecologic malignancies.},
}
@article {pmid41532605,
year = {2026},
author = {Chen, Q and Gou, H and Xu, C and Wang, S and Zhang, H and Song, M and Wang, M and Ji, X and Wei, X and Tan, Y and Quan, H and Luo, P and Shou, H and Liu, P and Liang, Y and Zhu, JK},
title = {Structure-Guided Engineering of a Cas12i Nuclease Unlocks Near-PAMless Genome Editing.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {17},
pages = {e16670},
pmid = {41532605},
issn = {2198-3844},
support = {KJZD20240903102703005//Shenzhen Science and Technology Program/ ; JCYJ20241202125332043//Shenzhen Science and Technology Program/ ; 2024B1111130001//Guangdong S&amp;T Program/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; *Protein Engineering/methods ; },
abstract = {The therapeutic and research applications of CRISPR-Cas nucleases are constrained by their reliance on specific Protospacer Adjacent Motifs (PAMs), which limit the accessible sites in the genome. To overcome this critical barrier, we performed structure-guided engineering of SF01, a compact Cas12i nuclease. Using AlphaFold-predicted structural models, we identified and systematically mutagenized 38 residues at the PAM-interacting interface. This iterative engineering process yielded three superior variants-KR, IKRR, and STKRR-that exhibit dramatically relaxed PAM specificity, enabling efficient editing at a broad spectrum of 5'-NNTN-3' sites. Importantly, while the most broad-spectrum variant (STKRR) shows a trade-off at canonical sites, the IKRR variant retains high activity at canonical 5'-NTTN-3' PAMs while simultaneously enabling efficient editing at 5'-NNTN-3' sites. This near-PAMless activity expands the targetable portion of the genome to over 25%, a four-fold increase over the parental nuclease. Furthermore, adenine base editors (ABEs) constructed with these variants achieve high-efficiency editing (∼80%) at endogenous loci with expanded targeting scope. Comprehensive off-target analysis using GUIDE-tag and Digenome-seq revealed that the enhanced on-target activity of the SF01 variants is not accompanied by a loss of specificity. These engineered nucleases represent a powerful and versatile expansion of the genome editing toolkit, enabling applications previously inaccessible due to PAM constraints.},
}
@article {pmid41532790,
year = {2026},
author = {Cutts, WD and Flanagan, AW and Gorman, BK and Sweten, A and Estrada, BJ and Subash, VN and Klemp, BT and Seely, KN and Sandobal, AD and Stilen, KR and Vaghela, T and Mehvish, A and Wood, JF and Govert, AM and Hobson, KE and Hillebrand, GH and Hooven, TA and Kim, BJ},
title = {CRISPR interference in a Streptococcus agalactiae multi-locus sequence type 17 strain.},
journal = {Journal of bacteriology},
volume = {208},
number = {2},
pages = {e0037625},
pmid = {41532790},
issn = {1098-5530},
support = {R01AI182835//National Institute of Allergy and Infectious Diseases/ ; R21AI178067//National Institute of Allergy and Infectious Diseases/ ; R01 AI182835/AI/NIAID NIH HHS/United States ; R03AI185593//National Institute of Allergy and Infectious Diseases/ ; R15NS131921/NS/NINDS NIH HHS/United States ; R01 AI177991/AI/NIAID NIH HHS/United States ; R01AI177991//National Institute of Allergy and Infectious Diseases/ ; },
mesh = {*Streptococcus agalactiae/genetics/pathogenicity ; Humans ; *CRISPR-Cas Systems ; Streptococcal Infections/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; Virulence Factors/genetics ; Gene Knockdown Techniques ; Gene Expression Regulation, Bacterial ; Endothelial Cells/microbiology ; },
abstract = {UNLABELLED: Group B Streptococcus (GBS), a common colonizer of the human genital and gastrointestinal tracts, is a leading cause of neonatal bacterial meningitis, which can lead to severe neurological complications. The hypervirulent serotype III, sequence type 17 (ST-17) strain COH1 is strongly associated with late-onset disease due to its unique set of virulence factors. However, genetic manipulation of ST-17 strains remains challenging, limiting the ability to study key pathogenic genes. In this study, we developed a CRISPR interference (CRISPRi) system utilizing an endogenous catalytically inactivated Cas9 (dCas9) in the COH1 strain, enabling targeted and tunable gene expression knockdown. We confirmed the efficacy of this system through hemolysis assays, qPCR transcriptional analysis, and in vitro infection models using human brain endothelial cells. The CRISPRi system successfully produced phenotypic knockdowns of key virulence genes, including PI-2b, srr2, and iagA, reducing adhesion, invasion, and inflammatory responses at the blood-brain barrier (BBB). This platform enables rapid gene knockdowns for functional genomics in ST-17 GBS, enabling high-throughput screening and pathogenesis research.<br><br>IMPORTANCE: Group B Streptococcus (GBS) remains the world's leading cause of neonatal meningitis. GBS-host interactions at the blood-brain barrier (BBB) are dependent on bacterial factors, including surface factors and two-component systems. Multi-locus sequence type 17 (ST-17) GBS strains are highly associated with neonatal meningitis, and these strains harbor many virulence factors for infection at the BBB. Historically, these factors have been studied using traditional knockout mutagenesis, which has been challenging in the most common ST-17 lab strain, COH1. This study utilizes CRISPR interference (CRISPRi) to generate rapid expression knockdown. This study validates a CRISPRi-enabled COH1 dCas9 strain as a versatile tool for probing GBS pathogenesis at the BBB.},
}
@article {pmid41533153,
year = {2026},
author = {Ricci, ML and Fillo, S and Giordani, F and Ciammaruconi, A and Girolamo, A and Anselmo, A and Monte, A and Mentasti, M and Cusimano, V and Caporali, MG and Rota, MC and Petzold, M and Afshar, B and Lista, F and Luck, C and Scaturro, M},
title = {Genomic characterization of Legionella pneumophila serogroup 1 ST901 isolates responsible for recurrent travel-associated Legionnaires' disease cases and clusters.},
journal = {Pathogens and global health},
volume = {},
number = {},
pages = {1-12},
doi = {10.1080/20477724.2025.2610657},
pmid = {41533153},
issn = {2047-7732},
abstract = {Cases of travel-associated Legionnaires' disease (TALD) are frequently reported in Italy. From 1987 to 2021, 61 cases of TALD were linked to 22 hotels in a municipality in northern Italy. Legionella pneumophila serogroup 1 (Lp1) strains isolated from both patients and hotel water systems were identified as sequence type (ST) 901, a genotype rarely associated with travel-related infections in Italy or elsewhere. Whole-genome sequencing was used to analyze 41 isolates, and phylogenetic relationships were inferred by core genome multilocus sequence typing (cgMLST), single nucleotide polymorphisms (SNP) and pangenome analyses. The Lp ST901 isolates were found to form a clade characterized by some accessory genomic islands (AGI) already described in other epidemic strains, such as Alcoy, Corby, Paris and Philadelphia; other islands, containing either transposase/recombinase or transcriptional regulator factors or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-cas systems, were specific to Lp ST901. Lp ST901 also appears to have undergone possible recombination with other strains, such as Lp ST47 (Lorraine strain). Additionally, CRISPR-Cas systems may have contributed to the protection of Lp ST901 from external dangers, while the colonized hotel water systems may have provided an ideal environmental protective niche. Our findings highlight that Lp ST901 has public health significance and deserves attention in Legionnaires' disease surveillance and risk assessment.},
}
@article {pmid41533202,
year = {2026},
author = {Kumar, P and Yadav, H and Mahakalkar, B and Mandlik, R and Vats, S and Thakral, V and Kumar, V and Nishad, SK and Nichal, S and Deshmukh, R and Sharma, TR and Sonah, H},
title = {Challenges and Opportunities with CRISPR-Based Genome Editing in Legume Crops.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {24},
pmid = {41533202},
issn = {1438-7948},
support = {HSCSIT/R&amp;D/2024/511//Haryana State Council for Science and Technology/ ; HSCSIT/R&amp;D/2024/511//Haryana State Council for Science and Technology/ ; Agril. Edn / 27/05/NP(VP)-2023-HRD//Indian Council of Agricultural Research/ ; BT/PR38279/GET/119/351/2020//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Fabaceae/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; Plant Breeding ; },
abstract = {Over the last couple of decades, tremendous progress has been made in legume genomics. Genomics information generated for legume crops is being explored through molecular breeding and transgenic approaches. However, the gap between knowledge generation and its utilization is increasing. In this regard, recent developments in genome editing techniques provide an excellent opportunity to utilize the available knowledge for the improvement of legume crops. This review highlights recent developments with Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based genome-editing approaches, including Cas variants/orthologs and Protospacer adjacent motif-less (PAMless) Genome Editing, multiplex genome editing, base editing, prime editing, transcriptional regulation, methylome editing, and DNA-free editing methods. Furthermore, the applications of non-homologous end joining (NHEJ) and homology-directed repair (HDR)- based editing, are addressed which enable targeted and precise genomic modifications. Moreover, virus-mediated genome editing, in planta transformation, and mobile guide RNAs are increasingly being leveraged to enhance the efficiency and heritability of genome editing. Additionally, the role of artificial intelligence in guide RNA design, off-target prediction, and the development of novel Cas variants is also discussed, which can speed up the legume improvement. This article highlights the successful examples of efforts utilizing CRISPR/Cas9 for the development of legume crops with biotic and abiotic stress tolerance, desirable plant architecture, improved nutrient uptake, and enhanced yield and quality. The biggest limitation in the genome editing of legume crops is their recalcitrance to both transformation and tissue culture. This article discusses how this particular limitation can be addressed in the context of genome editing of legume crops. Finally, the possibilities of integrating these recently developed tools with translational breeding have also been discussed, which will facilitate the legume production for sustainable agriculture and food security.},
}
@article {pmid41533573,
year = {2026},
author = {Kammerdiener, EK and Garcia, SK and Bales, MK and Klingeman, DM and Guss, AM and Giannone, RJ and Hettich, RL and Eckert, CA and Alexander, WG},
title = {Multilayered regulation by RNA thermometers enables precise control of Cas9 expression in E. coli.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533573},
issn = {1362-4962},
support = {ERKP886//U.S. Department of Energy/ ; },
mesh = {*Escherichia coli/genetics/metabolism ; *Gene Expression Regulation, Bacterial ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Promoter Regions, Genetic ; Temperature ; Escherichia coli Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; Heat-Shock Proteins/genetics/metabolism ; },
abstract = {Cas9-based genome editing technologies can rapidly generate mutations to probe a diverse array of mutant genotypes. However, aberrant Cas9 nuclease translation and activity can occur despite the use of inducible promoters to control expression, leading to extensive cell death. This background killing caused by promoter leakiness severely limits the application of Cas9 for generating mutant libraries because of the potential for population skew. We demonstrate the utility of temperature sensitive RNA elements as a layer of post-transcriptional regulation to reduce the impact of promoter leak. We observe significant temperature-dependent increases in cell survival when certain RNA thermometers (RNATs) are placed upstream of the cas9 coding sequence. We also show that the most highly repressing RNAT, hsp17rep, significantly reduces population skew with a library of characterized guide RNAs in Escherichia coli. This strategy should be applicable to all bacterial Cas9-based methods and technologies.},
}
@article {pmid41533581,
year = {2026},
author = {Sugimoto, Y and Kachi, T and Watanabe, Y and Kubokawa, M and Ogami, K and Kawamata, M and Yoshino, S and Suzuki, HI},
title = {Optimized CRISPR-Cas9 system for efficient engineering of ecDNA in cancer cells.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533581},
issn = {1362-4962},
support = {JP24H00614//Japan Society for the Promotion of Science/ ; JP22K06925//Japan Society for the Promotion of Science/ ; JP25K10464//Japan Society for the Promotion of Science/ ; 19K24694//Japan Society for the Promotion of Science/ ; JP22ama221111//Japan Agency for Medical Research and Development/ ; JP23ck0106791//Japan Agency for Medical Research and Development/ ; JP23tk0124003//Japan Agency for Medical Research and Development/ ; JP24ck0106875//Japan Agency for Medical Research and Development/ ; JP25ck0106019//Japan Agency for Medical Research and Development/ ; JP25ak0101291//Japan Agency for Medical Research and Development/ ; JP23kk0305026//Japan Agency for Medical Research and Development/ ; JP25kk0305028//Japan Agency for Medical Research and Development/ ; //Takeda Science Foundation/ ; 22-6304//oray Science Foundation/ ; //Inamori Research Institute for Science/ ; 24KJ1238//JSPS/ ; //Nagoya University/ ; //JST-SPRING/ ; },
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Neoplasms/genetics ; Cell Line, Tumor ; Gene Knock-In Techniques ; *DNA/genetics ; },
abstract = {Extrachromosomal DNA (ecDNA) amplification represents an emerging mechanism underlying oncogene amplification, tumor heterogeneity, and drug resistance in cancer. However, the biology of ecDNA remains poorly understood because tools to engineer ecDNAs and precisely monitor their dynamics are limited. In particular, genome engineering strategies have not been established for ecDNA, which exists in tens to hundreds of copies within a single cell. Here, we report a systematic validation of ecDNA editing using standard CRISPR-Cas9 system and optimized CRISPR-Cas9 system with safeguard single-guide RNAs (sgRNAs), in which the addition of cytosine extensions finely reduces excessive Cas9 activity. The conventional CRISPR-Cas9 system induced severe cytotoxicity and markedly reduced ecDNA copy number, together with frequent micronucleus formation. Knock-in efficiency was remarkably low, highlighting an intrinsic difficulty in editing ecDNA. In contrast, the safeguard sgRNA strategy not only alleviated cytotoxicity and ecDNA loss in a cytosine-length-dependent manner but also enabled efficient knock-in into multiple ecDNA per cell. Computational simulations suggested that the degree and temporal patterns of multiple DNA cleavage events shape cell death, micronucleus formation, and rapid expansion of knock-in ecDNA. Collectively, optimization of Cas9 activity using safeguard sgRNAs enables efficient and nondisruptive ecDNA engineering, providing a powerful tool to study ecDNA biology.},
}
@article {pmid41533584,
year = {2026},
author = {Zhang, J and He, X and Huang, J and Cheng, C and He, G and Xia, R and Yang, J and Chen, J and Guo, L and Xiang, D and Li, F and Shi, J and Li, P},
title = {Steric regulation of CRISPR/Cas12a trans-cleavage kinetics via split-activator extensions.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41533584},
issn = {1362-4962},
support = {2025ZDXM001//Chongqing Municipal Health Commission/ ; KJZD-K202400103//Chongqing Municipal Education Commission/ ; CSTB2024TIAD-CYKJCXX0031//Scientific and Technological Innovation Cooperation Program/ ; YXGD2025029//Chongqing Young and Middle-aged Medical High-level Talent Project/ ; YXQN2025049//Chongqing Youth Outstanding Medical Talent Project/ ; 2026MSXM018//Chongqing Science and Technology Bureau/ ; 2024YCXM010//2024 Hospital-level Cultivation Project of Chongqing University Jiangjin Hospital/ ; 2025qdjfxm001//Chongqing University Jiangjin Hospital/ ; 2025qdjfxm002//Chongqing University Jiangjin Hospital/ ; PS202511//2025 Key Research Project for Enhancing Medical Service Capabilities of County-level Medical Institutions/ ; CSTB2024NSCQ-KJFZMSX0018//Chongqing Science and Technology Development Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Kinetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism/chemistry ; MicroRNAs/genetics/analysis ; DNA/genetics/chemistry/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a holds substantial promise for molecular diagnostics, yet its rapid and uncontrolled activation often results in background leakage and disrupts the coordination of upstream reaction modules. Here, we established a steric-regulation framework that enables predictable tuning of Cas12a trans-cleavage kinetics through rationally engineered extensions on split activators. Systematic analysis of extension orientation, length, and hybridization state revealed quantitative and direction-dependent rules governing steric control of activator assembly and Cas12a activation. Guided by these insights, we integrated the sterically regulated split activator into an entropy-driven DNA circuit to construct a fully one-pot cascaded detection system. The engineered steric barriers effectively suppressed premature activation and established precise kinetic matching between the DNA circuit and Cas12a. The resulting platform achieved a detection limit of 1.24 pM for microRNA-21 and demonstrated high fidelity. This work defines a predictable steric-gating mechanism for Cas12a activation and delivers a nucleic-acid-only regulatory module that can be incorporated into diverse CRISPR architectures, supporting the development of robust, leakage-resistant one-pot diagnostic systems.},
}
@article {pmid41533833,
year = {2026},
author = {Chen, X and Mao, C and Gao, Y and Shi, C and Wang, Y and Jin, Z and Xia, B and Zhou, Y},
title = {Ultrasensitive Detection of Cardiac Troponin I via CRISPR/Cas12a-Mediated Liposomal Amplification Coupled with Electrospray Ionization Mass Spectrometry.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2183-2190},
doi = {10.1021/acs.analchem.5c05804},
pmid = {41533833},
issn = {1520-6882},
mesh = {*Troponin I/blood/analysis ; Humans ; *Spectrometry, Mass, Electrospray Ionization/methods ; *CRISPR-Cas Systems ; *Liposomes/chemistry ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Direct quantitative analysis of low-abundance protein biomarkers by electrospray ionization mass spectrometry (ESI-MS) remains challenging due to poor ionization efficiency and matrix interferences. Herein, we report an ultrasensitive analytical platform, termed CRISPR/Cas12a-mediated liposomal amplification coupled with electrospray ionization mass spectrometry (CMLA-MS), that overcomes this limitation by integrating CRISPR/Cas12a-mediated dual-cascade signal amplification with an ESI-MS readout. The strategy converts the detection of poorly ionizable protein molecules into the quantification of numerous, highly ionizable small-molecule reporters: proteins trigger Cas12a trans-cleavage (first amplification), which subsequently cleaves single-stranded DNA (ssDNA) probes anchored to signal-loaded liposomes, causing the burst release of thousands of MS-detectable reporters (second, physical amplification). This dual-amplification strategy enabled an exceptionally low limit of detection (LOD) of 10.8 fg/mL, and the method successfully quantified cardiac troponin I (cTnI) in clinical serum samples with high recoveries (90.3-101.6%).},
}
@article {pmid41533919,
year = {2026},
author = {Sun, X and Liang, Y and Liang, Y and Li, W and Chen, H and Ding, C and Duan, C and Zhou, Y and Zhang, R and Xu, H and Chen, J},
title = {PRPF8 Mutation-Induced Defects in Human iPSC-Derived RPE Are Rescued by Adenine Base Editing.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {67},
number = {1},
pages = {21},
pmid = {41533919},
issn = {1552-5783},
mesh = {Humans ; *Retinal Pigment Epithelium/metabolism/ultrastructure/pathology ; *Induced Pluripotent Stem Cells/metabolism ; *Mutation ; *Gene Editing/methods ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Adenine ; Cell Differentiation ; Cells, Cultured ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: The pathological effects of pre-mRNA processing factor 8 (PRPF8) mutations on the retinal pigment epithelium (RPE) are not fully understood. We aimed to identify disease-specific cellular and molecular phenotypes in PRPF8 retinitis pigmentosa (RP) patient-derived induced pluripotent stem cell (iPSC)-RPE and to test whether adenine base editing (ABE), which corrects the PRPF8 mutation in iPSCs, can reverse abnormal RPE phenotypes.<br><br>METHODS: We obtained patient-derived iPSCs with the heterozygous PRPF8 (c.5792C>T) mutation and created an induced mutation iPSC line by introducing the same mutation into wild-type iPSCs using CRISPR/Cas9. These cells were differentiated into RPE cells. We measured PRPF8 expression, barrier integrity, and apicobasal polarity. Electron microscopy examined apical microvilli and pigment granules. RNA sequencing quantified splicing events and affected pathways. ABE corrected the PRPF8 mutation in patient iPSCs, and the corrected clones were re-differentiated into RPE cells for evaluation.<br><br>RESULTS: PRPF8-mutant RPE cells exhibited decreased PRPF8 mRNA and protein levels, weakened barrier function, and disrupted cell polarity. Ultrastructural analysis showed loss of apical microvilli and pigment granules. Transcriptomic analysis identified abnormal splicing events, with enrichment in cilium assembly and melanosome pathways. ABE correction restored PRPF8 expression, normalized barrier integrity, apicobasal polarity, and rescued the defects in apical microvilli and pigment granules.<br><br>CONCLUSIONS: PRPF8 mutations in patient-derived iPSC RPE cause functional and ultrastructural defects driven by splicing abnormalities. ABE correction of the PRPF8 mutation in iPSCs can restore PRPF8 expression and alleviate cellular and molecular defects in RPE and highlights the therapeutic potential of precise gene editing correction strategies for RP.},
}
@article {pmid41533983,
year = {2026},
author = {Zhang, RR and Wang, YH and Peng, XF and Sun, YJ and Xu, ZS and Liu, H and Xiong, AS},
title = {Expansion protein DcEXP22 regulates taproot enlargement via mediating root cell extension in carrot.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70677},
doi = {10.1111/tpj.70677},
pmid = {41533983},
issn = {1365-313X},
support = {KYLH2025002//Fundamental Research Funds for the Central Universities/ ; 2023-SYS-02//Open Research Project of Key Laboratory of Biotechnology of Qinghai-Tibet Plateau Biotechnology of the Ministry of Education/ ; //Priority Academic Program Development of Jiangsu Higher Education Institutions Project PAPD/ ; //Bioinformatics Center of Nanjing Agricultural University/ ; },
mesh = {*Daucus carota/genetics/growth &amp; development/metabolism ; *Plant Roots/growth &amp; development/genetics/cytology/metabolism ; *Plant Proteins/genetics/metabolism/physiology ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Carrot (Daucus carota L.) is an important root vegetable crop of the Daucus genus in the Apiaceae. As the main product organ of carrot, the taproot has high nutritional and economic value. Expansins, a class of proteins involved in plant cell wall relaxation and cell extension, are mainly found in growing tissues and organs. Expansins play an important role in plant root development. Here, the DcEXP22 gene with a length of 789 bp was cloned from the carrot cultivar 'Kurodagosun'. Based on the stable genetic transformation system and CRISPR/Cas9 gene-editing technology, the DcEXP22 gene was overexpressed and knocked out in carrots. The results indicated that overexpression of the DcEXP22 gene increased carrot root fresh weight, root diameter, and root-shoot ratio, and enlarged the perimeter and area of taproot phloem cells. In contrast, knockout of the DcEXP22 gene inhibited the development of carrot taproots and the extension of phloem cells, suggesting that the DcEXP22 gene might promote the enlargement of carrot taproots by regulating the size of phloem cells. RNA-seq analysis identified several genes that were co-expressed with DcEXP22, including DcCYP734A1, DcERF1, DcMAP2K1, and DcSAD9. It was hypothesized that the DcEXP22 gene might influence the enlargement of carrot taproot by participating in the signal transduction of phytohormones such as brassinosteroids, cell wall synthesis and modification, and fatty acid metabolisms. These findings will advance our knowledge of the molecular mechanisms of carrot taproot enlargement.},
}
@article {pmid41534496,
year = {2026},
author = {Gogate, A and Chahrour, MH},
title = {Recent advances in the neurogenomics of autism spectrum disorder.},
journal = {Current opinion in genetics &amp; development},
volume = {96},
number = {},
pages = {102431},
doi = {10.1016/j.gde.2025.102431},
pmid = {41534496},
issn = {1879-0380},
mesh = {Humans ; *Autism Spectrum Disorder/genetics/pathology ; *Gene Regulatory Networks/genetics ; *Genomics ; Induced Pluripotent Stem Cells/metabolism ; *Genetic Predisposition to Disease ; CRISPR-Cas Systems/genetics ; Gene Editing ; },
abstract = {Neurogenomics has provided exceptional insights into the genetic architecture underlying autism spectrum disorder (ASD), which is increasingly understood as a collection of individually rare disorders. This review synthesizes current advancements in the field, examining how both rare and common genetic variants contribute to ASD etiology. To functionally interpret the convergence on biological pathways that has emerged despite this genetic heterogeneity, multiomic approaches have been applied to identify gene regulatory networks disrupted in ASD. High-throughput technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR) editing and massively parallel reporter assays, have been employed in human induced pluripotent stem cells and organoids to bridge the gap between genetic association and biological function. Finally, machine learning methods play a pivotal role in integrating and leveraging these complex datasets to inform personalized interventions.},
}
@article {pmid41535129,
year = {2026},
author = {Du, J and Pu, X and Yuan, T and Peng, F and Hu, J and Li, H and Chen, B and Luo, J and Li, S and Teng, Y and Zhu, X and Chen, W and Xie, Q and Jiang, L and Xiong, E and Yang, R},
title = {Plug-and-Play Photo-Initiated CRISPR-Cas12a One-Pot Nucleic Acid Detection via Universal Repeat RNA Acylation Strategy.},
journal = {Analytical chemistry},
volume = {98},
number = {3},
pages = {2136-2145},
doi = {10.1021/acs.analchem.5c05769},
pmid = {41535129},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Acylation ; *RNA/genetics/analysis/chemistry ; Humans ; Gene Editing ; CRISPR-Associated Proteins/metabolism ; Photochemical Processes ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Precise spatiotemporal control of CRISPR activity is central to both accurate gene editing and sensitive molecular diagnostics. However, current regulatory strategies are often sequence-specific, labor-intensive, and difficult to generalize. Here, we report a minimalist plug-and-play tactic: acylation of the repeat region (rRNA) of a split crRNA with photolabile groups. Because the modification is introduced post-synthesis and is independent of the spacer region (sRNA), every rRNA, regardless of its target sequence, can be activated by light irradiation alone, entirely eliminating the need for redesign or reoptimization. Integrating the photo-initiated CRISPR-Cas12a system with recombinase polymerase amplification into a one-pot format yields an upgraded platform, named POIROTv2 (PhotO-Initiated CRISPR-Cas12a system for Robust One-pot Testing, version 2). POIROTv2 achieves a 100-fold sensitivity gain over conventional always-on Cas12a-based one-pot assays and matches the analytical performance of a two-step assay while remaining a more streamlined and potentially faster detection process and avoiding the risk of aerosol contamination. In clinical validation with HCMV- and EBV-suspected samples, POIROTv2 delivered diagnostic accuracy statistically indistinguishable from that of gold-standard qPCR, highlighting its potential for robust and sensitive molecular diagnostics. Overall, the strategy opens up exciting possibilities for applications in infectious virus diagnostics and has broad prospects in the field of spatiotemporally controllable gene editing.},
}
@article {pmid41535270,
year = {2026},
author = {Taguchi, J and Kikuchi, M and Jeon, H and Shimizu, R and Mori, H and Ikawa, M and Yamada, Y and Sato, K and Ikeda, T and Yamazaki, S and Ozawa, M},
title = {A scalable two-step genome editing strategy for generating full-length gene-humanized mice at diverse genomic loci.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {356},
pmid = {41535270},
issn = {2041-1723},
support = {23K27084//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 25K18393//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05033//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21H05033//Japan Society for the Promotion of Science London (JSPS London)/ ; },
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; CRISPR-Cas Systems/genetics ; Gene Knock-In Techniques/methods ; Male ; Mice, Transgenic ; Alleles ; Homologous Recombination ; Embryonic Stem Cells/metabolism ; Genetic Loci ; Hematopoiesis/genetics ; Female ; },
abstract = {Full-length gene-humanized mice engineered by completely replacing mouse loci with human counterparts, including untranslated and regulatory regions, provide a robust in vivo platform for human gene function studies. However, reliably humanizing large genomic regions remains challenging due to limited DNA insert sizes, complex protocols, and specialized material requirements. This study introduces a streamlined approach that enables full-length gene humanization through two sequential CRISPR-assisted homologous recombination steps in embryonic stem cells. This method supports targeted knock-in of genomic fragments (> 200 kbp) and is applicable across multiple mouse strains. Humanized alleles generated using the developed method recapitulate human-like splicing isoforms and organ-specific gene expression while restoring essential functions in hematopoiesis, spermatogenesis, and survival. Furthermore, disease-associated mutations can be engineered into humanized alleles to model human genetic disorders in vivo. This versatile platform enables the creation of physiologically relevant, fully gene-humanized mouse models for broad applications in biomedical research.},
}
@article {pmid41535388,
year = {2026},
author = {Rolando, JC and Thieme, A and Weckman, NE and Kim, N and de Puig, H and Tan, X and Cotnoir, E and Chaturvedi, V and Collins, JJ and Walt, DR},
title = {Digital CRISPR-based diagnostics for quantification of Candida auris and resistance mutations.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {41535388},
issn = {2157-846X},
support = {WC-2019-01//New York State Department of Health - Wadsworth Center (Department of Health, Wadsworth Center)/ ; },
abstract = {Candida auris, an increasingly prevalent fungal pathogen, requires both rapid identification and antifungal susceptibility testing to enable proper treatment. This study introduces digital SHERLOCK (dSHERLOCK), a platform that combines CRISPR/Cas nucleic acid detection, single-template quantification and real-time kinetics monitoring. Assays implemented on this platform display excellent sensitivity to C. auris from major clades 1-4, while maintaining specificity when challenged with common environmental and pathogenic fungi. dSHERLOCK detects C. auris within 20 min in minimally processed swab samples and achieves sensitive quantification (1 c.f.u. µl[-1]) within 40 min. To address antifungal susceptibility testing, we develop assays that detect mutations that are commonly associated with azole and echinocandin multidrug resistance. We use machine learning and real-time monitoring of reaction kinetics to achieve highly accurate simultaneous quantification of mutant and wild-type FKS1 SNP alleles in fungal populations with mixed antifungal susceptibility, which would be misdiagnosed as completely susceptible or resistant under standard reaction conditions. Our platform's use of commercially available materials and common laboratory equipment makes C. auris diagnostics widely deployable in global healthcare settings.},
}
@article {pmid41535609,
year = {2026},
author = {Gottimukkala, KSV and Lane, DD and Cunningham, R and Malik, HS and Jwa, Y and Cassidy, ME and Castelli, JMP and Enstrom, MR and Poljakov, K and Gastelum, G and Ho, SH and Tassa, C and Adair, JE},
title = {CRISPR-AuNP: physicochemical optimization of a gold nanoparticle platform for cost-effective and modular non-viral gene editing in HSPCs.},
journal = {Gene therapy},
volume = {33},
number = {2},
pages = {188-202},
pmid = {41535609},
issn = {1476-5462},
support = {P30 CA015704/CA/NCI NIH HHS/United States ; R01 AI158728/AI/NIAID NIH HHS/United States ; R01 AI167009/AI/NIAID NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; },
mesh = {*Gene Editing/methods/economics ; *Gold/chemistry ; *CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Humans ; *Hematopoietic Stem Cells/metabolism ; Cost-Benefit Analysis ; Polyethylene Glycols/chemistry ; },
abstract = {Efficient delivery of CRISPR ribonucleoproteins into primary hematopoietic stem and progenitor cells (HSPCs) is essential for durable gene editing therapies but remains challenging. Here, we advance a modular, benchtop-assembled gold-polymer hybrid nanoparticle (CRISPR-AuNP) platform that enables non-viral delivery of multiple CRISPR systems into HSPCs. Guided by a mechanistic understanding of Cas9's interaction with gold surfaces, we engineered the formulation by conjugating pre-formed RNP-polymer complexes, assembled using thiolated polyethyleneimine-polyethylene glycol, to gold nanoparticles. This system achieved efficient editing in primary CD34+ HSPCs for Cas9, Cas12a, and Cas12a-M29-1 without compromising cell viability. Notably, the nanoformulation can be assembled in under 2 h in a PCR tube for less than $70/million HSPCs treated. This work establishes a scalable, cost-effective, and accessible gene editing system with the potential to democratize CRISPR applications in HSPC research and therapy.},
}
@article {pmid41536070,
year = {2026},
author = {Zeng, H and Liu, A and Daniel, TC and Golla, DA and Lu, Z and Serodio, R and Millette, BA and Lingineni, A and Gilberd, P and Chee, K and Talloo, K and Peddi, A and Park, SH and Bao, G and Gao, X},
title = {Precision A3G base editors for disease modeling and correction.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {2343-2360},
pmid = {41536070},
issn = {1525-0024},
support = {R01 HL157714/HL/NHLBI NIH HHS/United States ; R01 HL173243/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; CRISPR-Cas Systems ; *APOBEC-3G Deaminase/genetics/metabolism ; *Cystic Fibrosis/genetics/therapy ; Mutation ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Cytosine base editors (CBEs) enable efficient cytosine-to-thymine substitutions at targeted genomic loci without introducing double-stranded breaks. Among CBEs, APOBEC3G BEs (A3G-BEs) preferentially edit the second cytosine within a 5'-CC-3' motif in human cells, reducing potential bystander editing. However, A3G-BEs often unintentionally edit multiple CC motifs within their editing window and are limited by protospacer adjacent motif (PAM) constraints imposed by SpCas9, which restricts their applicability. Here, we engineered A3G-BE variants through linker optimization, rational mutagenesis, and the integration of SpG and SpRY Cas9 effectors with relaxed PAM constraints. These improvements enhanced the precision of single-cytosine editing within CC motifs and broadened the targeting scope to previously inaccessible genomic sites. We then validated the engineered A3G-BE variants by precisely installing and correcting cystic fibrosis-causing mutations in HEK293T cells. When applied to 16HBE14o-human bronchial epithelial cells, precise editing modulated cystic fibrosis transmembrane conductance regulator mRNA levels, protein expression, and channel function, establishing precision A3G-BE variants as powerful tools for modeling and treating cystic fibrosis and other human diseases.},
}
@article {pmid41536809,
year = {2026},
author = {Liou, RH and Urrutia-Cabrera, D and Liu, CF and Wu, S and Westin, IM and Golovleva, I and Liu, GS and Kumar, S and McLenachan, S and Chen, FK and Hsu, FT and Huang, CL and Edwards, T and Martin, KR and Cheng, AW and Wong, RCB},
title = {Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to target ABCA4 splice variants in Stargardt disease.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102789},
pmid = {41536809},
issn = {2162-2531},
abstract = {Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T>C and c.4773+3A>G in ABCA4 cause the skipping of exon 39-40 and exon 33-34, respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C, and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T>C variants and up to 78% reduction of the ABCA4 c.4773+3A>G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A>G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.},
}
@article {pmid41536960,
year = {2026},
author = {van der Wilt, CN and Veltrop, RJA and Janssens, MH and Bakker, I and Stillitano, F and Sluijter, JPG and van Laake, LW and van der Velden, J and Villard, E and Montag, J and Denning, C and van Tintelen, JP and Te Riele, ASJM and van der Harst, P and Schurgers, LJ and van Steenbeek, FG and Harakalova, M},
title = {Setting the stage for cardiomyopathy gene editing trials: a systematic review of isogenic pair use in human induced pluripotent stem cell-derived cardiomyocyte research.},
journal = {European heart journal open},
volume = {6},
number = {1},
pages = {oeaf161},
pmid = {41536960},
issn = {2752-4191},
abstract = {In vitro gene editing using isogenic pairs of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has demonstrated the feasibility of introducing or correcting specific pathogenic variants. These successes represent a key first step towards therapeutic genome editing for cardiomyopathies, showing that precise, variant-specific interventions are achievable. To translate in vitro findings to the clinic, it is essential to develop robust disease models that yield meaningful, translatable data. The next challenge is systematically identifying disease-causing variants amenable to gene editing with strong pre-clinical support. Therefore, we conducted a systematic search of published studies on isogenic hiPSC-CM pairs in cardiomyopathy research with specific criteria, including (likely) pathogenic variants causing cardiomyopathy, correction and/or introduction of variants, differentiation into CMs, and functional follow-up. We systematically assessed 785 papers and highlighted 101 studies meeting our inclusion criteria reporting 69 patients carrying 56 unique variants across 31 genes, most commonly MYH7, MYBPC3, and DMD. This expanded to 91 variants across 38 genes upon inclusion of the introduced variants in a donor line. However, reported clinical data were often incomplete, underscoring the need for standardized phenotypic documentation. We reveal a lack of patient details, which creates an incomplete picture of underlying disease variables that hinder the design of targeted personalized treatments. Omitted key clinical data can lead to misinterpretations or overlooked variables that impact treatment outcomes. This systematic review integrates current evidence from successful in vitro studies using isogenic hiPSC-CM models and proposes a reporting framework for variant prioritization and the rigorous application of isogenic controls in cardiomyopathy research.},
}
@article {pmid41538311,
year = {2026},
author = {Xiao, Z and Sun, Y},
title = {Illuminating the genome: emerging approaches in CRISPR-Cas live-cell imaging.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41538311},
issn = {1362-4962},
support = {2022YFA1303103//National Key Research and Development Program of China/ ; 2022YFA3401100//National Key Research and Development Program of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome ; Gene Editing/methods ; Animals ; },
abstract = {CRISPR-Cas-based live-cell imaging has rapidly become a central technology for studying genome dynamics with high specificity and flexibility. By coupling nuclease-deactivated Cas (dCas) with programmable guide RNAs, genomic loci can be tracked in living cells, providing direct insights into nuclear organization and chromatin behavior. While repetitive regions such as telomeres and centromeres are readily visualized, labeling non-repetitive loci remains more challenging due to weak signals and high background. Recent advances, including multicolor labeling strategies, innovative amplification systems based on dCas9 and single-guide RNA (sgRNA) engineering, and integration with novel fluorescent reporters, have markedly expanded the applicability of CRISPR imaging across the genome. These developments have expanded the multiplexing capacity of CRISPR imaging, improved signal-to-background ratios, and even enabled the visualization of non-repetitive genomic loci. Nonetheless, key challenges remain, including cellular toxicity, replication stress, and genomic instability associated with prolonged CRISPR expression. In this review, we summarize recent advances in CRISPR live-cell imaging and highlight key design trade-offs and biological constraints.},
}
@article {pmid41538317,
year = {2026},
author = {Ham, DT and Browne, TS and Zhang, CQ and Foo, GW and Uruthirapathy, AS and Gloor, GB and Edgell, DR},
title = {Machine learning reveals sequence and methylation determinants of SaCas9-PAM interactions in bacteria.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41538317},
issn = {1362-4962},
support = {PJT 159708/CAPMC/CIHR/Canada ; PJT 191939/CAPMC/CIHR/Canada ; },
mesh = {*Machine Learning ; Staphylococcus aureus/genetics/enzymology ; *CRISPR-Associated Protein 9/metabolism/genetics ; Escherichia coli/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems ; Nucleotide Motifs ; DNA Methylation ; Methylation ; Gene Editing/methods ; },
abstract = {Cas9 nucleases defend bacteria against invading DNA and can be used with single guide RNAs (sgRNAs) as antimicrobials and genome-editing tools. However, bacterial applications are limited by incomplete knowledge of Cas9-target interactions. Here, we generated large-scale Staphylococcus aureus Cas9 (SaCas9)/sgRNA activity datasets in bacteria and trained a machine learning model (crispr macHine trAnsfer Learning) to predict SaCas9 activity. Incorporating downstream sequences flanking the canonical NNGRRN protospacer adjacent motif (PAM) at positions [+1] and [+2] improved predictive performance, with T-rich dinucleotides at these positions correlating with higher in vivo activity. Crucially, SaCas9 showed $\sim$10-fold reduced activity at sites containing a 5$^{\prime}
$-NNGGAT[C]-3$^\prime$ PAM [+1] sequence in pooled sgRNA experiments in Escherichia coli and Citrobacter rodentium. Plasmid cleavage assays in DNA adenine methyltransferase (DAM)-deficient E. coli confirmed that adenine methylation at GATC motifs inhibited SaCas9 activity. Removal of a DAM site within a PAM sequence enhanced cleavage, while introduction of a site reduced activity, directly linking adenine methylation to SaCas9 activity. These findings demonstrate that machine learning can uncover biologically relevant determinants of Cas9 activity. Avoidance of methylated PAMs may reflect an evolutionary adaptation by SaCas9 to discriminate self from nonself or to counter methylation as a phage and plasmid antirestriction strategy.},
}
@article {pmid41538882,
year = {2026},
author = {Loubat, A and Wolfender, C and Calabre, M and Beaude, N and Tavares, P and Planson, AG and Jules, M},
title = {Advancing Fast-Track Genome Engineering in Bacillus subtilis Phages.},
journal = {ACS synthetic biology},
volume = {15},
number = {2},
pages = {687-700},
doi = {10.1021/acssynbio.5c00727},
pmid = {41538882},
issn = {2161-5063},
mesh = {*Bacillus subtilis/virology/genetics ; *Genome, Viral/genetics ; CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacillus Phages/genetics ; *Genetic Engineering/methods ; },
abstract = {Phage genome engineering methods accelerate the study of phage biology, the discovery of new functions, and the development of innovative genetic engineering tools. Here, we present QuickPhage, a rapid, technically accessible, precise, and cost-effective method for engineering Bacillus subtilis phages. Our approach uses CRISPR-Cas9 as a counter-selection system to isolate mutants of the model lytic siphovirus phage, SPP1. Efficient genome editing was achieved using homologous repair patches as short as 40 nucleotides, enabling streamlined patch construction and parallel engineering, resulting in highly accurate genome edits within a day. We applied QuickPhage to delete both essential and nonessential phage genes and to insert reporter genes. Protein production, such as GFP, was synthetically regulated using inducible systems without significantly affecting phage fitness, achieving induction levels of up to 400-fold. Time-series coinfection experiments with fluorescent protein expressing phages also revealed a highly efficient superinfection arrest mechanism that prevents reinfection as early as 13 min after initial infection. These findings highlight the potential of phages for protein production, opening new opportunities for metabolic engineering. This work also lays the foundation for systematic phage genome refactoring workflows and the development of phage-based tools for efficient DNA delivery, thereby expanding the synthetic biology toolbox for B. subtilis.},
}
@article {pmid41539085,
year = {2026},
author = {Dong, T and Zhao, Y and Jin, HF and Pan, HM and Yue, LL and Lin, Y and Shen, L},
title = {The establishment of a GPD1L knockout human embryonic stem cell line (WAe009-A-80) using the CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103910},
doi = {10.1016/j.scr.2026.103910},
pmid = {41539085},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Glycerolphosphate Dehydrogenase/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Differentiation ; },
abstract = {The GPD1L gene is located on 3p22.3. It encodes the glycerol phosphate dehydrogenase 1-like protein with homology to glycerol phosphate dehydrogenase (GPD1L), but the function of this enzyme is unclear. Mutations in GPD1L have been associated with BrS (Brugada syndrome) and SIDS (sudden infant death syndrome) and reduce Na[+] inward current through an unknown mechanism in human cardiomyocytes. Here, a GPD1L knockout human embryonic stem cell line was generated using CRISPR/Cas9 system. The GPD1L knockout human embryonic stem cell maintains the pluripotency, differentiation into three germ layers, forming normal EBs.},
}
@article {pmid41540035,
year = {2026},
author = {Zhang, H and Shang, R and Zhang, Z and Zhou, M and Bigot, A and Cai, Y and Zhao, Y and Wang, Y and Deshmukh, A and Kudryashova, E and Kudryashov, DS and He, C and Mouly, V and Bi, P},
title = {Development of a split-toxin CRISPR screening platform to systematically identify regulators of human myoblast fusion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {547},
pmid = {41540035},
issn = {2041-1723},
support = {R21 AR080330/AR/NIAMS NIH HHS/United States ; R35 GM147209/GM/NIGMS NIH HHS/United States ; GM147209//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; AR080330//U.S. Department of Health &amp; Human Services | NIH | National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)/ ; },
mesh = {Humans ; *Myoblasts/metabolism/cytology ; *Muscle Development/genetics ; Cell Differentiation/genetics ; Cell Fusion ; *CRISPR-Cas Systems/genetics ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Muscle, Skeletal/metabolism ; High-Throughput Screening Assays/methods ; Single-Cell Analysis ; Cell Line ; },
abstract = {Muscle defects are common in human developmental disorders and often cause severe functional impairment. These defects arise from intricate tissue crosstalk and rare genetic mutations, underscoring the need to systematically identify cell-autonomous mechanisms regulating human myogenesis. Here we show a rationally designed, high-throughput genetic screening platform that integrates human myoblast models, customized CRISPR libraries, and a split-toxin strategy that enables quantitative selection of fusion-defective myocytes. Leveraging this platform, our initial screen uncovers a large group of hits essential for human myoblast fusion. The majority of these hits converge into 23 protein complexes. Notably, mutations in 41 screen hits are associated with human diseases marked by abnormal skeletal-muscle morphology. Applying a new single-cell CRISPR &amp; RNA-seq approach, we show that majority of these hits control human myoblast fusion as well as influence early-stage myogenic differentiation. This work establishes a scalable approach to identify cell-autonomous regulators of human muscle differentiation and fusion.},
}
@article {pmid41540063,
year = {2026},
author = {Port, F and Buhmann, MA and Zhou, J and Stricker, M and Vaughan-Brown, A and Michalsen, AC and Roßmanith, E and Pöltl, A and Großkurth, L and Huber, J and Menendez Kury, LB and Weberbauer, B and Hübl, M and Puscher, E and Heigwer, F and Boutros, M},
title = {Improved in vivo gene knockout with high specificity using multiplexed Cas12a sgRNAs.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {877},
pmid = {41540063},
issn = {2041-1723},
support = {ERC-DECODE//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; SFB1324//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; Drosophila melanogaster/genetics ; Gene Targeting/methods ; *Bacterial Proteins/genetics/metabolism ; Drosophila/genetics ; },
abstract = {CRISPR nuclease-mediated gene knock-out is limited by suboptimal sgRNAs, inaccessible target sites, and undesired repair outcomes. Here, we present a Cas12a-based system in Drosophila that targets each gene with four sgRNAs to overcome these limitations. Multiplexed sgRNAs act through redundancy and synergism, frequently creating deletions between target sites and increasing the fraction of loss-of-function mutations. We show that multiplexed gene targeting is well tolerated and does not cause widespread proximity effects. To visualize CRISPR-nuclease activity in living animals, we developed a screening assay and used it to assess Cas12a activity across 33% of the Drosophila genome in combination with over 2000 sgRNAs. This revealed remarkably high on-target (>99%) and very low (<1%) off-target activity of multiplexed Cas12a sgRNA arrays. Quantitative side-by-side comparisons with current Cas9-based systems targeting over 100 genes in parallel demonstrate that multiplexed Cas12a gene targeting achieves superior performance and reveals phenotypes missed by established methods. The system described here provides a framework for reliable gene knock-out in multicellular systems.},
}
@article {pmid41541975,
year = {2025},
author = {Hummel, L and Carr, C and Biow, S and Asher, K and Sauer, T and Maylin, M},
title = {Re-examining the Diagnostic Criteria for Wilson's Disease: A Case Report and Literature Review.},
journal = {Cureus},
volume = {17},
number = {12},
pages = {e99271},
pmid = {41541975},
issn = {2168-8184},
abstract = {Wilson's disease (WD) is a heterogeneous genetic disorder for which diagnosis is challenging. We present the case of a 54-year-old woman with a complicated medical history, including hepatitis C, cirrhosis, hepatic encephalopathy, and extensive psychiatric disease, who was transferred to our hospital for management of a spinal epidural abscess. Further findings suggested undiagnosed WD, including bilateral rings around Descemet's membrane and modestly low ceruloplasmin but normal urinary copper excretion. Many algorithms have been proposed for Wilson disease diagnosis, including clinical, laboratory, imaging, and genetic findings; however, no single test is diagnostic. The European Association for the Study of the Liver (EASL) guidelines are the most commonly employed algorithm. Updated 2022 guidelines by both the American Association for the Study of Liver Diseases (AASLD) and the British Association for the Study of the Liver (BASL) build upon this algorithm. Our patient scored 5 points on the EASL scale, which is sufficient for diagnosis. Nevertheless, we believe that she met the diagnostic criteria without having the actual disease. Newer guidelines by AASLD and BASL do not provide additional conclusivity. Our case demonstrates the need for re-evaluation of the diagnostic criteria of WD, where uncertainty can mean permanent hepatic and brain damage. Diagnostic guidelines should incorporate new biomarkers, ophthalmological techniques, and advanced technologies such as next-generation sequencing or CRISPR-Cas-based tools.},
}
@article {pmid41542957,
year = {2026},
author = {Lee, SY and Park, HH},
title = {Structural insights into promoter recognition by Aca7.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70405},
pmid = {41542957},
issn = {1742-4658},
support = {RS-2025-02316334//NRF/ ; RS-2025-16065724//NRF korea/ ; },
abstract = {CRISPR-Cas systems provide adaptive immunity to bacteria, although bacteriophages counter these defenses with anti-CRISPR (Acr) proteins. Acr expression is frequently regulated by anti-CRISPR associated (Aca) proteins, which repress transcription by binding inverted repeat (IR) sequences in operon promoters. Here, we report the first identification of an IR motif within the AcrIF11-Aca7 operon promoter from Halomonas caseinilytica and present the crystal structure of Aca7 bound to this IR DNA. Biochemical assays demonstrated that Aca7 specifically recognizes the IR element, and structural analysis revealed a symmetric Aca7 dimer engaging both major grooves via helix-turn-helix motifs while stabilizing DNA bending through minor groove contacts. Residue-level interactions, including those mediated by R38, Q42, K46, and K49, establish a detailed basis for sequence-specific recognition. Comparison with Aca2 highlights distinct dimer architectures and DNA deformation strategies among Aca proteins. Our findings uncover the molecular mechanism by which Aca7 represses AcrIF11 expression and broaden the understanding of Aca-mediated transcriptional regulation.},
}
@article {pmid41543170,
year = {2026},
author = {Romanowski, JS and Myles, KM and Adelman, ZN},
title = {Microhomology-mediated end joining is the predominant form of DNA repair in the mosquito Aedes aegypti with implications for gene editing, gene drive, and transgene removal.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41543170},
issn = {1362-4962},
support = {//National Institute of Allergies and Infectious Diseases/ ; AI148787/NH/NIH HHS/United States ; //NIAID/ ; },
mesh = {Animals ; *Aedes/genetics ; *Gene Editing/methods ; *DNA End-Joining Repair ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; Transgenes ; Gene Drive Technology ; DNA Repair ; },
abstract = {Programmable site-specific nucleases have revolutionized the field of genetics, and in the field of mosquito vector control, gene editing by these tools has inspired a new wave of population control approaches that aim to prevent disease transmission. Little is known of how DNA repair is prioritized in mosquitoes, which diverged from the nearest model system (Drosophila) by >200 million years, despite site-specific gene editing now being commonplace. Here, we report a scalable, high-throughput platform for studying DNA double-stranded DNA break (DSB) repair in mosquitoes by delivering CRISPR/Cas9, I-SceI, or other nucleases to Aedes aegypti embryos, capable of measuring single-strand annealing (SSA), non-homologous end joining, and microhomology-mediated end-joining (MMEJ) repair outcomes. We find CRISPR/Cas9 can induce deletions of up to 8.6 kb through SSA repair and is tolerant of resection distances of 3.5 kb. Indel events were insensitive to lig4 knockouts, and across 20 synthetic guide RNAs (sgRNAs) representing 5 locations in 2 transgenic strains were almost exclusively attributed to MMEJ repair, establishing MMEJ as the dominant form of repair in A. aegypti at CRISPR/Cas9 DSBs. This information is critical to our understanding of how DNA repair shapes processes required for genetic control strategies involving gene drive action/resistance as well as transgene stability.},
}
@article {pmid41543272,
year = {2026},
author = {Lummerstorfer, M and Lächelt, U},
title = {Non-Viral CRISPR carriers: transient delivery with lasting effects.},
journal = {Drug delivery},
volume = {33},
number = {1},
pages = {2614125},
pmid = {41543272},
issn = {1521-0464},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Gene Transfer Techniques ; Animals ; Genetic Vectors ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-Cas9 has revolutionized the field of genome editing. While conventional gene supplementation therapies and the market of related gene therapy products are dominated by viral vectors, non-viral delivery strategies are increasingly being explored for in vivo CRISPR applications. Given the permanent nature of genome editing, prolonged expression of the CRISPR machinery is not required, and transient delivery nevertheless can achieve lasting therapeutic effects. In contrast, short-term availability of genome editing components is rather considered advantageous to reduce the risk of off-target effects in a 'hit-and-run' fashion. In this article, we provide a systematic survey of the current clinical trial landscape with focus on in vivo CRISPR therapies and discuss utilized delivery strategies. As of December 2025, 136 CRISPR trials are ongoing, including 36 based on in vivo delivery of CRISPR components which show a clear shift towards non-viral vectors. The article describes the clinically employed CRISPR technologies and non-viral delivery platforms, highlighting both the present opportunities and key challenges associated with CRISPR delivery in the future.},
}
@article {pmid41543528,
year = {2026},
author = {O'Connor-Moneley, J and Lange, T and Flanagan, PR and Brunke, S and Fischer, L and Sharma, R and Puri, S and Hube, B and Sullivan, DJ and Moran, GP},
title = {Depletion of the Candida albicans TLO gene family reveals a requirement for alpha TLO genes for wild-type virulence.},
journal = {Microbiology (Reading, England)},
volume = {172},
number = {1},
pages = {},
doi = {10.1099/mic.0.001654},
pmid = {41543528},
issn = {1465-2080},
mesh = {*Candida albicans/genetics/pathogenicity/growth &amp; development ; Animals ; Virulence/genetics ; Mice ; *Fungal Proteins/genetics/metabolism ; Hyphae/growth &amp; development/genetics ; Disease Models, Animal ; Candidiasis, Oral/microbiology ; Humans ; Macrophages/microbiology ; Female ; Gene Expression Regulation, Fungal ; CRISPR-Cas Systems ; *Mediator Complex/genetics/metabolism ; Multigene Family ; },
abstract = {Candida albicans uniquely possesses an expanded family of genes (the TLO gene family) that encodes 10-15 paralogues of the Med2 component of the transcriptional regulator Mediator. Previous studies have shown that TLO null mutants are unable to form hyphae and are hypersensitive to environmental stress. However, the reason for the TLO gene expansion remains unclear, and the current study aimed to determine if reduction in the TLO family copy number affected virulence. In order to investigate this, we used CRISPR-Cas9 mutagenesis to generate two TLO-depleted mutants: one mutant retaining only TLOβ2 (CaTLO2) and the second mutant containing only TLOγ5 (CaTLO5). Both TLO-depleted mutants exhibited increased filamentous growth, increased susceptibility to specific stresses and reduced virulence in a murine model of oropharyngeal candidiasis (OPC). In vitro, the CaTLO5 mutant also exhibited impaired hyphal escape from macrophages and reduced hyphal invasion of oral keratinocytes. We then investigated if complementation with TLOα1, a gene previously shown to restore wild-type growth in a Δtlo null mutant, could restore virulence. In vitro infection models showed that TLOα1 could restore true hypha formation, epithelial invasion and hyphal escape from macrophages in the CaTLO5 background. The murine OPC model showed that TLOα1 could restore wild-type virulence in both CaTLO2 and CaTLO5 strains, suggesting an essential role for α-TLO in oral mucosal infection. Together, these findings highlight the functional specialization between the α, β and γ TLO gene groups and establish α-TLO as a major regulator of virulence in C. albicans.},
}
@article {pmid41543903,
year = {2026},
author = {Yin, S and Jarosz, DF and Ting, AY},
title = {Towards CRISPR-based editing of the mitochondrial genome in yeast.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {3},
pages = {e2505894123},
pmid = {41543903},
issn = {1091-6490},
support = {R01 HG012366/HG/NHGRI NIH HHS/United States ; NA//Chan Zuckerberg Initiative (CZI)/ ; NA//Stanford University (SU)/ ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Gene Editing/methods ; *Genome, Mitochondrial/genetics ; *CRISPR-Cas Systems/genetics ; Mitochondria/genetics/metabolism ; DNA, Mitochondrial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Mitochondria, which evolved from symbiotic bacteria, possess their own genomes (mtDNA) and support independent transcription and translation within the organelle. Given the essential role of mtDNA in energy production, metabolism, as well as cellular homeostasis, and the high density of confirmed pathogenic mutations that map to mtDNA, there is a pressing need for versatile methods to study and manipulate this genome. Although CRISPR technology has revolutionized the editing of nuclear genomes, it has not been successfully extended to mtDNA, primarily due to the challenge of delivering single guide RNAs (sgRNAs) across both outer and inner mitochondrial membranes. Here we develop a survival-based reporter in Saccharomyces cerevisiae to screen for potential RNA import motifs. We identify a 40-nucleotide aptamer (IM83) that facilitates sgRNA entry into the mitochondrial matrix, enabling CRISPR editing by a mitochondrially-localized adenine base editor. We show that mitochondrial import of IM83 is ATP-dependent and enhanced by the tRNA synthetase Msk1. Further investigations identify barriers to efficient CRISPR editing of mtDNA, including loss of membrane potential associated with mitochondrial targeting of the base editor. These insights lay the groundwork for future improvements in CRISPR-based editing of mtDNA in eukaryotes.},
}
@article {pmid41544897,
year = {2026},
author = {Hossain, NS and Tasnim, N and Ferdoush, J and Roy, A and Saha, SC and Saha, A},
title = {Next-generation lung-cancer-on-a-chip: Toward personalized therapy, AI, and CRISPR-driven models.},
journal = {Drug discovery today},
volume = {31},
number = {2},
pages = {104604},
doi = {10.1016/j.drudis.2026.104604},
pmid = {41544897},
issn = {1878-5832},
mesh = {Humans ; *Lung Neoplasms/genetics/drug therapy/pathology/therapy ; *Precision Medicine/methods ; *Artificial Intelligence ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Editing ; Animals ; CRISPR-Cas Systems ; *Lab-On-A-Chip Devices ; Tumor Microenvironment ; },
abstract = {Lung-cancer-on-a-chip (LCOC) technologies have advanced rapidly, yet most models evaluate mechanical strain, patient-derived tumors, multi-organ interactions, artificial intelligence (AI) analytics, and clustered regularly interspaced short palindromic repeats (CRISPR) editing in isolation. In this review, we uniquely integrate these emerging components into a unified framework centered on the breathing LCOC. We highlight how embedding patient-derived lung tumor fragments into cyclically stretched microenvironments, then linking them to downstream organ compartments, enables patient-specific mapping of metastatic routes under physiologically relevant mechanics. We further describe how continuous high-resolution imaging from these platforms can feed AI pipelines for automated drug-response prediction and metastatic trajectory simulation, and how on-chip CRISPR editing enables accurate investigation of metastatic drivers within dynamic, strain-modulated microenvironments. By synthesizing these technologies, we outline a next-generation, personalized multi-organ-on-chip architecture capable of predicting individual disease progression without direct patient risk. We also address practical barriers, including tumor fragility under strain, imaging domain shift, and gene-editing delivery challenges, and how to overcome such barriers.},
}
@article {pmid41545355,
year = {2026},
author = {Tian, T and Zhang, T and Zhang, W and Qiu, Z and Guo, X and Chen, Y and Lin, M and Qi, W and Shen, Y and Hao, M and Xiao, H and Xiang, B and Pang, F and Song, J and Sun, B and Cheng, M and Zhou, X},
title = {Identification of thermotolerant non-canonical PAMs for robust one-pot CRISPR-Cas12a detection.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1771},
pmid = {41545355},
issn = {2041-1723},
support = {32150019//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82502830//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M741238//China Postdoctoral Science Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Humans ; *Thermotolerance ; Nucleotide Motifs/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The canonical PAM site TTTV (where V = A, G, or C) is widely used in the design of CRISPR-Cas12a systems for both genome editing and diagnostic applications. Although several non-canonical protospacer-adjacent motifs (PAM) have been identified, they generally exhibit weak Cas12a cleavage activity. In this study, we find that increasing the reaction temperature to 45 °C or higher allows the identification of numerous non-canonical PAMs with trans-cleavage activity comparable to that of canonical PAMs, while displaying only weak cis-cleavage activity. Moreover, we observe that combining these non-canonical PAMs with elevated temperatures significantly enhances the Cas12a system's ability to discriminate highly similar sequences. Based on these findings, we develop a non-canonical PAM-mediated, poikilothermal, one-pot CRISPR-Cas12a detection platform (POP-CRISPR), which demonstrates substantial improvements in sensitivity, specificity, speed, and target adaptability for nucleic acid detection compared to existing methods. These advantages are validated through the reliable detection of clinical samples, including those of Human papillomavirus (HPV), Mycoplasma pneumoniae (MP), and its drug-resistant strains. Additionally, we show that POP-CRISPR enables rapid, on-site pathogen detection within 20 min, using a fast sample processing protocol and a miniaturized detection device.},
}
@article {pmid41546459,
year = {2026},
author = {Niu, C and Fang, S and Zeng, B and Liu, D and Huang, K and Zhang, G and Qian, N and Li, D and Ye, C},
title = {Development of a PCR-Cas12a-LFD visual detection system for highly sensitive and specific detection of Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp. in tobacco.},
journal = {Pest management science},
volume = {82},
number = {5},
pages = {4169-4182},
doi = {10.1002/ps.70528},
pmid = {41546459},
issn = {1526-4998},
support = {//Youth Support Program of Zhengzhou Tobacco Research Institute, China National Tobacco Corporation (CNTC) (No. 702024CR0070)/ ; },
mesh = {*Nicotiana/microbiology ; *Plant Diseases/microbiology ; *Pseudomonas/isolation &amp; purification/genetics ; *Alternaria/isolation &amp; purification/genetics ; *Phytophthora/isolation &amp; purification/genetics ; *Ralstonia/isolation &amp; purification/genetics ; *Polymerase Chain Reaction/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; },
abstract = {Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp. are the major pathogens responsible for tobacco diseases and severe threats to the sustainable development of the tobacco industry. Traditional detection methods, such as plate isolation and polymerase chain reaction (PCR), have improved in sensitivity and timeliness but remain limited by operational complexity, heavy reliance on high expertise requirements for result interpretation. This study focuses on the trans-cleavage activity of the CRISPR-Cas12a system and develops a series of novel detection methods for Ralstonia sp., Phytophthora sp., Alternaria sp., and Pseudomonas sp., respectively. By screening specific genomic targets, designing highly sensitive primers and CRISPR RNA (crRNA), and integrating PCR with lateral flow dipstick (LFD), the PCR-Cas12a-LFD detection system was established with a sensitivity of 100 pg μL[-1] for Ralstonia sp., 10 pg μL[-1] for Phytophthora sp., 5000 pg μL[-1] for Alternaria sp., and 0.1 pg μL[-1] for Pseudomonas sp., respectively, and excellent specificity without cross-reactivity. Furthermore, the combination of this method with LFD enables visual interpretation of results. This study provides an efficient and visualized diagnostic tool for the early prevention and control of tobacco diseases during field cultivation while offering a theoretical reference for the molecular detection of other plant pathogens. © 2026 Society of Chemical Industry.},
}
@article {pmid41546796,
year = {2026},
author = {Borgohain, T and Suma, R and Muttappagol, M and Saikia, B and Keithellakpam, A and Laskar, A and Hiremath, SS and Basu, U and Velmurugan, N and Palakolanu, SR and Chikkaputtaiah, C},
title = {Precision breeding in a changing climate: unlocking resilience through omics and gene editing.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {26},
pmid = {41546796},
issn = {1438-7948},
support = {31/0025 (15358) 2022-EMR-I//Human Resource Development Centre, Council of Scientific And Industrial Research/ ; DST/WISE-PDF/LS-112/2024(G)//Department of Science and Technology, Ministry of Science and Technology, India/ ; MMP025301//Council of Scientific and Industrial Research, India/ ; },
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Climate Change ; *Crops, Agricultural/genetics/growth &amp; development ; *Genomics ; Genome-Wide Association Study ; },
abstract = {Climate change, rising global food demand, and shrinking resources require transformative innovations in crop breeding. This review outlines recent advances in new breeding technologies (NBTs), including molecular markers, genome-wide association studies (GWAS), genomic selection (GS), next-generation sequencing (NGS), and gene editing (GE) tools such as the clustered regularly interspaced short palindromic repeat (CRISPR/Cas), base editing, and prime editing. These methods enable the accurate improvement of traits, thereby accelerating the development of crops resistant to both abiotic and biotic stresses. The integration of multi-omics platforms, including genomics, transcriptomics, proteomics, metabolomics, and phenomics, provides a comprehensive framework for deciphering and manipulating complex trait architectures. Artificial intelligence (AI) and machine learning (ML) enhance precision breeding by providing data-driven insights and enabling the forecasting of traits. Emphasis is also placed on combining gene editing with other strategies, such as speed breeding, to accelerate the development of traits. This review underscores the importance of an integrated systems biology approach that combines multi-omics, gene editing, AI, and speed breeding to accelerate the development of climate-resilient, high-yielding, and nutritionally enhanced crops. The integration of these innovative technologies holds great promise for addressing global food security, environmental sustainability, and agricultural resilience in the face of climate change. A strategic framework for the future of plant breeding is outlined, emphasizing the importance of interdisciplinary collaboration in building a sustainable agricultural future.},
}
@article {pmid41546867,
year = {2026},
author = {Nebenfuehr, B and Sanford, L and Taylor, ER and Ball, K and Woods-Killam, CE and Ghasemi, HI and Proctor, B and Ortega, R and Sempeck, C and Dowell, RD and Arnoult, N},
title = {Uncovering genetic interactions in the DNA repair network in response to endogenous damage and ionizing radiation.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116850},
pmid = {41546867},
issn = {2211-1247},
support = {R01 CA266100/CA/NCI NIH HHS/United States ; R01 AI156739/AI/NIAID NIH HHS/United States ; T32 GM008759/GM/NIGMS NIH HHS/United States ; R35 GM143108/GM/NIGMS NIH HHS/United States ; T32 GM142607/GM/NIGMS NIH HHS/United States ; },
mesh = {*Radiation, Ionizing ; *DNA Repair/genetics/radiation effects ; Humans ; *DNA Damage/genetics ; Ku Autoantigen/metabolism/genetics ; CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/radiation effects ; MRE11 Homologue Protein/metabolism/genetics ; Ubiquitin-Protein Ligases/metabolism/genetics ; },
abstract = {Genomic integrity relies on a complex network of DNA damage response (DDR) pathways that repair endogenous and exogenous lesions, yet how individual factors operate within this broader landscape remains unclear. We performed a large-scale combinatorial CRISPR-Cas9 knockout screen targeting 461 DNA repair genes, disrupting over 100,000 gene combinations under basal conditions and after ionizing radiation (IR). This approach uncovered thousands of genetic interactions spanning pathways that respond to endogenous damage and those specific to double-strand break repair. From this dataset, we validated both positive and negative interactions under basal and irradiated conditions, including a synthetic lethal relationship between MRE11A and the E3 ligase UBR5, a role for Ku70/80 in preventing unscheduled nuclease activity at telomeres, an IR-specific vulnerability upon co-disruption of CYREN and PARG, and a link between CYREN-mediated radioresistance and innate immunity. This resource enables mechanistic insight and reveals therapeutic vulnerabilities in DNA-repair-deficient cancers.},
}
@article {pmid41546923,
year = {2026},
author = {Patil, S and Das, A and Inamdar, MS},
title = {Generation of a Brachyury reporter cell line (BJNhem20 Brachyury (TBXT)-2A-EGFP) in human embryonic stem cells using CRISPR-Cas9 gene targeting.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103907},
doi = {10.1016/j.scr.2026.103907},
pmid = {41546923},
issn = {1876-7753},
mesh = {Humans ; Brachyury Protein ; *T-Box Domain Proteins/genetics/metabolism ; *Fetal Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Line ; *Green Fluorescent Proteins/metabolism/genetics ; Genes, Reporter ; Cell Differentiation ; },
abstract = {Brachyury is a key transcription factor, which is required for mesoderm lineage development. Here, we have generated a Brachyury/TBXT-2A-EGFP knock-in Reporter line in the BJNhem20 human embryonic stem cell line, using CRISPR/Cas9-based gene editing. Successful gene editing was verified by DNA sequencing and comparing endogenous gene expression to reporter gene expression. This reporter line represents an important tool for tracking and assessing mesoderm differentiation.},
}
@article {pmid41547318,
year = {2026},
author = {Beckley, J and Barrangou, R},
title = {Phage-mediated delivery of CRISPR payloads.},
journal = {Current opinion in microbiology},
volume = {89},
number = {},
pages = {102704},
doi = {10.1016/j.mib.2025.102704},
pmid = {41547318},
issn = {1879-0364},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics/virology ; Genetic Engineering/methods ; },
abstract = {Over the past decade, improvements in sequencing technologies and computational tools have advanced our understanding of the composition and function of microbial communities in various environments. Now, in order to manipulate and engineer these communities, we need technologies that enable broadly applicable and specific alterations to establish and modulate the molecular basis for their functional roles. Recent advances in bacteriophage engineering strategies, synthetic biology techniques, and in silico approaches have greatly expanded our ability to perform in situ perturbations. Clustered regularly interspaced short palindromic repeats-Cas systems in particular can provide an efficient means of engineering phages, and can also be delivered as a recombinant payload to perform precision genome editing directly in the host environment. Modified Cas effectors have been developed that allow for increasingly diverse edits with applications in the fields of medicine, food, and agriculture. In this review, we discuss recent advances in using bacteriophages to deliver various clustered regularly interspaced short palindromic repeats-Cas effectors. While challenges remain regarding the phylogenetic breadth of deployment, recombinant phages generally present a unique and effective means to rationally engineering microbial community function and composition.},
}
@article {pmid41547662,
year = {2026},
author = {Mu, S and Li, Q and Chen, M and Li, Z and Ma, Y and Li, Y and Song, Y and Hou, S and Ding, Y and Ju, J and Lin, Y and Zhang, J and Yang, Y and Ren, X and Li, N and Jin, Q and Lai, L and Wang, K and Shi, H},
title = {Coiled-coil heterodimer-mediated split base editing systems enable flexible and robust nucleotide substitutions.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1765},
pmid = {41547662},
issn = {2041-1723},
support = {32570625//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Humans ; Animals ; Mice ; Dependovirus/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; Proprotein Convertase 9/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Cytidine/metabolism ; Genetic Vectors/genetics ; *Nucleotides/genetics ; },
abstract = {Base editors (BEs) enable precise base substitutions, but their size exceeds the packaging capacity of adeno-associated virus (AAV), impeding in vivo applications. Here we design a split BE system that recruits deaminases to Cas9 nickase via coiled-coil heterodimers, resulting in various coiled-coil heterodimers-mediated base editors (CC-BEs), including cytidine base editor (CC-CBE), adenine base editor (CC-ABE), and their derivatives. We reveal that CC-BEs maintain and even improve the editing efficiency of the original unsplit BEs across various cell types and editing scopes, achieving maximum enhancements of 9.6-fold in human immortalized cells and 12.4-fold in primary somatic cells for CC-CBE. Using CC-ABE, we validate in vivo editing efficiency and successfully achieve A-to-G conversion in the Pcsk9 and Dmd genes via dual-AAV vectors in mice. Altogether, we develop a simple and universal strategy to address the challenges posed by the large size of BEs without compromising editing efficiency for base substitutions in vivo.},
}
@article {pmid41547832,
year = {2026},
author = {Wang, X and Deng, X and Qiu, L and Liu, J and Shen, H and Du, H and Li, W and Song, L and Deng, W and Dong, X and Han, Y and Liu, B and Huang, J and Li, Z and Zhang, Y},
title = {Af-CUT&amp;Tag: a sensitive and antibody-free chromatin profiling method using genetically encoded tags and high-affinity binders fused to Tn5.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1746},
pmid = {41547832},
issn = {2041-1723},
mesh = {*Chromatin/metabolism/genetics ; Humans ; Animals ; YAP-Signaling Proteins ; Mice ; MicroRNAs/metabolism/genetics ; *Transposases/metabolism/genetics ; Transcription Factors/metabolism/genetics ; Liver Regeneration/genetics ; Adaptor Proteins, Signal Transducing/metabolism/genetics ; Chromatin Assembly and Disassembly ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Conventional chromatin profiling techniques are often limited by antibody availability and performance. Here, we introduce Af-CUT&amp;Tag, a target antibody-free method that overcomes these limitations by using CRISPR-integrated peptide tags (HiBiT/ALFA-tag) recognized by engineered binders (LgBiT/NbALFA) fused to a Tn5 transposase. Af-CUT&amp;Tag eliminates dependence on traditional target antibodies, achieving robust specificity and sensitivity with as few as 500 cells. It provides high-quality chromatin profiles, with improved signal-to-noise ratios and library quality compared with conventional antibody-based counterparts, while also enabling single-cell resolution (scAf-CUT&amp;Tag). Applying Af-CUT&amp;Tag to Hippo effectors (YAP1/TAZ) during liver regeneration reveals dynamic chromatin remodeling, including YAP1/TAZ-mediated control of lipid metabolism (e.g., Lpin1, Fasn) and heme clearance (Hpx, Trf). We further identify miR-122 as a critical regulator of these processes, impacting liver regeneration. The versatility of Af-CUT&amp;Tag in cell lines, bulk tissues, and single nuclei establishes it as a powerful tool for studying gene regulation in development, disease, and regeneration.},
}
@article {pmid41548240,
year = {2026},
author = {Fan, Y and Shen, S and Su, M and Yan, H and Dong, J and Li, Y and Li, N and Gao, Z and Xia, F},
title = {Synergistic CRISPR/Cas12a-Nanozyme System for Iontronic Sensing of Site-Specific Septin9 Methylation.},
journal = {Analytical chemistry},
volume = {98},
number = {4},
pages = {2870-2881},
doi = {10.1021/acs.analchem.5c05637},
pmid = {41548240},
issn = {1520-6882},
mesh = {*Septins/metabolism/genetics ; Humans ; *DNA Methylation ; *CRISPR-Cas Systems ; Gold/chemistry ; *DNA, Catalytic/chemistry/metabolism ; *Biosensing Techniques/methods ; *Endodeoxyribonucleases/metabolism ; Electrochemical Techniques ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Platinum/chemistry ; Colorectal Neoplasms/diagnosis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Methylated septin9 (septin9-mC) is a well-validated biomarker for colorectal cancer screening, and accurate detection of such site-specific methylated DNA holds significant clinical value for early disease diagnosis. However, conventional methods suffer from cumbersome pretreatment, DNA degradation risks, and poor performance in low-abundance samples. Herein, we report a synergistic iontronic sensing platform integrating methylation-sensitive restriction enzyme (AciI), CRISPR/Cas12a, Ag-DNAzyme, and Au/Pt heterometallic nanozyme for highly sensitive and specific detection of septin9-mC. AciI selectively cleaves unmethylated septin9 (septin9-C) while sparing septin9-mC, and intact septin9-mC activates Cas12a trans-cleavage activity to trigger catalytic hairpin assembly (CHA), generating Ag-DNAzyme. Activated Ag-DNAzyme induces detachment of Au/Pt nanoparticles from anodic aluminum oxide membranes, reducing the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to positively charged oxTMB and altering ion transport fluxes in nanochannels, which is read out via current-voltage characteristics. The linear range is 100 aM to 10 nM with a detection limit of 34.0 aM. This method effectively distinguishing colorectal cancer cells from human colonic epithelial cells and colorectal cancer patients from healthy individuals, showing excellent performance in real sample analysis. The proposed method provides a dependable tool for site-specific methylation detection with promising applications in biological research and clinical diagnosis.},
}
@article {pmid41548978,
year = {2026},
author = {Zheng, SH and Liu, Y and Xia, XX and Liu, YM},
title = {Advances in base editing technology and the construction of precise zebrafish disease models.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {1},
pages = {46-60},
doi = {10.16288/j.yczz.25-157},
pmid = {41548978},
issn = {0253-9772},
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Disease Models, Animal ; Humans ; },
abstract = {Single nucleotide variants (SNVs) are among the primary pathogenic factors of human genetic diseases, accounting for a significant proportion of all mutation types. Conducting in-depth research on the pathogenic significance of these mutations in animal models is essential for understanding disease mechanisms and developing therapeutic strategies. The progress of such research largely depends on the continuous innovation and advancement of gene editing technologies. In recent years, base editing technology based on the CRISPR/Cas9 system has emerged, enabling precise conversion of individual nucleotides. Owing to its efficiency and convenience, base editing has been widely applied in gene therapy, the construction of animal models, and molecular breeding, bringing new breakthroughs and opportunities to life sciences and medical research. Zebrafish, with their advantages of small size, high fecundity, transparent embryos, and external development, have become an ideal model organism for studying disease mechanisms and drug screening. In this review, we summarize the development of CRISPR/Cas9-based base editing technologies, highlight the emergence of novel editing tools, and explore the application and progress of base editing in constructing precise zebrafish disease models.},
}
@article {pmid41549062,
year = {2026},
author = {Mukherjee, P and Benicky, J and Panigrahi, A and Ailles, L and Goldman, R},
title = {SULF1 in Cancer Associated Fibroblasts Promotes Invasion in Head and Neck Cancer Cell Lines.},
journal = {Cancer medicine},
volume = {15},
number = {1},
pages = {e71540},
pmid = {41549062},
issn = {2045-7634},
support = {S10 OD028623/OD/NIH HHS/United States ; R01 CA238455/CA/NCI NIH HHS/United States ; S10 OD032420/OD/NIH HHS/United States ; R01CA238455/GF/NIH HHS/United States ; S10OD032420//NIH Office of the Director/ ; 1S10OD028623-01A1//NIH Office of the Director/ ; //School of Medicine and Health Sciences/ ; P30 CA051008/CA/NCI NIH HHS/United States ; 2P30CA051008/CA/NCI NIH HHS/United States ; //George Washington University/ ; },
mesh = {Humans ; *Cancer-Associated Fibroblasts/metabolism/pathology ; *Head and Neck Neoplasms/pathology/genetics/metabolism ; *Sulfotransferases/metabolism/genetics ; Cell Movement ; Neoplasm Invasiveness ; Cell Line, Tumor ; *Squamous Cell Carcinoma of Head and Neck/pathology/genetics/metabolism ; Cell Proliferation ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Expression Regulation, Neoplastic ; },
abstract = {BACKGROUND: Cancer-associated fibroblasts (CAFs) significantly influence tumor behavior in head and neck squamous cell carcinoma (HNSCC) and other malignancies. We identified the extracellular sulfatase SULF1 as a key stromal factor highly expressed in CAFs and associated with poor prognosis.<br><br>METHODS AND RESULTS: Using CRISPR/Cas9-edited SULF1-knockout primary HNSCC CAFs, we demonstrate that loss of SULF1 reduces fibroblast proliferation and markedly impairs cancer cell migration and invasion in&#xa0;vitro. Two-photon microscopy in 3D spheroid cocultures revealed that SULF1-deficient CAFs fail to support invasiveness of Cal33 cells, resulting in spheroids with fewer invasive projections and altered morphology. Proteomic analysis confirmed the absence of SULF1 in the knockout cell cultures and revealed that SULF2, expressed in tumor cells, does not compensate for its loss.<br><br>CONCLUSION: These findings highlight the importance of CAF-derived SULF1 in regulating tumor invasion and suggest that SULF1 is a promising therapeutic target in HNSCC.},
}
@article {pmid41549285,
year = {2026},
author = {Peng, X and Huang, J and Lv, S and Zhang, J and Zhang, L and Liu, H and Liu, Y and Qiao, ZA and Wei, F and Su, BL},
title = {TriCON: A Carbon-Based Triple-Modal Nanoplatform for Pancreatic Cancer Therapy.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {17},
pages = {e12978},
pmid = {41549285},
issn = {2198-3844},
support = {81972273//National Natural Science Foundation of China/ ; 82273276//National Natural Science Foundation of China/ ; 82473314//National Natural Science Foundation of China/ ; 20200201546JC//Natural Science Foundation of Jilin Province/ ; JJKH20211208KJ//Science and Technology Project of Jilin Provincial Education Science Department/ ; 2018J039//Health and Innovation Technology Project of Jilin Province/ ; 2024KC142//2025 Doctoral Research Innovation Enhancement Project of Jilin Province/ ; },
mesh = {*Pancreatic Neoplasms/therapy/genetics/immunology/drug therapy ; Animals ; Mice ; Humans ; Doxorubicin/pharmacology/administration &amp; dosage ; *Immunotherapy/methods ; Tumor Microenvironment/immunology/drug effects ; Cell Line, Tumor ; Gene Editing/methods ; CRISPR-Cas Systems ; *Carbon/chemistry ; *Carcinoma, Pancreatic Ductal/therapy ; },
abstract = {Pancreatic cancer, recognized for its high malignancy and tumor immunosuppressive microenvironment, has been refractory to conventional therapeutic modalities, necessitating the exploration of novel treatment strategies. Among these, reprogramming of the tumor immunosuppressive microenvironment is a promising strategy to enhance the efficacy of tumor immunotherapy. The CRISPR/Cas9 system-based gene editing further offers a viable approach for precise regulation of endogenous gene expression associated with tumor immunosuppression. Current delivery vectors face a trilemma between biosafety profiles, expansion capacity, and targeting accuracy. To this end, we developed a triple-modality therapeutic platform, termed TriCON (Triple Convergent Oncology Nanotherapy), characterized by three core mechanistic attributes: spatiotemporal convergence, stimuli-responsive controllability, and tumor-microenvironment modulatory conductivity. This orchestrated combination of rationally designed gene editing (targeting poliovirus receptor), nano-encapsulated doxorubicin (DOX) chemotherapy, and checkpoint blockade immunotherapy demonstrated enhanced synergistic antitumor activity in pancreatic ductal adenocarcinoma (PDAC) models, achieving tumor regression through enhanced chemotherapy, immunogenic cell death induction, and natural killer (NK) cells activation. The platform achieved superior in vivo gene editing (14.2% PVR editing efficiency) via optimized endosomal escape and CRISPR system release. This triaxial approach establishes a programmable nanotherapeutic paradigm that synergizes gene editing precision with chemo-immunotherapy, offering a novel framework for PDAC treatment.},
}
@article {pmid41549804,
year = {2026},
author = {Yang, J and Yang, J and Liu, K and Wang, S and Hu, Y and Li, J and Wang, H and Jiang, D and Han, X},
title = {Microfluidic-Assisted Metal-Polyphenol Cloaked Bacteria Enable CRISPR/dCas9-Mediated Pyroptosis Cascade Amplification for Effective Tumor Immunotherapy.},
journal = {Advanced healthcare materials},
volume = {15},
number = {13},
pages = {e04837},
doi = {10.1002/adhm.202504837},
pmid = {41549804},
issn = {2192-2659},
support = {2023CSJZN0600//Yangtze River Delta Joint Sci-Tech Innovation and Research Projects/ ; NZYSKL240201//Supporting the Innovation Projects of State Key Laboratory on Technologies for Chinese Medicine Pharmaceutical Process Control and Intelligent Manufacture/ ; XPT 82202347//project of Nanjing University of Chinese Medicine to NSFC/ ; //Jiangsu Key Discipline Construction Fund of the 14th Five-Year Plan (Biology)/ ; 82202347//National Natural Science Foundation of China/ ; 82204361//National Natural Science Foundation of China/ ; 32471475//National Natural Science Foundation of China/ ; },
mesh = {*Pyroptosis/drug effects ; Animals ; *CRISPR-Cas Systems/genetics ; Humans ; *Immunotherapy/methods ; *Salmonella typhimurium/metabolism ; Mice ; Phosphate-Binding Proteins/metabolism/genetics ; Tumor Microenvironment ; Cell Line, Tumor ; *Neoplasms/therapy/immunology ; Tannins/chemistry ; *Microfluidics/methods ; Intracellular Signaling Peptides and Proteins/metabolism/genetics ; },
abstract = {Pyroptosis triggered by pore-forming Gasdermin proteins in cancer cells facilitates anti-tumor immune activation by releasing pro-inflammatory cytokines and immunogenic contents following cellular rupture. However, selectively triggering pyroptosis in tumors still remain limited in clinical applications. Here, it is reported a microfluidic-assisted bacterial delivery system using attenuated Salmonella typhimurium VNP20009 encapsulated with metal-phenolic networks composed of ferric ions (Fe[3] [+]) and tannic acid (TA) to enhance intracellular gasdermin D (GSDMD) expression through targeted CRISPR/dCas9 delivery, thereby inducing robust tumor pyroptosis. Mechanistically, this system achieves cascade amplification of pyroptotic cell death through coordinated multi-modal mechanisms. Following systemic administration, VNP20009 specifically accumulates in hypoxic tumor regions while the coated Fe[3] [+]-TA nanofilm undergoes pH-responsive dissolution in the acidic tumor microenvironment (TME), simultaneously generating ROS through Fenton reaction and releasing CRISPR/dCas9 system to upregulate GSDMD expression. Concurrently, the abundant flagella of VNP activate caspase-1, which in turn cleaves the overexpressed GSDMD proteins into its active form, thereby triggering robust pyroptosis in tumor cells. Taken together, by coupling bacterial adjuvanticity with ROS-mediated stress and CRISPR-driven GSDMD upregulation, this strategy achieves efficient amplification of pyroptosis and promotes antitumor immune activation.},
}
@article {pmid41550334,
year = {2025},
author = {Lin, S and Li, H and Bai, S and Han, X},
title = {Mobile RNAs as systemic signaling beyond boundaries for plant stress resistance.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1712714},
pmid = {41550334},
issn = {1664-462X},
abstract = {Plant mobile RNAs-including small RNAs (miRNAs, siRNAs), mRNAs, lncRNAs, and tRNA fragments-function as systemic signaling molecules that traverse cellular, tissue, and species boundaries to coordinate plant adaptation to environmental stresses. Here, we summarize the critical roles of mobile RNAs in mediating systemic adaptation to abiotic challenges and biotic interactions. Crucially, we highlight the diverse transport mechanisms enabling their movement and discuss the emerging functional versatility of mobile RNAs, which extends beyond transcriptional regulation to encompass epigenetic modifications, resource allocation, and cross-species communication. These fundamental insights are driving transformative applications: Mobile RNAs provide the foundation for developing systemic RNAi-based biopesticides and are being integrated with CRISPR-Cas technologies to overcome delivery barriers and enable heritable, transgene-free genome editing in crops. Understanding and harnessing mobile RNA networks offers unprecedented potential for engineering resilient crops and implementing precise, sustainable crop protection strategies to address global food security challenges.},
}
@article {pmid41550651,
year = {2025},
author = {Yan, W and Weng, X},
title = {Targeted RNA base editing for therapeutic: mechanisms and advances.},
journal = {Pharmaceutical science advances},
volume = {3},
number = {},
pages = {100089},
pmid = {41550651},
issn = {2773-2169},
abstract = {RNA base editing, which enables RNA base modification through effector proteins guided by targeting systems, is a powerful technology to correct disease-associated point mutations. Although overshadowed by CRISPR-based genome editing, RNA editing has seen rapid development in recent years, with significant improvements in efficiency and precision. In this review, we summarize the core components of RNA base editing systems (RNA-targeting systems and effector proteins) and describe major RNA editing types, including A-to-I, C-to-U, A-to-m[6]A/m[6]A-to-A, and U-to-Ψ base editors, along with their research progress. In addition, we systematically summarize the delivery methods of the developed RNA editors and their initial exploration in treating diseases caused by nonsense mutations. Finally, combining the current development status of the RNA editing related field, we reflect on the problems encountered in the current development of the RNA editing field and offer our own insights on the future development direction.},
}
@article {pmid41551929,
year = {2026},
author = {Mir-Pedrol, J and Kuhlburger, L and Sanvicente-García, M and Yazar, M and Ryan, CJ and Krakau, S and Gabernet, G and Güell, M and Bonfanti, M and Nf-Core Community, and Nahnsen, S},
title = {nf-core/crisprseq: a versatile pipeline for comprehensive analysis of CRISPR gene editing and screening assays.},
journal = {NAR genomics and bioinformatics},
volume = {8},
number = {1},
pages = {lqaf214},
pmid = {41551929},
issn = {2631-9268},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Software ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {In recent years, CRISPR technology has become widely applied in scientific research, being simpler, cheaper, and more precise than previous gene-editing techniques. This editing technology can be used for various applications, such as gene knockout, gene knock-in, CRISPR activation (CRISPRa), CRISPR interference (CRISPRi), CRISPR screens, base editing, and prime editing. The share of pipelines to analyze the variety of CRISPR editing methods is low, and until now, none of them caters to both gene editing and CRISPR-based functional genomics. Here, we introduce nf-core/crisprseq, a Nextflow DSL2 pipeline for the assessment of CRISPR gene editing and screening assays. The workflow is written in a modularized fashion to allow the easy incorporation of new steps. nf-core/crisprseq is the first generic pipeline enabling the analysis of the broad spectrum of CRISPR designs. We show the performance and usability of the software using publicly available datasets.},
}
@article {pmid41552883,
year = {2026},
author = {Xu, Q and Ji, M},
title = {A Bst-driven Cas12a cascade amplification strategy for microRNA detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {4},
pages = {899-906},
doi = {10.1039/d5ay01957e},
pmid = {41552883},
issn = {1759-9679},
mesh = {*MicroRNAs/analysis/genetics/blood ; Humans ; *Nucleic Acid Amplification Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {Quantification of trace microRNAs is crucial for early disease diagnosis but remains technically challenging. Herein, we developed an ultrasensitive fluorescence platform for microRNA-21 (miR-21) detection by integrating Bst DNA polymerase - assisted target recycling with CRISPR/Cas12a-mediated signal amplification. In this design, the target miRNA triggers toehold-mediated opening of a hairpin probe, followed by Bst-driven primer extension that enables efficient target recycling and the generation of abundant DNA duplex activators. Subsequently, these activators induce strong trans-cleavage activity of Cas12a, producing markedly enhanced fluorescence responses. Benefiting from the dual amplification of enzymatic recycling and Cas12a activation, the proposed assay exhibits high sensitivity toward miR-21 with a detection limit down to 9.25 × 10[-12] M. Furthermore, the platform exhibited excellent sequence selectivity and was successfully applied to monitor miR-21 in both cell lysates and clinical serum samples. Considering its convenient operation, strong analytical performance, and simple readout mode, this method holds great potential for trace biomarker analysis in clinical diagnostics.},
}
@article {pmid41553775,
year = {2026},
author = {Zheng, I and Learn, B and Bailey, S},
title = {Structural basis for inhibition of SpyCas9 by the anti-CRISPR protein AcrIIA26.},
journal = {The Biochemical journal},
volume = {483},
number = {3},
pages = {289-300},
pmid = {41553775},
issn = {1470-8728},
support = {R01 GM097330/GM/NIGMS NIH HHS/United States ; GM097330//HHS | National Institutes of Health (NIH)/ ; },
mesh = {*Streptococcus pyogenes/genetics/enzymology/metabolism ; *CRISPR-Associated Protein 9/chemistry/metabolism/antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; *Viral Proteins/chemistry/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/chemistry/genetics ; Cryoelectron Microscopy ; *Bacteriophages/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Models, Molecular ; },
abstract = {CRISPR-Cas9 systems provide adaptive immunity in prokaryotes by targeting and cleaving invading phage DNA. In response, phages have evolved anti-CRISPR (Acr) proteins to inhibit Cas9 and evade this immune response. AcrIIA26 is a type II-A anti-CRISPR protein that inhibits Streptococcus pyogenes Cas9 (SpyCas9) DNA binding, but its molecular mechanism remains unclear. Here, we determined the 3.0 Å resolution cryo-EM structure of AcrIIA26 in complex with SpyCas9-single-guide RNA, revealing a dual inhibition mechanism. AcrIIA26 adopts a novel fold comprising a central β-sheet flanked by two α-helical bundles. The 5-helix bundle, which features a negatively charged surface whose shape mimics duplex DNA, occupies the same position as the protospacer adjacent motif (PAM) duplex in target-bound Cas9. This directly blocks PAM recognition by burying critical residues R1333 and R1335 in the PAM-interacting domain. Mutagenesis confirmed that residues E49 and D50 in AcrIIA26 are essential for this interaction. Simultaneously, the 4-helix bundle binds the Cas9 REC lobe and sterically prevents the conformational changes required for Cas9 activation, with mutation of AcrIIA26 F121 completely eliminating inhibitory activity. Structural comparisons reveal that despite diverse folds, multiple anti-CRISPRs convergently evolved to block PAM recognition, highlighting this as a critical vulnerability in Cas9 function. Our findings provide mechanistic insights into AcrIIA26 inhibition and offer a foundation for engineering improved Cas9 off-switches for genome editing applications.},
}
@article {pmid41553913,
year = {2026},
author = {Garnica, M and San Martin-Uriz, P and Rodriguez-Marquez, P and Calleja-Cervantes, ME and Rodriguez-Diaz, S and Martinez-Turrillas, R and Hernaez, M and Prosper, F and Rodriguez-Madoz, JR},
title = {Improving CRISPR-Cas9 Screens in CAR T Cells: A Refined Method for Library Preparation.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {227},
pages = {},
doi = {10.3791/69721},
pmid = {41553913},
issn = {1940-087X},
mesh = {Humans ; *CRISPR-Cas Systems ; *T-Lymphocytes/immunology ; *Receptors, Chimeric Antigen/genetics/immunology ; *Gene Library ; Immunotherapy, Adoptive/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Chimeric antigen receptor (CAR) T cell therapies have demonstrated remarkable efficacy in several hematological malignancies, yet their success has not been fully replicated in solid tumors. Moreover, even in hematological cancers, relapse after CAR T cell infusion continues to compromise long-term outcomes. These challenges highlight the urgent need to develop strategies that enhance CAR T cell efficacy, persistence, overcoming tumor and microenvironment-mediated resistance. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-based screening platforms provide a powerful approach to systematically identify genes that regulate CAR T cell function. By linking genetic perturbations to phenotypic outcomes, these assays enable the discovery of pathways controlling activation, proliferation, memory formation, and cytotoxicity. Standard workflows involve transduction of substantial numbers of cells with a single guide RNA (sgRNA) library, Cas9-mediated editing, selection of edited cells, and PCR amplification of sgRNA cassettes from genomic DNA (gDNA) prior to sequencing. However, PCR amplification using large amounts of gDNA poses significant challenges and often fails to selectively amplify and retrieve sgRNAs. Here, we describe an optimized CRISPR-Cas9 knockout screening protocol, which we have tested on primary human CAR T cells. The method here incorporates an intermediate step during sgRNA library preparation that reduces gDNA carryover through enzymatic digestion and selective pulldown of the sgRNA cassette, thereby increasing the efficiency of the first PCR amplification. This modification allowed us to retrieve sgRNA information across our CAR T cell screens, which had remained elusive in our previous attempts using traditional 1 and 2-step PCR amplification protocols. In conclusion, this optimized workflow facilitates CRISPR screening library preparation in challenging samples and enables the identification of key genetic determinants that can be targeted to improve therapeutic efficacy.},
}
@article {pmid41554027,
year = {2025},
author = {Ireri, SW and Cao, M},
title = {CRISPR-Cas9-based Mutagenesis in the Entomopathogenic Nematode Steinernema hermaphroditum and the Maintenance of Mutant Lines.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {226},
pages = {},
doi = {10.3791/68932},
pmid = {41554027},
issn = {1940-087X},
mesh = {Animals ; *CRISPR-Cas Systems ; Mutagenesis ; *Rhabditida/genetics ; Microinjections/methods ; Gene Editing/methods ; },
abstract = {Entomopathogenic nematodes (EPNs) in the genus Steinernema and Heterorhabditis maintain mutualistic interactions with Xenorhabdus and Photorhabdus symbiotic bacteria, respectively. Together, these nematode-bacterium pairs infect and kill insect hosts that are primarily larvae from the orders of Lepidoptera and Coleoptera, forming a tractable tripartite system for dissecting the molecular basis of mutualism and parasitism. A key step towards fully utilizing this model is the development of stable and transgenerational genetic tools in EPNs. Here, we demonstrate a reliable CRISPR-Cas9 genome editing platform in the emerging model Steinernema hermaphroditum, a species that is readily maintained in vivo and in vitro, and is highly amenable to gonadal microinjection. Importantly, its hermaphroditic reproduction greatly streamlines the generation and maintenance of homozygous mutant lines. We provide a detailed protocol for efficient, targeted gene disruption using microinjection-based delivery of Cas9 ribonucleoprotein complexes. As a proof of concept, we modified the conserved muscle-associated gene unc-22, generating a characteristic twitching phenotype that validates targeted mutagenesis in this system. This CRISPR-Cas9 platform opens the door to stable genetic manipulation in S. hermaphroditum, such as transgene expression, and provides a framework that can be extended to additional EPN species of agricultural and ecological importance.},
}
@article {pmid41554314,
year = {2026},
author = {Paulo, BS and Romanowski, SB and Kadjo, AE and Lourenzon, VB and Eustáquio, AS},
title = {Genome minimization of a Burkholderia bacterial host.},
journal = {Metabolic engineering},
volume = {94},
number = {},
pages = {305-314},
pmid = {41554314},
issn = {1096-7184},
support = {R01 GM129344/GM/NIGMS NIH HHS/United States ; T32 AT007533/AT/NCCIH NIH HHS/United States ; },
mesh = {*Burkholderia/genetics/metabolism ; *Genome, Bacterial/genetics ; Plasmids/genetics ; *Metabolic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {Genome minimization, including the deletion of endogenous gene clusters that encode natural products, is a common strategy to improve the yield of heterologous products. We have been interested in developing Burkholderia sp. FERM BP-3421 as an alternative bacterial host. Instead of indiscriminately deleting gene clusters, which may have deleterious effects, we guided our efforts using transcriptomics data from production cultures. The genome of FERM BP-3421 is subdivided into two chromosomes and two plasmids. The top transcribed gene clusters were those encoding polyketide-nonribosomal peptide spliceostatins on plasmid p1 and nonribosomal peptide selethramide on chromosome 1. Deletion of the spliceostatin cluster had been shown to improve titers of the ribosomal peptide capistruin, whereas we showed that deletion of the selethramide cluster had no effect on capistruin titers. We next targeted the two endogenous plasmids using a CRISPR-Cas12a strategy, resulting in an 11 % reduction in genome size. The plasmid cured strains showed improved growth and 20-40 % increased production of capistruin depending on whether one or both plasmids were deleted. However, deletion of p2 alone negatively affected the heterologous production of two distinct polyketide-nonribosomal peptides. The p2[-] strain produced only 5-23 % of the glidobactin A and megapolipeptin A titers compared to the wild type, respectively, whereas titers were restored to wild type levels in the p1[-] p2[-] strain. The observation that p2 appears to contain functions that support polyketide-nonribosomal peptide biosynthesis was unexpected and sets the stage for future studies aimed at identifying these functions and further enabling engineering efforts that may be widely applicable to other strains.},
}
@article {pmid41554741,
year = {2026},
author = {Li, S and Xu, K and Li, G and Jiang, H and Wu, Z and Gao, P and Yue, Y and Chen, Y and Liu, Z and Zhou, B and Zhou, M and Chen, Y and Liu, X and Wang, X and Wu, Z and Wei, Y},
title = {Engineering the MmeFz2-ωRNA system for efficient genome editing through an integrated computational-experimental framework.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41554741},
issn = {2041-1723},
support = {32441080, 32301251//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22207074//National Natural Science Foundation of China (National Science Foundation of China)/ ; //China Agricultural Research System/ ; },
mesh = {*Gene Editing/methods ; Animals ; Humans ; Mice ; Muscular Dystrophy, Duchenne/genetics/therapy ; Male ; Dystrophin/genetics/metabolism ; HEK293 Cells ; Protein Engineering/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Dependovirus/genetics ; CRISPR-Cas Systems ; },
abstract = {Eukaryotic Fanzor proteins are compact, programmable RNA-guided nucleases with substantial potential for genome editing, although their efficiency in mammalian cells remains suboptimal. Here, we present a combinatorial engineering strategy to optimize a representative Fanzor system, MmeFz2-ωRNA. AlphaFold3-powered rational redesign produced a minimized ωRNA scaffold that is 30% smaller while maintaining up to 82.2% efficiency. Synergistic structure-guided and AI-augmented protein engineering generated two variants, enMmeFz2 and evoMmeFz2, which exhibited an average ~32-fold increase in activity across 38 genomic loci. Moreover, fusion of the non-specific DNA-binding domain HMG-D further enhanced editing performance (enMmeFz2-HMG-D and evoMmeFz2-HMG-D). Notably, evoMmeFz2-HMG-D demonstrated robust in vivo genome editing activity, enabling dystrophin restoration in humanized male Duchenne muscular dystrophy mouse models via single adeno-associated virus (AAV) delivery. This study establishes Fanzor2 as a gene editing platform for genome engineering and therapeutic applications, and underscores the power of AI-guided engineering to accelerate genome editor development while reducing experimental burden.},
}
@article {pmid41554880,
year = {2026},
author = {Bot, JF and Zhao, Z and Li, M and Kammeron, D and Shang, P and Geijsen, N},
title = {Temporal dynamics of collateral RNA cleavage by LbuCas13a in human cells.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {233},
pmid = {41554880},
issn = {2399-3642},
support = {NNF21CC0073729//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; W.OR18-11//Prinses Beatrix Spierfonds/ ; 15804//Netherlands Organisation for Scientific Research | Stichting voor de Technische Wetenschappen (Technology Foundation STW)/ ; 201706890022//China Scholarship Council (CSC)/ ; 202206300033//China Scholarship Council (CSC)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *RNA Cleavage ; *CRISPR-Associated Proteins/metabolism/genetics ; *RNA/metabolism/genetics ; HEK293 Cells ; Ribonucleoproteins/metabolism/genetics ; Apoptosis/genetics ; },
abstract = {CRISPR-Cas13 exclusively targets RNA. In prokaryotes, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. Here we report that LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage starts within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulate genes associated with the stress and innate immune response, ultimately leading to apoptotic cell death. This enables us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, using both total RNA sequencing and Nanopore sequencing, we find that LbuCas13a activation leads to rapid and near-global depletion of cytoplasmic RNAs, and that cleavage occurs at specific nucleotide positions.},
}
@article {pmid41554885,
year = {2026},
author = {Zhang, L and Cai, C and Chen, Q and Tan, X and Chen, S and Zhang, K and Cheng, F},
title = {A CRISPR-based sequence proximity binding protein labelling system for scanning upstream regulatory proteins.},
journal = {Nature plants},
volume = {12},
number = {2},
pages = {277-283},
pmid = {41554885},
issn = {2055-0278},
support = {31972411//National Science Foundation of China | National Natural Science Foundation of China-Yunnan Joint Fund (NSFC-Yunnan Joint Fund)/ ; 32302570//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; Oryza/genetics/metabolism ; Promoter Regions, Genetic ; *DNA-Binding Proteins/genetics/metabolism ; Brassica/genetics/metabolism ; Gene Expression Regulation, Plant ; *CRISPR-Cas Systems ; *Plant Proteins/genetics/metabolism ; *Transcription Factors/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Arabidopsis Proteins/genetics/metabolism ; },
abstract = {Transcriptional regulation involves complex and dynamic protein-DNA interactions, which alter chromatin states and, consequently, regulate gene expression. In plants, current technologies face challenges in efficiently capturing dynamically DNA-binding proteins, especially transcription factors. Here, by leveraging the binding ability of dead Cas9 to specific DNA fragments and the labelling capacity of the TurboID protein for adjacent proteins, we have developed a CRISPR-based sequence proximity binding protein labelling system (CSPL) to detect promoter-binding proteins. Using this approach, we identified both known and novel upstream binding proteins on the PIF4 promoter in Arabidopsis, cabbage and rice. This demonstrates the powerful capabilities and broad potential applications of CSPL for detecting promoter-binding proteins in plants.},
}
@article {pmid41555077,
year = {2026},
author = {Yu, L and Yin, M and Zhu, Y and Lu, Z and Xiao, B and Zhou, F and Yu, Y and Huang, Z},
title = {An anti-CRISPR targets the sgRNA to block Cas9 and guides the design of enhanced genome editors.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {2},
pages = {318-329},
pmid = {41555077},
issn = {1545-9985},
mesh = {*Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; Humans ; Cryoelectron Microscopy ; *CRISPR-Associated Protein 9/metabolism/antagonists &amp; inhibitors/chemistry/genetics ; *Viral Proteins/chemistry/metabolism/genetics ; *Bacteriophages/genetics/metabolism ; Models, Molecular ; },
abstract = {Bacteriophages have evolved anti-CRISPR (Acr) proteins to combat the adaptive immunity provided by bacterial CRISPR-Cas systems. Here, we report the cryo-electron microscopy structure of an anti-Cas9 protein AcrIIA27 bound to SpyCas9-sgRNA (single guide RNA) complex. Our structure reveals that AcrIIA27 binds the solvent-exposed phosphate backbone of the sgRNA, acting as a potent inhibitor of diverse Cas9 orthologs. AcrIIA27 in the structure is positioned near the protospacer-adjacent motif DNA-binding pocket on SpyCas9, causing steric hindrance that prevents substrate DNA recognition. This mechanism suggests solvent-exposed regions of sgRNAs (PTP RNAs), prone to nonspecific binding of positively charged components, may compromise CRISPR-Cas genome-editing efficiency. Indeed, truncations of the PTP RNAs in different editing systems significantly enhance genome-editing efficiency in human cells. Overall, our findings reveal a previously uncharacterized inhibition mechanism of an anti-Cas protein and offers a general strategy for developing more efficient genome-editing tools.},
}
@article {pmid41555514,
year = {2026},
author = {Xiao, B and Zhang, J},
title = {A Simple Programmable Cas12a/crRNA Induced Walking System for Sensitive Methicillin-Resistant Staphylococcus aureus Detection via Integrated cis- and trans-Cleavage Activity.},
journal = {Journal of microbiology and biotechnology},
volume = {36},
number = {},
pages = {e2511026},
pmid = {41555514},
issn = {1738-8872},
mesh = {*Methicillin-Resistant Staphylococcus aureus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; Aptamers, Nucleotide/genetics ; *Staphylococcal Infections/diagnosis/microbiology ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Biosensing Techniques/methods ; Humans ; },
abstract = {Methicillin-resistant Staphylococcus aureus (MRSA) represents a serious threat to public health due to its strong antibiotic resistance, wide dissemination, and high infection rates. Rapid identification of MRSA strains is essential for accurate diagnosis and timely treatment of related infections. In this study, we propose an analytical method for MRSA that employs a hairpin-structured locker-probe to directly regulate the trans-cleavage activity of Cas12a. This designed locker-probe connects a target-specific aptamer to an inhibitory aptamer of the CRISPR/Cas12a system. Upon binding to the specific target, the probe undergoes a conformational change that abolishes its inhibitory effect on Cas12a. As a result, the structure-switchable probe modulates Cas12a activity in a target-dependent manner. Additionally, the sensing substrate combines a "cis-cleavage trigger" and a "trans-cleavage trigger" to integrate both cis- and trans-cleavage activities of Cas12a/crRNA within a single probe. This design significantly simplifies the probe architecture while maintaining high signal amplification efficiency. The proposed method was successfully applied to detect MRSA, achieving a detection limit as low as 2.5 CFU/ml with high specificity. By exploiting the inhibitory aptamer of Cas12a as a regulatory element for MRSA analysis, this work expands the toolbox of CRISPR/Cas12a-based methodologies and offers a promising strategy for bacterial detection.},
}
@article {pmid41556260,
year = {2026},
author = {Neherin, K and Holloway, K and Song, Y and Houston, A and Chen, F and Ding, L and Zhang, H},
title = {Introducing Cellular Senescence in Human Induced Pluripotent Stem Cells and Differentiated Neural Lineage for Modeling of Age-Associated Diseases.},
journal = {Advanced biology},
volume = {10},
number = {1},
pages = {e00468},
doi = {10.1002/adbi.202500468},
pmid = {41556260},
issn = {2701-0198},
support = {R21 AG086894/AG/NIA NIH HHS/United States ; RF1 AG056302/AG/NIA NIH HHS/United States ; R21AG086894/AG/NIA NIH HHS/United States ; RF1AG056302/AG/NIA NIH HHS/United States ; U54 AG075934/AG/NIA NIH HHS/United States ; U54AG075934//NIH Common Fund/ ; },
mesh = {*Cellular Senescence ; Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Lineage ; *Cell Differentiation ; *Neural Stem Cells/cytology/metabolism ; Aging ; Telomeric Repeat Binding Protein 2/genetics/metabolism ; CRISPR-Cas Systems ; DNA Damage ; Signal Transduction ; Cell Line ; Gene Expression Regulation ; },
abstract = {Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) resets the epigenetic landscapes that mark the aging clock, and consequently cells differentiated from iPSCs resemble fetal cells rather than adult or aged cells. The lack of proper cellular aging in cells differentiated from iPSCs presents a unique challenge in iPSC-based modeling of age-associated diseases such as neurodegeneration. To address this challenge, we seek to introduce cellular senescence, a hallmark of aging, into iPSC-based models in a robust and temporally controlled manner. An inducible CRISPR interference (CRISPRi) is used to suppress the expression of TERF2, a key component of the telomere protecting Shelterin complex. We demonstrate that suppression of TERF2 robustly activates the DNA damage response, p53/p21 signaling, and cellular senescence in iPSCs in a highly homogeneous and synchronous manner. Applying this inducible CRISPRi-TERF2 system to differentiation of iPSCs to neural progenitor cells (NPCs), we show efficient activation of senescence-associated phenotypes in NPCs. This inducible cell model allows isogenic comparisons of the same cell populations over the course of differentiation with or without the activation of cellular senescence in a synchronous and homogeneous manner, and has broad applications in investigating the role of cellular senescence in the progression of age-related diseases.},
}
@article {pmid41556308,
year = {2026},
author = {Xie, Z and Jin, YS and Miller, MJ},
title = {Exploiting the Endogenous Type II-A CRISPR-Cas System for Functional Engineering of Probiotic Lacticaseibacillus rhamnosus GG.},
journal = {Microbial biotechnology},
volume = {19},
number = {1},
pages = {e70303},
pmid = {41556308},
issn = {1751-7915},
support = {//University of Illinois at Urbana-Champaign/ ; },
mesh = {*CRISPR-Cas Systems ; *Lacticaseibacillus rhamnosus/genetics ; *Gene Editing/methods ; *Probiotics ; Glucuronidase/genetics/metabolism ; },
abstract = {Lacticaseibacillus rhamnosus GG (LGG) is one of the most extensively studied probiotic strains, widely used in food and health applications. However, the absence of efficient, precise genome editing methods has limited its broader potential and functional versatility. Here, we present an endogenous type II-A CRISPR-Cas genome editing workflow for LGG designed for functional strain construction. Using a plasmid interference assay together with single-nucleotide substitutions, we confirm the precise PAM requirement as 5'-NGAAA-3'. We pair a synthetic sgRNA cassette with homology-directed repair donors to enable targeted deletions and insertions across multiple loci, achieving modest but practically relevant editing efficiencies (11.1-25.0% of recovered transformants) that support routine strain construction. Using this optimised and precise genome engineering method, we generated a β-glucuronidase (GUS)-expressing LGG strain for robust strain tracking within complex microbial communities. This work removes barriers to LGG engineering, expands the probiotic CRISPR toolkit, and provides broadly applicable strategies for designing next-generation probiotics with applications in food biotechnology and microbial therapeutics.},
}
@article {pmid41557883,
year = {2026},
author = {Jiang, Y and Gao, H and Ran, Q and Xian, Y and Zhang, C},
title = {Near-Infrared Light-Inducible CRISPR-Cas12a/Tetrahedral DNA Nanosystem for Spatiotemporal Control of mtDNA Mutation Imaging and Gene Therapy.},
journal = {Nano letters},
volume = {26},
number = {4},
pages = {1438-1448},
doi = {10.1021/acs.nanolett.5c05621},
pmid = {41557883},
issn = {1530-6992},
support = {//National Natural Science Foundation of China/ ; //Natural Science Foundation of Shanghai/ ; //Shanghai Magnolia Talent Plan Pujiang Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Infrared Rays ; *DNA, Mitochondrial/genetics ; Mice ; Humans ; *Genetic Therapy/methods ; *Nanoparticles/chemistry ; Mutation ; Gene Editing/methods ; Cell Line, Tumor ; *Neoplasms/therapy/genetics ; },
abstract = {Mitochondrial DNA (mtDNA) mutations are associated with tumor progression and metabolic dysregulation, yet their spatiotemporal visualization and selective therapeutic modulation remain challenging owing to their low abundance and mitochondrial double-membrane barrier. We report a near-infrared (NIR) light-inducible CRISPR-Cas12a/tetrahedral DNA nanosystem for spatiotemporally resolved imaging and editing of mtDNA mutations. The nanosystem integrates upconversion nanoparticles as NIR to ultraviolet converters, photocleavable DNA linkers, and Cas12a/crRNA modules. NIR irradiation triggers linker cleavage, activates Cas12a trans-cleavage, and generates amplified fluorescence for mtDNA mutation detection with a detection limit of 0.83 pM. In living cells and tumor-bearing mice, the nanosystem enables high-spatiotemporal resolution imaging of mtDNA mutations while inducing DNA self-assembly and CRISPR-based mtDNA editing. These disrupt the mitochondrial membrane potential, increase the level of reactive oxygen species, and promote apoptosis, resulting in effective tumor suppression. This nanosystem enables the controllable imaging of mutant mtDNA and gene modulation, offering new opportunities for spatiotemporal cancer theranostics.},
}
@article {pmid41560288,
year = {2026},
author = {Zou, R and Lu, X and Liu, Y and Yang, P and Sun, S and Liu, Y and Mo, Y and Zhu, G and Lee, JS and Guo, Y},
title = {Quantitative Proteomics and CRISPR/Cas9 Editing Reveal UPR-Mediated Control of Immunoglobulin Homeostasis in Hybridomas.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {17},
pages = {e14140},
pmid = {41560288},
issn = {2198-3844},
support = {32302389//National Natural Science Foundation of China/ ; 32272574//National Natural Science Foundation of China/ ; 2023M743075//China Postdoctoral Science Foundation/ ; 2024T170787//China Postdoctoral Science Foundation/ ; RS-2024-00397714//National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Unfolded Protein Response/genetics/immunology ; Animals ; *Proteomics/methods ; Gene Editing/methods ; Mice ; *Homeostasis/genetics/immunology ; *Hybridomas/metabolism/immunology ; *Immunoglobulins/metabolism/genetics ; Antibodies, Monoclonal/genetics ; },
abstract = {Despite their tremendous economic value, monoclonal antibodies are often compromised by the loss of immunoglobulin (Ig) chains, which disrupts antibody homeostasis and quality control. Through subclone screening and characterization, we identified that the loss of Ig production impaired the recognition ability of hapten-specific hybridomas. Proteomic analysis further highlighted the critical role of the unfolded protein response (UPR) pathway in regulating aberrant Ig chain production. Using CRISPR/Cas9-mediated knockout and rescue experiments, we revealed the importance of the UPR pathway in facilitating hybridoma antibody production by targeting Xbp1s, an active transcription factor downstream of UPR signaling. With CRISPR/HDR, we inserted a fluorescent mGFP tag into the endogenous Hspa5 gene (encoding BiP, the master regulator of the UPR pathway), enabling in situ and real-time monitoring of UPR activation. A strong negative correlation (R[2] = 0.86) was observed between intracellular mGFP signals and IgG levels in the engineered system, indicating a close relationship between UPR activation and Ig production. Fluorescence-activated cell sorting of high-mGFP populations identified two dysfunctional subclones that failed to secret Ig, validating the system's effectiveness in tracing Ig homeostasis. In summary, this study provides new insights into UPR-mediated regulation of Ig synthesis and offers a novel UPR-based reporter system for monitoring antibody stability.},
}
@article {pmid41560876,
year = {2026},
author = {More, R and Yadav, V and Vadakedath, N},
title = {Calyptranema fuscum gen. sp. nov.: a novel cyanobacterial genus within Oculatellaceae based on polyphasic and genomic characterization.},
journal = {Current research in microbial sciences},
volume = {10},
number = {},
pages = {100542},
pmid = {41560876},
issn = {2666-5174},
abstract = {This study presents a comprehensive characterization and classification of a novel cyanobacterial isolate, strain S582, proposed as Calyptranema fuscum gen. sp. nov. within the family Oculatellaceae using an integrated polyphasic approach. Strain S582 was isolated from a lake in the Botanical Garden, Sarangpur, Chandigarh, India. Initial molecular characterization with the 16S rRNA gene revealed ≤ 94.90% of similarity with related genera and showed unique 16S-23S ITS secondary structures, indicating its delineation as a novel genus. Morphological assessment highlighted the presence of a cap-like structure called calyptra at the terminal cells, further distinguishing it from related genera. Furthermore, whole genome sequencing yielded an assembly of 7962,515 bp with GC content of 48.27%. Genome-based analysis encompassing average nucleotide identity (ANI), average amino acid identity (AAI), percentage of conserved proteins (POCP) was subsequently performed. The observed values for ANI (71.15% to 73.00%) and AAI (63.30% to 69.62%) were below the established genus-level thresholds. Phylogenetic analysis using maximum-likelihood and Bayesian inference along with phylogenomic reconstruction based on 1434 single copy core genes supported its taxonomic novelty. Functional classification revealed the presence of biosynthetic gene clusters (BGCs), tRNAs, insertion elements, CRISPR/Cas systems, and genes associated with metabolism, carbon fixation and photosynthesis. Additionally, the pangenome was constructed to study the genomic diversity of the studied isolate and related genera among the Oculatellaceae family and identified core, accessory, and singleton gene clusters. Collectively, these findings establish Calyptranema fuscum gen. sp. nov. as a novel genus within Oculatellaceae while expanding our understanding of cyanobacterial diversity and genomic potential.},
}
@article {pmid41561390,
year = {2026},
author = {Fehrenbach, A and Mitrofanov, A and Backofen, R and Baumdicker, F},
title = {The complexity of multiple CRISPR arrays in strains with (co-occurring) CRISPR systems.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf042},
pmid = {41561390},
issn = {2633-6693},
abstract = {CRISPR and their associated Cas proteins provide adaptive immunity in prokaryotes, protecting against invading genetic elements. These systems are categorized into types and are highly diverse. Genomes often harbor multiple CRISPR arrays varying in length and distance from Cas loci. However, the ecological roles of multiple CRISPR arrays and their interactions with multiple Cas loci remain poorly understood. We present a comprehensive analysis of CRISPR systems that uncovers variation between diverse Cas types regarding the occurrence of multiple arrays, the distribution of their lengths and positions relative to Cas loci, and the diversity of their repeat sequences. Some types tend to occur as the sole Cas locus present in the genome, but typically have two or more associated arrays, especially for types I-E and I-F. Multiple Cas types are also common, with some systems showing a preference for specific co-occurrence. Distinct array distributions and orientations around Cas loci indicate substantial differences in functionality and transcriptional behavior among Cas types. Our analysis suggests that arrays with identical repeats in the same genome acquire new spacers at comparable rates, irrespective of their proximity to the Cas locus. Furthermore, repeat similarities indicate that arrays of systems that often co-occur with other systems tend to have more diverse repeats than those mostly appearing alongside solitary systems. Our results indicate that co-occurring Cas-type pairs might not only collaborate in spacer acquisition but also maintain independent and complementary functions and that CRISPR systems distribute their defensive spacer repertoire equally across multiple CRISPR arrays.},
}
@article {pmid41561392,
year = {2026},
author = {Choudhary, DK and Turgeman-Grott, I and Robinzon, S and Gophna, U},
title = {CRISPR-Cas targeting in Haloferax volcanii promotes within-species gene exchange by triggering homologous recombination.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf047},
pmid = {41561392},
issn = {2633-6693},
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated genes) systems provide adaptive immunity in bacteria and archaea against mobile genetic elements, but the role they play in gene exchange and speciation remains unclear. Here, we investigated how CRISPR-Cas targeting affects mating and gene exchange in the halophilic archaeon Haloferax volcanii. Surprisingly, we found that CRISPR-Cas targeting significantly increased mating efficiency between members of the same species, in contrast to its previously documented role in reducing interspecies mating. This enhanced mating efficiency was dependent on the Cas3 nuclease/helicase and extended beyond the targeted genomic regions. Further analysis revealed that CRISPR-Cas targeting promoted biased recombination in favor of the targeting strain (the strain containing the CRISPR-Cas system) during mating, resulting in an increased proportion of recombinant progeny that are positive for CRISPR-Cas. To test whether an increase in recombination is sufficient to increase mating efficiency, we tested whether strains lacking the Mre11-Rad50 complex, which are known to have elevated recombination activity, also exhibited higher mating success. Indeed, these strains showed higher mating, as did cells that were exposed to DNA damage using methyl methanesulfonate. These findings suggest that CRISPR-Cas systems in archaea play roles beyond their canonical immune function. They may contribute to speciation by facilitating within-species gene exchange while limiting between-species genetic transfer, thereby maintaining species boundaries.},
}
@article {pmid41562083,
year = {2025},
author = {Liu, J and Hong, W and Sun, Z and Zhang, S and Xue, C and Dong, N},
title = {The gut-lung axis: effects and mechanisms of gut microbiota on pulmonary diseases.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1693964},
pmid = {41562083},
issn = {1664-3224},
mesh = {Humans ; *Gastrointestinal Microbiome/immunology ; Animals ; *Lung/immunology/metabolism/microbiology ; Dysbiosis/immunology ; *Lung Diseases/microbiology/immunology/metabolism/therapy/etiology ; Fatty Acids, Volatile/metabolism ; Fecal Microbiota Transplantation ; },
abstract = {The proposal of the gut-lung axis has profoundly reshaped our understanding of the mechanisms underlying respiratory diseases. As a crucial component of this axis, the gut microbiota plays a central role in pulmonary immune regulation through inter-organ communication mediated by metabolic products. However, a systematic integration of mechanisms explaining how gut microbes achieve precise cross-organ immune regulation remains elusive. Existing research predominantly focuses on descriptive observations, such as the association between early-life microbiota dysbiosis and an increased risk of asthma and chronic obstructive pulmonary disease (COPD), as well as the frequent occurrence of acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF), often accompanied by gut microbiome disruption. This paper focuses on three key gut microbial metabolites-short-chain fatty acids (SCFAs), tryptophan metabolites, and polyamines (PAs)-to examine their roles in immune regulation, maintenance of barrier function, and modulation of metabolic signaling networks. Based on the latest experimental and clinical evidence, this study systematically elucidates how dysbiosis of the gut microbiota, a key component of the gut-lung axis, crosses physiological barriers to exacerbate pulmonary inflammation. Regarding intervention strategies, probiotics, fecal microbiota transplantation (FMT), and CRISPR-Cas systems have demonstrated significant therapeutic potential in restoring gut microbial balance. Finally, this paper outlines future research directions, emphasizing the need to further explore non-invasive microbial sampling techniques, molecular interaction mechanisms of the gut-lung axis, and personalized microbiome-based diagnostic and therapeutic strategies to provide new insights for the prevention and treatment of respiratory diseases involving gut microbiota.},
}
@article {pmid41562237,
year = {2026},
author = {Mattivi, C and Wang, S and Ji, L and Xiao, Q and Cao, J},
title = {HLA-Knockout: Enabling Allele-Specific Knockout of HLA Class I Genes for Immunogenic Engineering.},
journal = {HLA},
volume = {107},
number = {1},
pages = {e70548},
pmid = {41562237},
issn = {2059-2310},
support = {P30 CA072720/CA/NCI NIH HHS/United States ; 82473308//National Natural Science Foundation of China Grant/ ; P30CA072720//Rutgers Cancer Institute of New Jersey/ ; //Nanjing University/ ; 2025300355//Fundamental Research Funds for the Central Universities/ ; //Science Fund Program for Distinguished Young Scholars (Overseas)/ ; },
mesh = {Humans ; *Alleles ; *Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Histocompatibility Antigens Class I/genetics/immunology ; Receptors, Antigen, T-Cell/genetics/immunology ; },
abstract = {The interaction between T-cell receptors (TCRs) and antigenic peptides presented by HLA molecules is fundamental to adaptive immunity. However, the extreme polymorphism of HLA genes poses major challenges for transplantation, antigen discovery, immunotherapy and studies of allele-specific function. Although CRISPR/Cas9 has transformed gene editing, existing sgRNA design tools are not optimised for knockout of HLA Class I genes due to their high rates of polymorphism. To address this, we developed HLA-Knockout (https://hlaknockout.rutgers.edu), a novel web-based tool that enables precise, allele-specific targeting of HLA Class I genes. HLA-Knockout retrieves user-defined HLA sequences from the IPD-IMGT/HLA database and applies stringent design criteria, including mismatch filtering and PAM disruption analysis, to ensure high specificity and minimal off-target effects on non-target HLA Class I alleles. Using HLA-Knockout, we achieved efficient single- and double-allele HLA Class I knockouts in human cells without disrupting non-target HLA Class I alleles. Functional assays confirmed allele-specific loss of antigen-specific TCR activation, validating the platform's utility. HLA-Knockout provides a unique resource for dissecting HLA-restricted immune interactions and has broad applications in transplantation biology, autoimmunity and cancer immunotherapy.},
}
@article {pmid41562469,
year = {2026},
author = {Wan, Z and Xu, C and Wang, Y and Song, L and Yuan, W and Chen, M and Gong, R and Zhang, XE},
title = {An AND-Logic Gate-Based Biosensor for Simultaneous Detection of SARS-CoV-2 Nucleic Acids and Nucleocapsid Proteins.},
journal = {Analytical chemistry},
volume = {98},
number = {4},
pages = {3275-3285},
doi = {10.1021/acs.analchem.5c07321},
pmid = {41562469},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *Coronavirus Nucleocapsid Proteins/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; *RNA, Viral/analysis/genetics ; *COVID-19/diagnosis/virology ; CRISPR-Cas Systems ; *Phosphoproteins/analysis ; *Nucleocapsid Proteins/analysis ; Limit of Detection ; },
abstract = {Nucleic acids and proteins are recognized as gold standard biomarkers for disease diagnosis and pathogen detection. However, conventional single-analyte detection methods remain susceptible to false positives caused by manual operational errors or sample contamination, thereby undermining diagnostic reliability and increasing the burden on healthcare systems. To address this limitation, we developed a one-pot isothermal amplification and CRISPR-Cas cooperative system (OIACS) that functions as an AND-logic gate biosensor for the simultaneous detection of SARS-CoV-2 RNA and nucleocapsid protein. Unlike conventional methods relying solely on CRISPR RNA (crRNA) recognition, the OIACS employs antibody-mediated target binding with blocker release for target recognition, offering increased flexibility in assay design for different targets. A universal Cas12a-targetable DNA barcode is generated via strand displacement isothermal amplification, enabling signal amplification upon dual-target recognition. The OIACS assay exhibited practical utility by reliably detecting SARS-CoV-2 transcription- and replication-competent virus-like-particles at 5000 copies/mL, and the limit of detection was determined to be as low as 1698 copies/mL, highlighting its robustness and potential for clinical diagnosis.},
}
@article {pmid41563484,
year = {2026},
author = {Pardy, F and Reblova, K and Svozilova, H and Tichy, B and Pospisilova, S and Kotaskova, J and Navrkalova, V},
title = {Assessment of long-read strategies for the enrichment of clinically relevant breakpoints in lymphomas: towards a diagnostic implementation.},
journal = {Annals of hematology},
volume = {105},
number = {2},
pages = {47},
pmid = {41563484},
issn = {1432-0584},
mesh = {Humans ; *Translocation, Genetic ; *Lymphoma/genetics/diagnosis ; *Chromosome Breakpoints ; Cell Line, Tumor ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; },
abstract = {Recurrent chromosomal translocations are hallmarks of many hematological malignancies, including lymphomas and leukemias. Accurate breakpoint detection is essential for diagnostics, treatment optimization, and disease monitoring. Long-read sequencing (Oxford Nanopore Technologies) enables unambiguous mapping and translocation identification. We designed a Cas9-based enrichment panel targeting common translocations in lymphoid malignancies. To accommodate both well-defined and promiscuous translocation partners, we employed single-side and dual-side sequencing strategies. Using well-established lymphoid cell lines, we benchmarked three enrichment approaches: (i) Cas9 read-out, (ii) Cas9 excision with multiplexing, and (iii) adaptive sampling. Cas9-mediated enrichment achieved superior on-target coverage, particularly in densely targeted regions (such as the IGH locus), while single-probe targets showed lower coverage depth. Adaptive sampling offered higher throughput, flexibility, and better pore occupancy, however with limited breakpoint detection. Cas9 excision has been demonstrated as a fast and reliable method to detect canonical translocation partners in clinical lymphoma samples. Our findings indicate that long-read enrichment strategies are suitable for targeting breakpoint hotspots, although the choice of approach depends heavily on the laboratory's specific goal. We propose a decision algorithm for selecting the optimal method based on experimental and clinical needs: Cas9-mediated enrichment suits focused diagnostic intent, while adaptive sampling is preferable for broader research use.},
}
@article {pmid41564380,
year = {2026},
author = {Myojin, Y and Kodama, T and Takahashi, R and Nagasawa, H and Kondo, Y and Yusa, K and Yoshida-Hashidate, T and Shindou, H and Furuta, K and Murai, K and Saito, Y and Hikita, H and Takehara, T},
title = {Genome-wide CRISPR screen identifies ACSL3 as a regulator of lipotoxicity and progression of MASLD.},
journal = {Hepatology communications},
volume = {10},
number = {2},
pages = {},
pmid = {41564380},
issn = {2471-254X},
mesh = {Humans ; *Coenzyme A Ligases/genetics/metabolism ; Long-Chain-Fatty-Acid-CoA Ligase ; Hepatocytes/metabolism ; Disease Progression ; Endoplasmic Reticulum Stress/genetics ; Apoptosis/genetics ; *Fatty Liver/genetics/metabolism/pathology ; CRISPR-Cas Systems ; Animals ; Oxidative Stress ; Mice ; Male ; Liver/pathology/metabolism ; Lipid Metabolism ; Lipogenesis ; },
abstract = {BACKGROUND: Metabolic dysfunction-associated steatotic liver disease (MASLD) and its progressive form, metabolic dysfunction-associated steatohepatitis, are highly prevalent and lack effective pharmacotherapies. Hepatocellular lipotoxicity-driven by the accumulation of saturated fatty acids (eg, palmitate)-promotes disease progression; however, the determinants of hepatocyte susceptibility remain incompletely defined.<br><br>METHODS: We performed a genome-wide CRISPR-Cas9 loss-of-function screening to identify the regulators of palmitate-induced lipotoxicity. The top candidates were validated using genetic perturbation and pharmacological inhibition. Lipid handling, endoplasmic reticulum/oxidative stress, apoptosis, and lipogenic transcriptional programs were also quantified. Human MASLD liver tissues were analyzed for ACSL3 expression in relation to histology and aminotransferases. Single-cell and spatial transcriptomics were used to localize ACSL3 expression and the associated pathway signatures in metabolic dysfunction-associated steatohepatitis.<br><br>RESULTS: The screen recovered established mediators (CASPASE-8, AGPAT9, RNF213) and identified ACSL3 as a novel determinant of hepatocyte survival under lipotoxic stress. Genetic deletion or pharmacological inhibition of ACSL3 renders hepatocytes resistant to palmitate-induced apoptosis and endoplasmic reticulum stress, accompanied by reduced lipid-droplet accumulation, decreased incorporation of saturated fatty acids into neutral lipids and phospholipids, and blunted induction of lipogenic programs. In human MASLD, hepatic ACSL3 expression positively correlated with histological severity and aminotransferase levels. Single-cell transcriptomics localized ACSL3 predominantly to hepatocytes in advanced metabolic dysfunction-associated steatohepatitis displaying oxidative and endoplasmic reticulum stress signatures, whereas spatial transcriptomics showed ACSL3-high hepatocyte regions enriched for apoptotic and inflammatory pathways and spatially coupled to macrophage-rich and plasma cell-rich niches.<br><br>CONCLUSIONS: ACSL3 is a central regulator of lipotoxic hepatocyte injury and MASLD progression, mechanistically linking lipid-droplet biogenesis to apoptosis and inflammatory niche formation. These data suggest that ACSL3 is a promising therapeutic target and support further translational studies to evaluate ACSL3 modulation in steatotic liver disease.},
}
@article {pmid41564857,
year = {2026},
author = {Balke-Want, H and Keerthi, V and Del Carmen Arenas, M and Chen, Y and Malipatlolla, M and Klysz, DD and Xu, P and Ho, K and Asano, K and Stahl, D and Huang, J and Retherford, A and Patel, S and Fowler, C and Maas, L and Gkitsas-Long, N and Jiang, Q and Liu, X and Ullrich, R and George, J and Heitzeneder, S and Tunuguntla, R and Sage, J and Sotillo, E and Mackall, CL and Feldman, SA},
title = {c-JUN enhances CRISPR knockin anti-B7-H3 CAR T cell function in small cell lung cancer and thoracic SMARCA4-deficient undifferentiated tumors.},
journal = {Cell reports. Medicine},
volume = {7},
number = {1},
pages = {102549},
pmid = {41564857},
issn = {2666-3791},
support = {R35 CA283888/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *DNA Helicases/deficiency/genetics/metabolism ; *Lung Neoplasms/immunology/pathology/genetics/therapy/metabolism ; *Nuclear Proteins/deficiency/genetics/metabolism ; *Transcription Factors/deficiency/genetics/metabolism ; *Small Cell Lung Carcinoma/immunology/pathology/therapy/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *T-Lymphocytes/immunology/metabolism ; Cell Line, Tumor ; *Proto-Oncogene Proteins c-jun/metabolism/genetics ; *Receptors, Chimeric Antigen/metabolism/immunology ; Immunotherapy, Adoptive/methods ; Animals ; },
abstract = {Small cell lung cancer (SCLC), a highly lethal disease, limits T cell responses by downregulating major histocompatibility (MHC) class I molecules. Because chimeric antigen receptor (CAR) T cells are not MHC restricted, they may provide a powerful strategy against SCLC. However, few CAR targets for SCLC are known. Here, we show that B7-H3/CD276 is expressed in SCLC and thoracic SMARCA4-deficient undifferentiated tumors (UTs) that can clinicopathologically mimic SCLC. Thoracic SMARCA4-deficient UTs limit killing by B7-H3 CAR T cells via secretion of transforming growth factor β1 (TGF-β1). To overcome tumor-driven CAR T cell suppression, we knock in c-JUN alongside a B7-H3 CAR into the TRAC locus of primary human T cells utilizing CRISPR-Cas9. Non-viral c-JUN+B7-H3 CAR T cells show enhanced killing of both SCLC cells with low antigen density and thoracic SMARCA4-deficient UTs, providing a platform to address these highly aggressive entities. We also provide evidence that good manufacturing practice (GMP) clinical-scale manufacturing is feasible for c-JUN+B7-H3 CAR T cells.},
}
@article {pmid41564866,
year = {2026},
author = {Watterson, A and Picco, G and Veninga, V and Samarakoon, Y and Cattaneo, CM and Vieira, SF and Karakoc, E and Bhosle, S and Battaglia, TW and Consonni, S and Halim, TYF and Voest, EE and Garnett, MJ and Coelho, MA},
title = {CRISPR screens in the context of immune selection identify CHD1 and MAP3K7 as mediators of cancer immunotherapy resistance.},
journal = {Cell reports. Medicine},
volume = {7},
number = {1},
pages = {102565},
pmid = {41564866},
issn = {2666-3791},
mesh = {Humans ; Animals ; *Immunotherapy/methods ; Mice ; *MAP Kinase Kinase Kinases/genetics/metabolism ; *DNA Helicases/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *DNA-Binding Proteins/genetics/metabolism ; Cell Line, Tumor ; Immune Checkpoint Inhibitors/pharmacology/therapeutic use ; CD8-Positive T-Lymphocytes/immunology ; *Drug Resistance, Neoplasm/genetics ; *Neoplasms/immunology/therapy/genetics ; Interferon-gamma ; Melanoma/immunology/genetics ; Mice, Inbred C57BL ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Cancer immunotherapy is only effective in a subset of patients, highlighting the need for effective biomarkers and combination therapies. Here, we systematically identify genetic determinants of cancer cell sensitivity to anti-tumor immunity by performing whole-genome CRISPR-Cas9 knockout screens in autologous tumoroid-T cell co-cultures, isogenic cancer cell models deficient in interferon signaling, and in the context of four cytokines. We discover that loss of CHD1 and MAP3K7 (encoding TAK1) potentiates the transcriptional response to IFN-γ, thereby creating an acquired vulnerability by sensitizing cancer cells to tumor-reactive T cells. Immune checkpoint blockade is more effective in a syngeneic mouse model of melanoma deficient in Chd1 and Map3k7 and is associated with elevated intra-tumoral CD8[+] T cell numbers and activation. CHD1 and MAP3K7 are recurrently mutated in cancer, and reduced expression in tumors correlates with response to immune checkpoint inhibitors in patients, nominating these genes as potential biomarkers of immunotherapy response.},
}
@article {pmid41565063,
year = {2026},
author = {Wu, Q and Du, F and Zhang, X and Lu, Z and Zheng, X and Li, A and Zhang, X and Zhang, R and Wang, Q},
title = {Development and evaluation of a one-pot CRISPR/Cas13-based assay for syphilis detection across multiple clinical sample types.},
journal = {International journal of infectious diseases : IJID : official publication of the International Society for Infectious Diseases},
volume = {164},
number = {},
pages = {108414},
doi = {10.1016/j.ijid.2026.108414},
pmid = {41565063},
issn = {1878-3511},
mesh = {Humans ; Sensitivity and Specificity ; *Treponema pallidum/genetics/isolation &amp; purification ; *Syphilis/diagnosis/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; },
abstract = {OBJECTIVES: To develop and evaluate a rapid, one-pot molecular assay for the detection of Treponema pallidum subspecies pallidum (TPA), addressing the limitations of current diagnostic methods influenced by sample type and pathogen load.<br><br>METHODS: A one-pot assay integrating recombinase polymerase amplification (RPA) and Cas13a-based collateral cleavage activity was established for isothermal detection of TPA. The assay targeted the tpp47/tp0574 gene and was validated using 186 clinical specimens, including whole blood, lesion exudate, and cerebrospinal fluid (CSF) samples.<br><br>RESULTS: The one-pot RPA-Cas13a assay demonstrated high analytical sensitivity and specificity for TPA detection. Clinical sensitivities were 58.97% in whole blood, 84.21% in lesion exudate, and 57.14% in CSF, with 100% specificity across all sample types.<br><br>CONCLUSION: This one-pot isothermal assay enables rapid and accurate detection of T. pallidum directly from diverse clinical samples. Its high specificity and field-friendly format make it a promising complementary tool to conventional diagnostic approaches, particularly for point-of-care testing and screening in resource-limited or high-risk settings.},
}
@article {pmid41565761,
year = {2026},
author = {Beauchemin, KS and Supattapone, S},
title = {Genome-wide screens identify core regulators of cell surface prion protein expression.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5895},
pmid = {41565761},
issn = {2045-2322},
support = {P20 GM113132/GM/NIGMS NIH HHS/United States ; 1R37NS125431/NS/NINDS NIH HHS/United States ; T32 AI007519/AI/NIAID NIH HHS/United States ; R37 NS125431/NS/NINDS NIH HHS/United States ; R01 NS118796/NS/NINDS NIH HHS/United States ; R01 NS117276/NS/NINDS NIH HHS/United States ; 5T32AI007519/NH/NIH HHS/United States ; },
mesh = {*Neurons/metabolism/cytology ; Humans ; CRISPR-Cas Systems ; *Prion Proteins/genetics/metabolism ; Cell Differentiation/genetics ; *Gene Expression Regulation ; *PrPC Proteins/genetics/metabolism ; Animals ; Cell Line ; Cell Membrane/metabolism ; },
abstract = {Expression of the cellular prion protein, PrP[C], on the surface of neurons plays an important role in the pathogenesis of prion disease. We performed genome-wide CRISPR/Cas9 knockout screens in prion-infectible cells of neuronal origin (CAD5) to identify regulators of cell surface PrP[C] expression. We identified and validated 46 positive and 21 negative regulators of cell surface PrP[C] expression in undifferentiated CAD5 cells. Pathway analysis of the screening dataset showed that genes involved in the glycophosphatidylinositol (GPI) anchor and N-glycosylation biosynthetic pathways were overrepresented as positive regulators of cell surface PrP[C]. We also sought to determine whether the same or different genes regulate cell surface PrP[C] in CAD5 cells that have been differentiated to a more neuronal state and validated 41 positive and 13 negative regulators of CAD5 cell surface PrP[C] expression in the differentiated state. We identified 23 core genes as shared between the undifferentiated and differentiated cell states, including many positive regulators involved in GPI anchor biosynthesis. Intriguingly, unique regulators were also identified in the undifferentiated and differentiated cell states, suggesting that some mechanisms regulating cell surface PrP[C] expression in CAD5 cells are dependent on cell state. This list of core genes involved in regulating cell surface PrP[C] expression in a prion-susceptible, neuron-like cell type offers a valuable guide for future research and may help identify potential therapeutic targets for prion disease and other neurodegenerative diseases.},
}
@article {pmid41566394,
year = {2026},
author = {Xin, C and Xiang, G and Cao, S and Wang, Y and Yuan, S and Liu, X and Huo, Y and Sun, J and Wan, X and Liu, D and Hong, J and Hu, J and Wang, H},
title = {Comprehensive assessment of activity, specificity, and safety of hypercompact TnpB systems for gene editing.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {39},
pmid = {41566394},
issn = {1474-760X},
support = {24YF2703900//the "Rising-Star Program" of "Shanghai 2024 Science and Technology Innovation Action Plan/ ; 32101204//NSFC grant/ ; 31771485//NSFC grant/ ; 202423110050063//Science and Technology Major Project of Anhui Province/ ; 2024YFA0917301//the National Key R&amp;D Program of China/ ; 2022YFC3400201//National Key Research and Development Program of China/ ; 2023ZD04074//Biological Breeding-Major Projects of the ministry of Agriculture and Rural Affairs of China/ ; 2022FH122//Program of Beijing Institute for Stem Cell and Regenerative Medicine/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Genetic Therapy ; HEK293 Cells ; },
abstract = {BACKGROUND: As the ancestor of CRISPR-Cas12 nucleases, TnpB represents the most compact gene editing tool currently available. Recent studies have identified multiple TnpB systems with gene editing activity in mammalian cells, and the potential of TnpB in treating diseases has been demonstrated in animal models. However, the editing characteristics of various TnpB systems, comparable to CRISPR tools, require more extensive investigation.<br><br>RESULTS: Using a standardized evaluation framework, we conduct a thorough analysis of the editing properties of four TnpB variants alongside representative Cas12 and Cas9 tools applications. Overall, TnpBs exhibit intermediate editing activity and safety profiles among all tested systems, with ISYmu1 TnpB demonstrating a good performance in both editing activity and specificity. Considering its compact size, potent editing efficiency and high specificity, ISYmu1 TnpB represents a promising candidate for gene therapy.<br><br>CONCLUSIONS: By comprehensively analyzing genome editing outcomes, we characterize TnpB systems for genome editing and identify ISYmu1 TnpB as an optimal miniature RNA-guided genome editors with balanced performance, highlighting its potential for therapeutic applications.},
}
@article {pmid41568169,
year = {2026},
author = {Zhang, W and Wang, H and Liu, D and Mao, X and Zhang, Y and Yang, Y and Liu, Z and Pan, T and Liu, Y and Zhang, Q},
title = {Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for therapeutic applications.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102811},
pmid = {41568169},
issn = {2162-2531},
abstract = {The CRISPR-Cas13 system exhibits potent RNA cleavage activity and has been widely utilized for RNA-targeting applications. However, its collateral cleavage of bystander RNAs limits in vivo therapeutic applications. In this study, we generated a series of LwaCas13a mutants through structure-based design and site-directed mutagenesis strategies. A triple mutant enCas13a (Q521R/E796A/E810A) was obtained with significantly enhanced target RNA cleavage activity along with only slightly increased collateral activity. To reduce the collateral activity, we optimized crRNA terminal extensions and obtained M1crRNA and M3crRNA variants that, in combination with enCas13a, maintained or reduced collateral activity while preserving enhanced targeted cleavage activity. Thus, by optimizing the Cas protein and crRNA, we have created an improved CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity. This system demonstrated superior performance in targeting endogenous genes and antiviral applications. Mechanistic studies revealed that enhanced protein-crRNA interactions and altered complex conformations underlie the improved cleavage activity. This engineering approach provides a generalizable strategy for developing CRISPR-Cas systems with enhanced therapeutic potential.},
}
@article {pmid41569151,
year = {2026},
author = {Zhang, W and Kong, J and Zeng, Y and Su, Y and Zhang, S and Li, Y and Hu, C and Chen, Q and Xiao, Y and Lu, M},
title = {Structural plasticity enables broad cAn binding and dual activation of CRISPR-associated ribonuclease Cdn1.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41569151},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 2021ZD0203400//STI2030-Major Projects/ ; 31970547//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Protein Binding ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; Adenine Nucleotides/metabolism/chemistry ; Catalytic Domain ; *Ribonucleases/metabolism/chemistry/genetics ; Oligoribonucleotides ; },
abstract = {Prokaryotes have naturally evolved diverse RNA-guided defense systems against viral infections, with the type III CRISPR-Cas systems representing the most intricate. These systems feature accessory proteins activated by cyclic oligoadenylates (cOAs) produced upon target RNA recognition, synergizing with the CRISPR-Cas machinery to defend against exogenous invaders. Typically, each accessory protein is activated by only one specific cOA type. Here, we characterize Cdn1, a type III-B CRISPR accessory protein from Psychrobacter lutiphocae, which binds to cA3, cA4, and cA6, but activated by cA4 and cA6 with different efficacies to catalyze ssRNA cleavage. Combined structural and biochemical analyses reveal that cOA binding triggers dramatic conformational reorganization, including the formation of a dimerization interface of nuclease domains, the emergence of substrate binding cleft, and the reconstruction of a metal-dependent catalytic center essential for RNA cleavage. This dual activation mechanism illustrates evolutionary innovation within CRISPR-associated Rossman-fold nucleases. We propose that such structural plasticity evolved to maximize defensive resilience during microbial competition and horizontal gene transfer, while preserving broad-spectrum antiviral ability. These findings not only elucidate the activation mechanisms of Cdn1 within the type III systems but also underscore the functional complexity and adaptability of CRISPR-Cas ancillary proteins.},
}
@article {pmid41569163,
year = {2026},
author = {Luo, Y and Jiang, Q and Qu, Y and Li, W and Liu, R and Zhu, Y and Xie, Y and Jiang, C and Chen, C and Cong, L and Han, F and Bao, J and Wang, C},
title = {Compact bacterial recombination complexes drive efficient kilobase-scale knock-in in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {2},
pages = {},
pmid = {41569163},
issn = {1362-4962},
support = {2024YFC3408100//National Key Research and Development Program of China/ ; JSSCTD202450//Jiangsu Shuangchuang Project/ ; TJ-2023-005//Jiangsu Science and Technology Association Youth Science and Technology/ ; QYPY20230032//Center for Advanced Interdisciplinary Science and Biomedicine of IHM/ ; BK20240529//Natural Science Foundation of Jiangsu Province/ ; //Nanjing Medical University/ ; 82403421//National Natural Science Foundation of China/ ; 2408085J016//Anhui Provincial Natural Science Foundation/ ; },
mesh = {Animals ; Humans ; Mice ; *Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; *Gene Editing/methods ; *Recombinational DNA Repair ; Escherichia coli/genetics/enzymology ; *Escherichia coli Proteins/genetics/metabolism ; HEK293 Cells ; Neurons/metabolism ; *Rec A Recombinases/genetics/metabolism ; },
abstract = {Efficient homologous recombination, homology-directed repair (HDR), remains a major hurdle for precise genome editing in mammalian cells, particularly for kilobase-scale insertions. Bacterial recombineering proteins, such as RecE and RecT, offer potential solutions, but their activity in eukaryotic systems has been largely uncharacterized. Here, we identify Escherichia coli RecE (EcRecE) as a potent enhancer of HDR in mammalian cells. Targeted recruitment of EcRecE via CRISPR/Cas9 significantly increased HDR efficiency at multiple genomic loci across different cellular contexts, including human embryonic stem cells, achieving a 3-6-fold enhancement in the integration efficiency of kilobase-scale sequences. Furthermore, in combination with RecT and a catalytically inactive Cas9 (dCas9), applying functional domain engineering, we developed a dCas9-miniRecTE editor that enhances large-fragment integration without introducing double-strand breaks in human cells and primary mouse neurons, achieving ∼20% kilobase-scale knock-in efficiency. These results establish EcRecE as a versatile tool for improving precision genome engineering, with potential applications in therapeutic gene editing.},
}
@article {pmid41569521,
year = {2026},
author = {Yang, X and Wu, H and Zeng, Z and Chen, WN and Luan, GX and Zhang, QL and Chen, JM},
title = {Recent advances in highly sensitive and specific functional nucleic acid sensors for environmental pollutant detection: from mechanism to application.},
journal = {The Analyst},
volume = {151},
number = {3},
pages = {697-717},
doi = {10.1039/d5an01139f},
pmid = {41569521},
issn = {1364-5528},
mesh = {*Environmental Pollutants/analysis ; *Biosensing Techniques/methods ; *Nucleic Acids/chemistry ; DNA, Catalytic/chemistry ; *Environmental Monitoring/methods ; G-Quadruplexes ; Metals, Heavy/analysis ; Aptamers, Nucleotide/chemistry ; },
abstract = {Functional nucleic acids (FNAs) have emerged as a cutting-edge tool in environmental pollutant detection, attributed to their exceptional stability, robust specificity, and remarkable capacity for signal transduction and amplification. This review elaborates comprehensively on four pivotal categories of FNAs-aptamers, RNA-cleaving DNAzymes, G-quadruplex/hemin DNAzymes, and gRNAs-alongside their applications in monitoring a spectrum of pollutants. These encompass organic contaminants (e.g., pesticides and bisphenols), heavy metals (such as Pb[2+] and Hg[2+]), biotoxins, and pathogenic microorganisms. It also underscores the integration of FNAs with sophisticated technologies like nanomaterials and CRISPR/Cas systems to augment detection sensitivity and efficacy. Despite prevailing challenges, including susceptibility to environmental variables (pH and temperature) and intricate synthesis procedures, FNAs hold immense potential for advancing environmental monitoring and pollution control.},
}
@article {pmid41570230,
year = {2025},
author = {Demidova, NA and Klimova, RR and Kushch, AA and Karpov, DS},
title = {CRISPR-Cas genome editing system in the diagnosis and therapy of infection caused by herpes simplex virus type 1 (Orthoherpesviridae: Alphaherpesviridae: Simplexvirus: Simplexvirus humanalpha1).},
journal = {Voprosy virusologii},
volume = {70},
number = {6},
pages = {493-507},
doi = {10.36233/0507-4088-307},
pmid = {41570230},
issn = {2411-2097},
mesh = {Humans ; *Herpesvirus 1, Human/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Herpes Simplex/therapy/diagnosis/genetics/virology ; Animals ; Genome, Viral ; *Genetic Therapy/methods ; },
abstract = {Herpes simplex virus type 1 (HSV-1), newly named as Simplexvirus humanalpha1 is one of the most common pathogens in the human population, which can cause severe disease, often with fatal outcomes. Diagnostic methods currently in use are specific and sensitive, but time-consuming, require expensive laboratory equipment and highly qualified personnel. Existing therapeutic agents have a number of significant drawbacks. To successfully treat and prevent the spread of the infection, new rapid, easy-to-use, and highly sensitive diagnostic tools and effective therapeutic agents are required. One approach to achieve this goal is CRISPR-based technology. This review analyzes information obtained from a literature search in the Scopus, Web of Science and MedLine databases on the topics «HSV-1, structure, distribution, life cycle», «new methods for molecular diagnosis of HSV-1-infection», «classification of CRISPR-Cas systems», «nucleic acid amplification methods», «CRISPR-Cas effector proteins», «application of CRISPR-Cas systems in molecular diagnostics of HSV-1-infection», «application of CRISPR-Cas systems in therapy of HSV-1-infection». New approaches of CRISPR using effector proteins Cas12 and Cas13 in the diagnosis of HSV-1 infections are reviewed. The article discusses the progress in the development of CRISPR-Cas-based therapies against HSV-1-infection in vitro and in vivo. CRISPR gene therapy in vivo has a great clinical potential, but its safety and efficacy require further investigation. An analysis of the available data suggests that CRISPR-based technologies offer promising prospects for expanding the arsenal of diagnostic tools and antiviral drugs in the context of current and future outbreaks of viral diseases.},
}
@article {pmid41570748,
year = {2026},
author = {Gholami, S and Aghbash, PS and Ravanlo, ZZ and Rahimi, SB and Baghi, HB},
title = {Therapeutic targeting of the HPV E7 oncoprotein: Current advances and emerging strategies.},
journal = {International immunopharmacology},
volume = {172},
number = {},
pages = {116193},
doi = {10.1016/j.intimp.2026.116193},
pmid = {41570748},
issn = {1878-1705},
mesh = {Humans ; *Papillomavirus E7 Proteins/genetics/antagonists &amp; inhibitors/immunology/metabolism ; *Uterine Cervical Neoplasms/therapy/immunology/virology ; Female ; *Papillomavirus Infections/therapy/immunology ; Animals ; *Immunotherapy/methods ; Genetic Therapy/methods ; Gene Editing ; Oncogene Proteins, Viral/genetics ; CRISPR-Cas Systems ; },
abstract = {Cervical cancer is one of the most common malignancies among women, with persistent infection by high-risk human papillomavirus (HPV) types, particularly HPV16 and HPV18, being the primary etiological factor. The viral oncoproteins E6 and E7 play pivotal roles in carcinogenesis by inactivating the tumor suppressor proteins p53 and pRb, respectively. E7 has emerged as a promising therapeutic target due to its continuous expression in transformed cells and its essential role in maintaining the malignant phenotype. Recent advances in molecular biology and nanotechnology have led to the development of novel therapeutic strategies aimed at silencing or inhibiting E7, such as immunotherapy, RNA interference (RNAi), CRISPR/Cas9-based genome editing, and the use of natural bioactive compounds. Immunotherapeutic approaches aim to elicit specific cytotoxic T-cell responses against E7, whereas RNAi and CRISPR/Cas systems enable precise suppression or disruption of the E7 oncogene. As a result, it leads to the reactivate of p53 and pRb pathways, cell cycle arrest, and apoptosis. Additionally, the design of innovative delivery systems, such as liposomal nanoparticles, polymeric carriers, and viral vectors, has improved the efficiency and safety of therapeutic gene delivery. Collectively, these targeted approaches offer promising prospects for the treatment of HPV-related cancers. However, further optimization of delivery platforms and minimization of off-target effects are essential for the successful clinical translation of E7-targeted therapies in cervical cancer.},
}
@article {pmid41571527,
year = {2026},
author = {Kaneko, Y and Kawabe, Y and Nishijima, KI and Kamihira, M},
title = {Evaluation of cellular characteristics and genome editing responses in chicken primordial germ cell lines.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {4},
pages = {290-299},
doi = {10.1016/j.jbiosc.2025.12.010},
pmid = {41571527},
issn = {1347-4421},
mesh = {Animals ; *Chickens/genetics/growth &amp; development ; Male ; Female ; *Germ Cells ; Organisms, Genetically Modified ; *Gene Editing ; *Chick Embryo/cytology ; CRISPR-Cas Systems ; Gene Knock-In Techniques ; *Gene Targeting/methods ; Green Fluorescent Proteins/analysis ; Cell Culture Techniques ; },
abstract = {Chicken primordial germ cells (cPGCs) hold great potential for genetic modification and germ cell research in chickens. In this study, we evaluated the cellular characteristics of three cPGC lines: cPGC-1, cPGC-2, and cPGC-3. cPGC-1 and cPGC-2 were derived from male chickens, whereas cPGC-3 was derived from a female chicken. We analyzed and compared cell proliferation rates, marker gene expression, and gonadal colonization abilities. Three different cell culture temperatures were assessed (37 °C, 39 °C, and 41 °C) and proliferation rates were highest for all cPGC lines at 39 °C. Additionally, cPGC-1 demonstrated a higher proliferation rate than cPGC-2. No significant differences were observed between cPGC-1 and cPGC-2 with regard to the expression of germ cell and pluripotency marker genes (Cvh, Dazl, Pou5f3, and Nanog). To assess changes in cellular characteristics before and after genetic modification, we performed a green fluorescent protein (GFP) gene knock-in using the CRISPR/Cas9 system, followed by site-specific integration of the scFv-Fc gene using the Cre-loxP system. Transplantation experiments revealed that cPGC-2/GFP exhibited higher gonadal colonization efficiency than cPGC-1/GFP. This study demonstrates differences in cellular characteristics among established cPGC lines and highlights the impact of genetic modification on cPGC function. Our findings emphasize the importance of selecting appropriate cell lines and optimizing culture conditions based on cPGC traits to achieve efficient and reproducible production of transgenic chickens. These insights will aid in the conservation of poultry genetic resources and the advancement of transgenic chicken production for both research and industrial applications.},
}
@article {pmid41572361,
year = {2026},
author = {Mulero-Sánchez, A and Bosma, A and Visuvasam, B and Pouliopoulou, N and van de Ven, M and Proost, N and Boeije, M and Lieftink, C and Beijersbergen, R and Bernards, R and Mainardi, S},
title = {CRISPR knockout screens reveal JUN as the master mediator of resistance to MAPK inhibition in KRAS-mutant pancreatic cancer.},
journal = {Journal of experimental &amp; clinical cancer research : CR},
volume = {45},
number = {1},
pages = {},
pmid = {41572361},
issn = {1756-9966},
support = {SU2C-AACR-PCC-01-18//Stand Up To Cancer/ ; KWF 12539//KWF Kankerbestrijding/ ; },
mesh = {Humans ; *Proto-Oncogene Proteins p21(ras)/genetics ; *Pancreatic Neoplasms/genetics/drug therapy/pathology/metabolism ; Animals ; Mice ; *Drug Resistance, Neoplasm/genetics ; Mutation ; Cell Line, Tumor ; *Protein Kinase Inhibitors/pharmacology ; CRISPR-Cas Systems ; *Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology ; Xenograft Model Antitumor Assays ; Gene Knockout Techniques ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is often driven by KRAS mutations, but inhibitors targeting the most frequent KRAS substitutions in PDAC are not yet approved in the clinic. We previously discovered that KRAS-mutant PDAC is sensitive to the combination of SHP2 and ERK inhibitors, recently investigated in the Phase I/Ib clinical trial NCT04916236. Lately, RAS(ON) multi-selective inhibitors have entered clinical development, representing a promise for mono or combination therapies in PDAC. However, resistance may arise even for combination therapies. Here, we aimed at anticipating mechanisms of resistance to SHP2 plus ERK or RAS(ON) multi-selective inhibitors.<br><br>METHODS: We performed a genome-wide CRISPR-KO screening, followed by four follow-up focused screenings, leading to the identification of resistance mediators, which were further validated through functional genetic and pharmacological experiments, both in vitro and in vivo.<br><br>RESULTS: Through unbiased CRISPR-based screenings, we identified mTOR and JUN hyperactivation as interconnected mechanisms that overcome MAPK suppression. Further investigation pointed at JUN as the most downstream resistance mediator, and indirect therapeutic target, using MAP2K4 inhibitors.<br><br>CONCLUSIONS: Alterations in the PI3K/AKT/mTOR and JUN pathways can induce resistance to multiple combinations of MAPK pathway inhibitors, and may serve as biomarkers for sensitivity/resistance in clinical trials exploring such combinations in KRAS-mutant PDAC.},
}
@article {pmid41574295,
year = {2025},
author = {Zhang, R and Zheng, Y and Ding, C and Wu, J and Zhu, W and Zhu, X and Xu, G and Chen, L},
title = {OxyR contributes to the oxidative stress capacity and virulence of hypervirulent Klebsiella pneumoniae ATCC 43816.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1661384},
pmid = {41574295},
issn = {2235-2988},
mesh = {*Oxidative Stress ; *Klebsiella pneumoniae/pathogenicity/genetics/growth &amp; development/drug effects ; Animals ; Virulence ; Klebsiella Infections/microbiology/pathology ; Biofilms/growth &amp; development ; Mice ; Hydrogen Peroxide/toxicity ; Disease Models, Animal ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Gene Deletion ; Moths/microbiology ; *Repressor Proteins/genetics/metabolism ; Genetic Complementation Test ; Virulence Factors/genetics ; Gene Expression Profiling ; CRISPR-Cas Systems ; Lepidoptera/microbiology ; },
abstract = {INTRODUCTION: Hypervirulent Klebsiella pneumoniae (hvKP) is an emerging pathogen associated with severe invasive infections and high mortality, in which resistance to host-derived reactive oxygen species (ROS) is critical for immune evasion and persistence. However, the mechanisms underlying oxidative stress resistance in hvKP remain poorly understood, and the role of the global regulator OxyR in this species has not been fully elucidated.<br><br>METHODS: In this study, VK055_RS16305 was first identified as an OxyR homologue in K. pneumoniae ATCC 43816 by sequence alignment. The oxyR deletion mutant was generated using a CRISPR/Cas9-based genome editing system, whereas the complemented strain was obtained using the pSTV28 plasmid carrying oxyR. We then compared their growth characteristics, susceptibility to H&#x2082;O&#x2082;, biofilm formation, and virulence in Galleria mellonella and mouse infection models, and performed RNA sequencing followed by qRT-PCR to characterize the OxyR regulon under oxidative stress.<br><br>RESULTS: Deletion of oxyR did not alter bacterial growth or colony morphology under non-stress conditions, but markedly increased susceptibility to H&#x2082;O&#x2082; and impaired biofilm formation. In vivo, the oxyR mutant exhibited attenuated virulence, with improved survival of Galleria mellonella and mice and significantly reduced bacterial burdens in blood, liver, lung, and spleen, all of which were restored by genetic complementation. Transcriptomic analysis revealed that OxyR positively regulates multiple oxidative stress-associated genes, including hemH, grxA, gsk, katG, and ahpC, in response to H&#x2082;O&#x2082;.<br><br>DISCUSSION: Together, these findings demonstrate that OxyR is a key regulator of oxidative stress defense, biofilm formation, and systemic virulence in hvKP, providing new insight into OxyR-mediated pathogenic mechanisms in K. pneumoniae.},
}
@article {pmid41575301,
year = {2026},
author = {Park, H and Bae, SM and Hong, T and Song, G and Lim, W},
title = {CRISPR/Cas9-mediated SLC2A1 gene knockout changes in energy metabolism and cellular behavior in human trophoblasts.},
journal = {Reproduction (Cambridge, England)},
volume = {171},
number = {2},
pages = {},
doi = {10.1093/reprod/xaag006},
pmid = {41575301},
issn = {1741-7899},
support = {//National Research Foundation of Korea/ ; RS-2024-00453204//Korea government/ ; RS-2025-02216962//Korea government/ ; },
mesh = {Humans ; *Trophoblasts/metabolism/cytology ; *CRISPR-Cas Systems ; *Glucose Transporter Type 1/genetics/metabolism ; *Energy Metabolism/genetics ; Endoplasmic Reticulum Chaperone BiP ; Female ; Pregnancy ; Gene Knockout Techniques ; Endoplasmic Reticulum Stress ; Mitochondria/metabolism ; Reactive Oxygen Species/metabolism ; Signal Transduction ; Autophagy ; },
abstract = {Glucose transport across the placenta is essential for fetal growth and development. Glucose transporter 1, encoded by the SLC2A1 gene, plays a central role in mediating maternal-fetal glucose exchange. Dysregulation of placental glucose transport is implicated in pregnancy-related complications, such as preeclampsia and fetal growth restriction; however, the mechanistic role of SLC2A1 in trophoblast function remains poorly defined. To functionally validate the role of SLC2A1 in human trophoblasts, we used clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9-mediated knockout of the SLC2A1 gene, enabling complete and permanent loss of SLC2A1 expression. In the resulting SLC2A1 knockout human trophoblast HTR8/SVneo cells, SLC2A1 depletion induced a metabolic shift from glycolysis to oxidative phosphorylation, leading to increased mitochondrial respiration, ATP production, mitochondrial calcium overload, and elevated mitochondrial reactive oxygen species generation. These changes were accompanied by enhanced endoplasmic reticulum stress, as shown by the upregulation of p-PERK, IRE1α, and GRP78, as well as increased autophagic activity indicated by LC3B-II and p62 accumulation. Notably, mTOR signaling was also upregulated, suggesting a feedback loop that regulates autophagy. The loss of SLC2A1 impaired the PI3K/AKT pathway, reduced trophoblast migration and 3D spheroid formation, and disrupted epithelial-mesenchymal transition-like properties. These findings demonstrate that SLC2A1 is essential for maintaining trophoblast energy homeostasis, redox balance, and invasive capacity; its deficiency triggers mitochondrial and endoplasmic reticulum stress responses that may contribute to placental dysfunction during early pregnancy.},
}
@article {pmid41575844,
year = {2026},
author = {Bourner, LA and Acken, KA and Long, H and Chung, LA and Roth, KD and Dorsey, FC},
title = {Protocol to study the role of endogenously produced itaconate using CRISPR-Cas9 technology in THP-1 cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104304},
pmid = {41575844},
issn = {2666-1667},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Succinates/metabolism/analysis ; THP-1 Cells ; Macrophages/metabolism ; Chromatography, Liquid/methods ; Mass Spectrometry ; Carboxy-Lyases ; },
abstract = {Itaconate mimetics inadequately represent endogenous itaconate, a negative regulator of innate immune-driven pro-inflammatory cytokines. We present a CRISPR-Cas9 protocol to delete a 4-nucleotide region in the immunoresponsive gene 1 (IRG1), ablating ACOD1 (the itaconate-producing enzyme) in THP-1 cells. We describe the functional validation of ACOD1 deletion using immunoblotting, ELISA, and liquid chromatography-mass spectrometry (LC-MS) quantification of itaconate, enabling the study of endogenous itaconate in THP-1 macrophages. For complete details on the use and execution of this protocol, please refer to Bourner et al.[1].},
}
@article {pmid41575845,
year = {2026},
author = {Priesmeier, L and Tiburcy, M and Zelarayán, LC},
title = {Protocol for differentiation of vascular smooth muscle cells from human iPSCs and their application in CRISPRa-mediated gene regulation.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104345},
pmid = {41575845},
issn = {2666-1667},
mesh = {Humans ; *Induced Pluripotent Stem Cells/cytology/metabolism ; *Cell Differentiation/genetics ; *Muscle, Smooth, Vascular/cytology ; *Myocytes, Smooth Muscle/cytology/metabolism ; *Cell Culture Techniques/methods ; *CRISPR-Cas Systems/genetics ; Cells, Cultured ; Gene Expression Regulation ; },
abstract = {Directed differentiation of human induced pluripotent stem cells (hiPSCs) holds major promise for the development of disease models, drug screening platforms, and regenerative medicine. Here, we provide a step-by-step highly reproducible protocol for differentiating vascular smooth muscle cells (vSMCs) from hiPSCs, including hiPSC culture, hiPSC differentiation, and vSMC passaging under chemically defined conditions. We also detail molecular and functional analysis procedures for hiPSC-derived contractile vSMCs along with endogenous transcriptional activation modulation ready for any downstream application.},
}
@article {pmid41576437,
year = {2026},
author = {Wei, L and Cheng, Z and Xu, M and Chen, H and Lan, W and Long, W and She, Y and Fu, H},
title = {Bulge DNA-driven CRISPR/Cas12a dynamic activation circuit enables highly sensitive and versatile biosensing.},
journal = {Biosensors &amp; bioelectronics},
volume = {298},
number = {},
pages = {118412},
doi = {10.1016/j.bios.2026.118412},
pmid = {41576437},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Methicillin-Resistant Staphylococcus aureus/isolation &amp; purification/genetics ; Limit of Detection ; *DNA/chemistry/genetics ; *Mycotoxins/analysis/isolation &amp; purification ; Aptamers, Nucleotide/chemistry/genetics ; Bacterial Proteins/genetics/chemistry ; *Endodeoxyribonucleases/chemistry/genetics ; Humans ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as an innovative biosensing tool; however, its intrinsically linear accumulation of cleavage signals limits detection sensitivity. Herein, we report a bulge DNA (BD)-driven CRISPR/Cas12a dynamic activation circuit, termed CBD, as a highly sensitive and versatile biosensing platform for both nucleic acid and non-nucleic acid targets. The BD structure was rationally engineered to undergo programmable structural and functional transformation upon bulge degradation, thereby initiating exponential, self-amplifying activation of the Cas12a circuit. For nucleic acid targets, direct Cas12a recognition triggers BD cleavage and a positive feedback loop, enabling highly sensitive detection with a limit of 14 CFU/mL for methicillin-resistant Staphylococcus aureus. For non-nucleic acid targets, a universal single-stranded DNA activator was linked to the aptamer-complementary strand, enabling target-responsive release and subsequent initiation of the CBD system without altering the crRNA or BD sequence. This strategy enabled the detection of pesticides and mycotoxins at the picogram-per-milliliter level. Furthermore, an "OR" logic gate was constructed for the simultaneous detection of dual mycotoxins, highlighting the platform's capability for multiplexed hazard monitoring. Overall, CBD demonstrates significant potential as a new paradigm for next-generation biosensing technologies.},
}
@article {pmid41576918,
year = {2026},
author = {Pinglay, S and Atwater, JT and Brosh, R and Shendure, J and Maurano, MT and Boeke, JD},
title = {Mammalian genome writing: Unlocking new length scales for genome engineering.},
journal = {Cell},
volume = {189},
number = {2},
pages = {356-374},
pmid = {41576918},
issn = {1097-4172},
support = {DP5 OD036167/OD/NIH HHS/United States ; R01 HG012743/HG/NHGRI NIH HHS/United States ; RM1 HG009491/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Gene Editing/methods ; *Genome ; *Genetic Engineering/methods ; *Mammals/genetics ; CRISPR-Cas Systems ; },
abstract = {The ability to design and engineer mammalian genomes across arbitrary length scales would transform biology and medicine. Such capabilities would enable the systematic dissection of mechanisms governing gene regulation and the influence of complex haplotypes on human traits and disease. They would also facilitate the engineering of disease models that more faithfully recapitulate human physiology and of next-generation cell therapies harboring sophisticated genetic circuits. Over the past decade, advances in genome editing have made small, targeted modifications at single sites routine. However, achieving multiple coordinated alterations across long sequence windows (>10 kb) or installing large synthetic DNA segments in mammalian cells remains a major challenge. Recent advances in mammalian genome writing-the bottom-up design, assembly, and targeted integration of large custom DNA sequences, independent of any natural template-offer a potential solution. Here, we review key technological developments, highlight emerging applications, and discuss current bottlenecks and strategies for overcoming them.},
}
@article {pmid41576935,
year = {2026},
author = {Hommersom, MP and Puvogel, S and Scheefhals, N and Carpentiero, E and Bouma, M and van Beusekom, E and Dillen, L and van de Warrenburg, BPC and Nadif Kasri, N and van Bokhoven, H},
title = {Human neuronal networks on micro-electrode arrays as a tool to assess genotype-phenotype correlation in CACNA1A-related disorders.},
journal = {Stem cell reports},
volume = {21},
number = {2},
pages = {102783},
pmid = {41576935},
issn = {2213-6711},
mesh = {Humans ; *Genetic Association Studies/methods ; *Nerve Net/metabolism ; *Neurons/metabolism ; *Calcium Channels/genetics ; Phenotype ; CRISPR-Cas Systems/genetics ; Electrodes ; Genotype ; Calcium Channels, N-Type ; },
abstract = {CACNA1A-related disorders constitute a diverse group of neurological conditions, including ataxia, migraine, and epilepsy. Despite extensive genetic studies, clear genotype-phenotype correlations remain elusive. Moreover, next-generation sequencing has identified many variants of uncertain significance (VUS). Here, we leveraged patient-derived and CRISPR-Cas9-engineered human neuronal networks to explore relationships between CACNA1A variants and neurophysiological activity. CACNA1A haploinsufficiency induced subtle alterations in glutamatergic network activity, whereas missense variants had a more pronounced effect on overall network function. Network fingerprints were most affected from patients where ataxia co-occurred with migraine or epilepsy. Furthermore, we analyzed the impact of CRISPR-Cas9-induced VUS on network developmental trajectories. Although functional changes could not be directly linked to clinical phenotypes, all tested variants induced measurable alterations in neuronal network function, supporting their classification as likely pathogenic. These findings highlight the potential of human neuronal networks as a translational model for evaluating CACNA1A variant effects and improving clinical variant interpretation.},
}
@article {pmid41577003,
year = {2026},
author = {Zade, NH and Jain, M and Garg, M and Checker, R and Ghosh, A and Khattar, E},
title = {Proteomics method for identifying POT1-associated complexes at telomeres using ChIP-Mass spectrometry.},
journal = {Methods (San Diego, Calif.)},
volume = {247},
number = {},
pages = {161-174},
doi = {10.1016/j.ymeth.2026.01.007},
pmid = {41577003},
issn = {1095-9130},
mesh = {Shelterin Complex ; *Telomere/metabolism/genetics/chemistry ; *Proteomics/methods ; Humans ; *Telomere-Binding Proteins/metabolism/genetics ; Mass Spectrometry/methods ; CRISPR-Cas Systems ; *Chromatin Immunoprecipitation/methods ; Protein Binding ; Gene Editing ; Tripeptidyl-Peptidase 1 ; },
abstract = {POT1 is the only single stranded telomere binding protein in the shelterin complex. Together with TPP1, POT1 plays a crucial role in regulating telomere length and protecting telomeres from DNA damage repair proteins. The activation of DNA damage repair proteins at telomeres can be detrimental to cells, so their activity must be suppressed. POT1 interacts with other telomeric proteins (TRF2, TRF1, TIN2 and RAP1) via its association with TPP1. These proteins function together to protect and maintain the telomeres. Despite extensive knowledge of POT1's role within the shelterin complex, the full spectrum of its interactors at the single-stranded telomeric overhang remains poorly defined. To study these interactions, we generated an endogenous Flag-tag knock-in of POT1 using the CRISPR-Cas9 gene editing system. To address the risk of unintended gene disruption associated with this technique, we conducted an in-depth characterization of the endogenously Flag-tagged POT1 clone to ensure that its telomere and TPP1 binding functions remained intact. Further, we performed proteomic profiling of the Flag-tagged POT1 within the chromatin fraction using ChIP-MS to explore its proteome. Our analysis uncovered a novel set of POT1-associated proteins at the extremes of telomeres. Given that POT1 exclusively binds to the single-stranded 3' overhang of telomeres, the proteomic data obtained indicates POT1 interactions occurring at the extreme ends of telomeres. In conclusion, our study reveals previously uncharacterized POT1 associated proteins using ChIP mass spectrometric approach, paving the way for further investigations into telomere biology and potential therapies targeting telomere regulation.},
}
@article {pmid41577144,
year = {2026},
author = {Mocchetti, A and Steelant, P and Hosseinkhani, M and De Rouck, S and Khajehali, J and Van Leeuwen, T},
title = {Knockout of nAChR subunits in spider mites and their phytoseiid predators confers spinosyn cross-resistance and reveals a conserved mode of action in mites.},
journal = {Insect biochemistry and molecular biology},
volume = {189},
number = {},
pages = {104498},
pmid = {41577144},
issn = {1879-0240},
mesh = {Animals ; *Receptors, Nicotinic/genetics/metabolism ; *Tetranychidae/genetics/drug effects/metabolism ; *Macrolides/pharmacology ; Drug Combinations ; Phylogeny ; *Insecticide Resistance/genetics ; *Acaricides/pharmacology ; },
abstract = {Spinosyns are allosteric modulators of nicotinic acetylcholine receptors (nAChRs) which in insects specifically target subunit α6. However, their mode of action in mites and compatibility with phytoseiid predators remain unclear. We combined phylogenetics with CRISPR/Cas-based reverse genetics to test whether α6-like subunits mediate spinosyn toxicity in mites and to assess prospects for resistance breeding in phytoseiids. The phylogenetic analysis identified seven α and three β subunits in multiple phytoseiids and in Tetranychus urticae. A single phytoseiid subunit clustered within the insect α6/α7 clade, whereas T. urticae possessed three (Tuα5/α6/α7) without strict one-to-one insect orthology. Using SYNCAS maternal delivery of CRISPR RNPs, we disrupted the putative α6 ortholog in Amblyseius swirskii (Asα6) and each of the three α6/α7-clade genes in T. urticae. In A. swirskii, all survivors of a discriminating spinosad dose carried Asα6 indels, and three independently edited lines exhibited insensitivity to both spinosad and spinetoram (no significant mortality at 10 000 mg a.i./L), whereas the wild type showed LC50 = 163 mg/L (spinosad) and 54 mg/L (spinetoram). In T. urticae, Tuα6 knockouts conferred high cross-resistance to both compounds, while Tuα5 knockouts slightly increased susceptibility and Tuα7 knockouts produced modest resistance. Our data demonstrate that α6-mediated spinosyn action is conserved in mites, with α6 loss conferring strong cross-resistance in a key phytoseiid predator and in a model tetranychid. These findings enable marker-assisted editing/selection of spinosyn-resistant phytoseiid strains to improve pesticide-biocontrol compatibility and establish α6 as a practical universal marker gene for genome editing in acarine systems.},
}
@article {pmid41577322,
year = {2026},
author = {Wang, L and Miao, M and Bao, L and Chu, J and Zhou, J and Song, W and Cai, P and Cheng, C and Xu, H and Wang, T and Zhao, R and Wang, H and Liu, F and Xu, M and Tian, G},
title = {CRISPR/Cas9-based genome-wide screen reveals a synergistic effect of Irinotecan and USP1 inhibitor in colorectal cancer.},
journal = {European journal of pharmacology},
volume = {1015},
number = {},
pages = {178558},
doi = {10.1016/j.ejphar.2026.178558},
pmid = {41577322},
issn = {1879-0712},
mesh = {Humans ; *Irinotecan/pharmacology/therapeutic use ; *Colorectal Neoplasms/drug therapy/genetics/pathology ; Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Ubiquitin-Specific Proteases/antagonists &amp; inhibitors/metabolism ; Drug Synergism ; Cell Proliferation/drug effects ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; Drug Resistance, Neoplasm/drug effects ; HCT116 Cells ; Gene Expression Regulation, Neoplastic/drug effects ; Antineoplastic Combined Chemotherapy Protocols/pharmacology ; },
abstract = {Irinotecan resistance remains a significant challenge in metastatic colorectal cancer (mCRC) therapy. To address this, we identified USP1 as a synthetic lethal partner of Irinotecan through genome-wide CRISPR/Cas9 screening in HCT-116 cells. Combining the USP1 inhibitor I-138 with Irinotecan in HCT-116, HT-29, and SW620 cell lines significantly reduced IC50, suppressed proliferation, and diminished colony formation compared to monotherapy, demonstrating a synergistic effect (combination index CI < 1). The synergistic therapeutic efficacy was further validated in the xenograft mouse model. Mechanistic studies revealed that I-138 significantly upregulated pCREB (Ser133), concurrently dynamically regulating the activity of USP1, FANCD2/FANCI, and PCNA upon DNA damage response and repair. RNA sequencing further highlighted the enrichment of cAMP, PI3K-AKT, and Wnt pathways, which are all linked to CREB activity in the combination group. These findings establish USP1 inhibition as a promising strategy to overcome Irinotecan resistance through the combination strategy, providing a novel therapeutic avenue for CRC.},
}
@article {pmid41577687,
year = {2026},
author = {Montagud-Martínez, R and Ruiz, R and Baldanta, S and Delicado-Mateo, R and Rodrigo, G},
title = {CRISPR-Cas9 trans-cleavage is hindered by a flanked R-loop, an elongated spacer, and an inactive HNH domain.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41577687},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; *R-Loop Structures ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Protein Domains ; DNA, Single-Stranded/metabolism/genetics ; Models, Molecular ; DNA/metabolism ; },
abstract = {Cas9 can process poly(T) single-stranded DNA molecules upon activation in an RNA-guided manner. Here, we uncover key determinants underlying this function. First, we show that unflanked R-loops in the RNA 5' side favor trans-cleavage activity, which occur when targeting short double-stranded DNA molecules. Second, we show that elongated guide RNA spacers beyond the canonical 20 bases, even by a few bases, severely impair this collateral activity. Third, although trans-cleavage is mediated by the RuvC domain, we show that a catalytically active HNH domain contributes to an efficient process. Analysis of structural models provides tentative mechanistic insights. Together, these findings illustrate that fine modulation of Cas9 function can be achieved.},
}
@article {pmid41577693,
year = {2026},
author = {Shankar, A and Olender, L and Hsu, I and Miyauchi, M and Pálovics, R and Meaker, GA and Kaito, S and Rizq, O and Khoo, HM and Bozhilov, Y and Igarashi, KJ and Bhadury, J and Munson, C and Mack, PK and Tan, TK and Rehwinkel, J and Iwama, A and Wyss-Coray, T and Nakauchi, H and Haney, MS and Wilkinson, AC},
title = {In vivo CRISPR screening identifies SAGA complex members as key regulators of hematopoiesis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1756},
pmid = {41577693},
issn = {2041-1723},
support = {KKL1378//Kay Kendall Leukaemia Fund (KKLF)/ ; 302479Z23Z//Wellcome Trust (Wellcome)/ ; K99HL150218//U.S. Department of Health &amp; Human Services | NIH | National Heart, Lung, and Blood Institute (NHLBI)/ ; 3385-19//Leukemia and Lymphoma Society (Leukemia &amp; Lymphoma Society)/ ; RG-202211-02958//European Hematology Association (EHA)/ ; },
mesh = {*Hematopoiesis/genetics ; Humans ; Hematopoietic Stem Cells/metabolism/cytology ; Animals ; Mice ; Histones/metabolism ; Acetylation ; Ubiquitination ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Mice, Inbred C57BL ; },
abstract = {The biological mechanisms that sustain the vast blood production required for healthy life remain incompletely understood. To search for cell intrinsic regulators of hematopoiesis, we perform a genome-wide in vivo hematopoietic stem and progenitor cell (HSPC)-based CRISPR knockout screen. We discover SAGA complex members, including Tada2b and Taf5l, as key regulators of hematopoiesis. Loss of Tada2b or Taf5l strongly inhibits hematopoiesis in vivo, causing a buildup of immature hematopoietic cells in the bone marrow. The SAGA complex deposits histone H3 lysine 9 acetylation (H3K9ac) and removes histone H2B ubiquitination (H2Bub). Loss of Tada2b leads to a reduction in H3K9ac levels and altered H2Bub enrichment in HSPCs, implicating disruption of SAGA complex activity. This is associated with upregulation of interferon pathway genes, reduced mitochondrial activity, and increased megakaryocyte progenitor cell commitment. Loss of these factors also enhances the cell outgrowth and the interferon pathway in an in vivo human myelodysplastic syndrome cell line model. In summary, this study identifies the SAGA complex as an important regulator of hematopoiesis.},
}
@article {pmid41578087,
year = {2026},
author = {Gautam, B and Jarvis, BA and Esfahanian, M and McGinn, M and Williams, D and Liu, S and Phippen, ME and Heller, NJ and Wesley, TL and Phippen, WB and Ulmasov, T and Marks, MD and Chopra, R and Sedbrook, JC},
title = {Creating a new oilseed crop, pennycress, by combining key domestication traits using CRISPR genome editing.},
journal = {Nature plants},
volume = {12},
number = {1},
pages = {74-87},
pmid = {41578087},
issn = {2055-0278},
support = {2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2018-67009-27374//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; 2019-69012-29851//United States Department of Agriculture | National Institute of Food and Agriculture (NIFA)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; DE-SC0021286//DOE | SC | Biological and Environmental Research (BER)/ ; },
mesh = {*Gene Editing ; *Domestication ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Seeds/genetics ; *Brassica/genetics ; },
abstract = {Considerable off-season farmland lies fallow because few crops can profitably fit between primary crops. As a remedy, we performed de novo domestication of the freeze-tolerant, rapid-cycling wild brassica Thlaspi arvense L. (field pennycress), identifying and stacking CRISPR-Cas9-induced mutations that have minimal impacts on seed yields. High-yielding varieties were created with seed compositions such as 'double-low' canola (low erucic acid and reduced glucosinolate) and reduced seed fibre content. Seed glucosinolate content was reduced by 75% by combining mutations in R2R3-MYB (MYB28/HAG1) and basic helix-loop-helix MYC (MYC3) transcription factors. Pennycress weediness was greatly reduced by knockout of the basic helix-loop-helix transcription factor TRANSPARENT TESTA8 (TT8), which lowered seed dormancy and seed coat protections, thereby mitigating re-emergence in fields. Domesticated pennycress offers farmers a low-carbon-intensity intermediate crop that fits between two full-season summer crops, resulting in three cash crops in 2 years, conferring cover-crop-like ecosystem benefits while producing grain for renewable fuels and enhanced food security.},
}
@article {pmid41578091,
year = {2026},
author = {Torres-Higuera, LD and Rojas-Tapias, DF and Jiménez-Velásquez, S and Renjifo-Ibáñez, C},
title = {Comprehensive genotyping and taxonomic analysis uncovers extensive distribution of intermediate Leptospira species in Colombia.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {2},
pages = {57},
pmid = {41578091},
issn = {1573-0972},
mesh = {Colombia/epidemiology ; *Leptospira/genetics/classification/isolation &amp; purification ; Phylogeny ; *Leptospirosis/microbiology/epidemiology/veterinary ; Humans ; Animals ; RNA, Ribosomal, 16S/genetics ; Genotype ; Genetic Variation ; DNA, Bacterial/genetics ; Multilocus Sequence Typing ; Whole Genome Sequencing ; Genome, Bacterial ; Ribotyping ; DNA-Directed RNA Polymerases/genetics ; Virulence Factors/genetics ; Minisatellite Repeats ; },
abstract = {Leptospirosis, a globally prevalent zoonosis caused by pathogenic and intermediate Leptospira species, poses significant threats to public health and livestock industries. Despite its substantial impact, knowledge gaps persist regarding the prevalence and genetic diversity of Leptospira strains in many regions, including South America. This study aimed to characterize a diverse collection of Leptospira strains isolated from various sources in Colombia to enhance our understanding of the genetic diversity within this genus. Using a tiered approach combining conventional and genomic methods, we genotyped 55 isolates from various sources using 16S rRNA and rpoB gene sequencing, DNA ribotyping, and Multiple-Locus Variable-Number Tandem Repeat Analysis (MLVA). Most isolates were classified into phylogenetic groups containing pathogenic and intermediate strains of L. interrogans and L. wolffii, respectively, which was corroborated by ribotyping and MLVA. Whole-genome sequencing of selected strains revealed distinct genomic characteristics compared to related strains. Pan-genome analysis identified strain-specific genes, primarily hypothetical, while virulence factor analysis distinguished species-specific patterns. Furthermore, CRISPR-Cas system analysis uncovered genetic variations among the isolates. This study provides a framework for understanding Leptospira genetic diversity in Colombia and its potential implications on human and animal health. Our findings highlight the need for improved diagnostic methods and surveillance strategies that encompass both pathogenic and intermediate Leptospira species, which could significantly impact public health policies and veterinary practices in the region.},
}
@article {pmid41578173,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M and Panahi, B},
title = {Genomic landscape of biosynthetic gene clusters in Iranian extremophiles reveals prolific metabolite potential, prophage associations, and integrated defensive-metabolic islands.},
journal = {BMC microbiology},
volume = {26},
number = {1},
pages = {},
pmid = {41578173},
issn = {1471-2180},
mesh = {*Prophages/genetics ; *Multigene Family ; Iran ; *Extremophiles/genetics/metabolism/virology ; Genome, Bacterial ; *Bacteria/genetics/metabolism/virology/isolation &amp; purification/classification ; Soil Microbiology ; *Genomic Islands ; *Biosynthetic Pathways/genetics ; Secondary Metabolism/genetics ; Phylogeny ; Bacteriocins/genetics ; Genomics ; },
abstract = {The extreme and underexplored ecosystems of Iran represent a significant reservoir of microbial diversity with profound biosynthetic potential. To systematically investigate this resource, we employed a comprehensive genome mining approach on 16 bacterial isolates from hypersaline, desert, and petroleum-contaminated soils. Our analysis revealed an extraordinary density and complexity of biosynthetic gene clusters (BGCs), identifying 229 BGCs in total. A substantial majority (56.8%) showed no significant similarity to known clusters, underscoring the extensive novelty encoded within these extremophiles. Notably, we discovered highly intricate "trio" and "quartet" hybrid BGCs, which encode the machinery for three or four distinct classes of secondary metabolites, pushing the boundaries of known biosynthetic complexity. Parallel analysis identified six novel, high-quality prophages, largely uncharacterized in public databases. These prophages were found to carry a putative bacteriocin cluster (UviB) indicating a direct role in enhancing host fitness. Furthermore, we uncovered a dynamic co-evolutionary arms race, with bacterial genomes fortified by diverse defense systems, including abundant CRISPR-Cas arrays, and prophages encoding a repertoire of counter-defense anti-CRISPR proteins. Genomic architecture analysis revealed widespread co-localization of BGCs, prophages, and defense systems into functional genomic islands, suggesting a synergistic linkage between secondary metabolism and phage resistance. This study illuminates the remarkable biosynthetic and defensive landscape of Iranian extremophiles, highlighting them as a premier resource for discovering novel natural products and understanding virus-host evolutionary dynamics.},
}
@article {pmid41578383,
year = {2026},
author = {Tao, T and Lin, L and Tang, Y and Liu, Z and Liu, Y and Xie, Y and Hu, X and Wang, J and Wang, T and Zhang, GF and Wang, Y and Zhu, S},
title = {Generation of an isogenic human induced pluripotent stem cell line with a mutant propionyl-CoA carboxylase α subunit.},
journal = {Orphanet journal of rare diseases},
volume = {21},
number = {1},
pages = {61},
pmid = {41578383},
issn = {1750-1172},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Methylmalonyl-CoA Decarboxylase/genetics/metabolism ; Myocytes, Cardiac/metabolism ; Cell Differentiation ; Propionic Acidemia/genetics ; Mutation/genetics ; Cell Line ; CRISPR-Cas Systems ; Gene Editing ; Propionyl-Coenzyme A Carboxylase ; },
abstract = {BACKGROUND: Propionic acidemia (PA) is a rare autosomal recessive metabolic disorder caused by defects in propionyl-CoA carboxylase (PCC), a mitochondrial enzyme composed of six alpha (PCCA) and six beta (PCCB) subunits. Mutations in PCCA/PCCB genes disrupt PCC function, leading to toxic metabolite accumulation and clinical manifestations. Current research is limited by inadequate patient-derived cellular models and ethical constraints in sample acquisition.<br><br>METHOD: Using CRISPR/Cas9-mediated gene editing, we established an isogenic human induced pluripotent stem cell (iPSC) line carrying the PCCA c.2002G>&#x2009;A mutation. The mutant iPSCs were further subjected to directed cardiac differentiation. Characteristic metabolites in the iPSC-derived cardiomyocytes (iPSC-CMs) culture medium were analyzed via untargeted metabolomics, and contractile function was assessed by video-based motion analysis under propionate challenge.<br><br>RESULTS: The mutant iPSCs showed sustained expression of pluripotency markers (OCT4, NANOG, SOX-2), maintained normal karyotype (46, XY), and retained trilineage differentiation capacity. Functional characterization demonstrated significantly reduced PCC enzyme activity, accurately modeling PA metabolic pathology. Furthermore, the mutant iPSCs successfully differentiated into cardiomyocytes and exhibited a PA-specific metabolic profile, including significantly elevated propionylcarnitine levels. Upon propionate treatment (2.5&#x2009;mM), the contractile function of mutant iPSC-CMs was significantly impaired, whereas wild-type iPSC-CMs showed the opposite response with enhanced contraction.<br><br>CONCLUSIONS: This isogenic iPSC line provides an ethically unconstrained platform to investigate PA molecular mechanisms and genotype-phenotype relationships. The model enables systematic drug screening and therapeutic development while overcoming patient sample limitations.},
}
@article {pmid41578398,
year = {2026},
author = {Ferreira de Vasconcellos, J and Friedman, L and Satapathy, I and Cubbage, N and Palmer, J and Majumder, S and Kono, M},
title = {A genome-wide CRISPR/Cas9 screen reveals novel positive regulators of FTY720 sensitivity in acute lymphoblastic leukemia cells.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {75},
pmid = {41578398},
issn = {1756-0500},
mesh = {Humans ; *Fingolimod Hydrochloride/pharmacology ; *CRISPR-Cas Systems/genetics ; *Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics/drug therapy ; Cell Line, Tumor ; },
abstract = {OBJECTIVE: Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer. Fingolimod (FTY720) is a sphingosine-1-phosphate (S1P) receptor agonist that prevents lymphocytes from egressing from lymphoid tissues and has shown a cytotoxic effect on T-cell ALL (T-ALL) cells. However, the mechanism of action of FTY720 cytotoxicity in hematological malignancies is still unclear, and cell-specific effects have been reported. Here, we investigated the mechanism of cytotoxicity of FTY720 in T-ALL cells using a CRISPR-Cas9 genomic screening. Our goal was to identify novel positive regulators for the cytotoxic effect of FTY720 in T-ALL.<br><br>RESULTS: Cells treated with FTY720 were enriched for single-guide RNAs (sgRNAs) such as ZNF575, GPX3, FBXL15, DNAJB5, UBE2D1, ATXN7, C6orf201, RIC8A, RAB13, and C10orf12 when compared to the DMSO (vehicle control) samples. Altogether, our study identified novel genes that, when silenced, were positively correlated with the survival of T-ALL cells treated with FTY720.},
}
@article {pmid41579593,
year = {2026},
author = {Jovanovic, VM and Rausch, R and DeRosa, MC and Castellano, D and McKee, C and Sen, C and Daly, F and Doege, CA and Tristan, CA},
title = {Generation and characterization of POMC-tdTomato reporter human pluripotent stem cell lines.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103905},
pmid = {41579593},
issn = {1876-7753},
support = {P30 DK026687/DK/NIDDK NIH HHS/United States ; R01 DK135938/DK/NIDDK NIH HHS/United States ; },
mesh = {Humans ; *Pro-Opiomelanocortin/metabolism/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Neurons/metabolism/cytology ; Cell Line ; Cell Differentiation ; Genes, Reporter ; CRISPR-Cas Systems ; Red Fluorescent Protein ; },
abstract = {Proopiomelanocortin (POMC) is a precursor polypeptide that undergoes extensive, tissue-specific post-translational processing. It is expressed in several tissues, including pituitary gland, hypothalamus, brain stem, and skin. The hypothalamic POMC neurons in the arcuate nucleus are major neuronal populations involved in the regulation of body weight. In these neurons, POMC is processed into several peptides, among them the anorexigenic alpha-melanocyte stimulating hormone. Thus, the POMC neurons in the ARC have been named "satiety" neurons and are highly desirable drug targets. Here, we performed CRISPR/Cas9-mediated insertion of tdTomato reporter at the endogenous POMC locus, enabling direct visualization of POMC expression through tdTomato fluorescence in human pluripotent stem cell (hPSC)-derived hypothalamic neurons. This reporter line enables real-time visualization of POMC neuron differentiation, and selective enrichment of these populations for molecular, functional, and pharmacological studies. This line is readily available as new alternative method (NAM) platform, to support disease modeling and drug discovery in metabolic and neuroendocrine disorders within a human context.},
}
@article {pmid41579807,
year = {2026},
author = {Zhang, Y and Wu, M and Geng, F and Wang, Y and Lü, J},
title = {Deciphering functional redundancy of lysine decarboxylases in probiotic E. coli Nissle 1917 via an integrated CRISPR-FTIR phenomics platform.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {351},
number = {},
pages = {127508},
doi = {10.1016/j.saa.2026.127508},
pmid = {41579807},
issn = {1873-3557},
mesh = {*Escherichia coli/enzymology/genetics ; Spectroscopy, Fourier Transform Infrared/methods ; *CRISPR-Cas Systems ; *Probiotics/metabolism ; *Carboxy-Lyases/metabolism/genetics ; Mutation ; *Escherichia coli Proteins/metabolism/genetics ; Phenotype ; },
abstract = {Fourier transform infrared (FTIR) microspectroscopy is a rapid, label-free tool for microbial metabolic phenotyping. Here, we integrate synchrotron-based FTIR microspectroscopy with CRISPR-Cas9 editing to decipher the functional redundancy of lysine decarboxylases (LdcC1 and LdcC2) in probiotic Escherichia coli Nissle 1917. Under lysine stress, isogenic mutants (ΔldcC1, ΔldcC1ΔldcC2) exhibited distinct FTIR fingerprints. Spectral analysis revealed: (i) CH shifts (2950-2850 cm[-1]) indicating ΔldcC1-specific membrane remodeling; (ii) Amide I band profile alterations (∼1650 cm[-1]) suggesting protein structural perturbations; and (iii) a constitutive elevation in 1220-1260 cm[-1] band area in the double mutant, revealing a basal state of metabolic frailty. Principal component analysis of second-derivative spectra revealed clear separation trends among strain phenotypes. We establish LdcC2 as a crucial functional complement, while LdcC1 uniquely contributes to membrane homeostasis. The compensatory stress response activated in the double mutant underscores metabolic redundancy as a cornerstone of intrinsic cellular robustness. Collectively, this work validates a CRISPR-FTIR phenomics platform that bridges targeted genetics with global biochemistry, offering a rapid alternative for functional genomics and metabolic engineering in microbes.},
}
@article {pmid41581366,
year = {2026},
author = {Yu, Y and Zhang, J and Xie, G and Lin, Y and Huang, Y and Rao, C and Hou, Y and Chen, H},
title = {A DNAzyme-CRISPR cascade strategy for preamplification-free detection of Mycobacterium tuberculosis.},
journal = {Biosensors &amp; bioelectronics},
volume = {298},
number = {},
pages = {118428},
doi = {10.1016/j.bios.2026.118428},
pmid = {41581366},
issn = {1873-4235},
mesh = {*Mycobacterium tuberculosis/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *DNA, Catalytic/chemistry/genetics ; *Tuberculosis/diagnosis/microbiology ; Humans ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; *DNA, Bacterial/genetics/isolation &amp; purification ; Nucleic Acid Amplification Techniques ; Reproducibility of Results ; },
abstract = {Tuberculosis (TB) remains one of the most fatal infectious diseases worldwide, successful treatment is often limited by insufficient diagnostic capabilities. It creates a pressing need for diagnostic methods that combine high sensitivity, specificity, and operational robustness. In this work, we developed a DNAzyme-CRISPR cascade strategy (Dz-CRISPR) for the specific identification of the IS6110 sequence of Mycobacterium tuberculosis (MTB). This system integrated a thermodynamically stabilized hairpin probe, an Arch-shaped signal transduction switch, and an allosterically activated CRISPR-Cas12a cascade. The design enabled direct target recognition and subsequent signal amplification without a preamplification step, offering a simplified workflow with enhanced stability. The assay demonstrated a detection limit of 211.3 fM and exhibited high specificity by accurately discriminating the IS6110 from specific DNA sequence of non-tuberculous mycobacteria and other common respiratory pathogens. Validation using clinical bronchoalveolar lavage fluid samples further confirmed the method's reliable performance, reproducibility, and satisfactory recovery rates. Current Dz-CRISPR detection strategy provides a reliable and practical solution for tuberculosis diagnosis with high sensitivity, high specificity, and operational robustness, thus demonstrating potential for practical use in resource-constrained areas.},
}
@article {pmid41581373,
year = {2026},
author = {Akla, N and Boudah, A and Bertomeu, T and Chatr-Aryamontri, A and Desjarlais, M and Annabi, B},
title = {CRISPR-based chemogenomic profiling reveals redox vulnerabilities to epigallocatechin-3-gallate and green tea polyphenol extract.},
journal = {Redox biology},
volume = {90},
number = {},
pages = {104047},
pmid = {41581373},
issn = {2213-2317},
mesh = {*Catechin/analogs &amp; derivatives/pharmacology ; Humans ; Oxidation-Reduction/drug effects ; *Tea/chemistry ; *CRISPR-Cas Systems ; *Polyphenols/pharmacology ; *Plant Extracts/pharmacology/chemistry ; Oxidative Stress/drug effects ; Antioxidants/pharmacology ; Cell Line, Tumor ; },
abstract = {Green tea polyphenols, particularly epigallocatechin-3-gallate (EGCG), are widely recognized for their beneficial preventive effects against chronic diseases including cancer and obesity. These effects are traditionally attributed to EGCG's antioxidant, anti-inflammatory, and metabolic regulatory properties. In conditions characterized by persistent oxidative stress, the disrupted redox signaling further creates a unique vulnerability that EGCG may exploit through a dual redox mechanism. Emerging evidence therefore suggests that EGCG not only mitigates oxidative damage but could also induce selective pro-oxidant stress in cancer cells, enhancing its therapeutic potential. To investigate this duality, we performed a genome-wide CRISPR/Cas9 knockout screen to identify genetic determinants of EGCG sensitivity and resistance. Our chemogenomic analysis revealed that loss of key antioxidant genes, including PRDX1, CAT, GSS, GCLM, and GCLC, significantly heightened cellular susceptibility to EGCG and green tea extract (GTE), underscoring the critical role of glutathione biosynthesis and redox homeostasis in mediating cytotoxicity. In contrast, knockouts of Kelch-like ECH-associated Protein 1 (KEAP1) and peroxisome-associated PEX genes conferred resistance, implicating in part NRF2 (also known as nuclear factor erythroid-derived 2-like 2; NFE2L2) activation and peroxisomal reactive oxygen species clearance in protective responses. Comparative profiling with gallic acid (GA), which lacks EGCG's catechin structure, further highlighted the gallate moiety's contribution to glutathione-dependent antioxidant mechanisms. Altogether, these findings illuminate the complex redox biology of EGCG and identify novel genetic vulnerabilities that may be leveraged to enhance its anticancer efficacy, particularly in obesity-associated cancers. Clinically, this work could support the development of EGCG-based interventions tailored to individual redox profiles, offering a precise chemopreventive strategy for patients at high risk of malignancies driven by metabolic and oxidative dysregulation. Furthermore, the identification of new genetic markers of EGCG sensitivity and resistance may inform future exploration of patient stratification.},
}
@article {pmid41581816,
year = {2026},
author = {Xiao, Y and Yang, J and Yang, W and Yuan, M and Zhang, Y and Liu, J and Zhang, Y and Zhu, H and Luo, G},
title = {A rapid on-site diagnostic method for goose parvovirus disease based on recombinase polymerase amplification and CRISPR/AsCas12a.},
journal = {International journal of biological macromolecules},
volume = {343},
number = {Pt 2},
pages = {150496},
doi = {10.1016/j.ijbiomac.2026.150496},
pmid = {41581816},
issn = {1879-0003},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Geese/virology ; *Parvoviridae Infections/diagnosis/veterinary/virology ; *Parvovirus/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; *Poultry Diseases/diagnosis/virology ; Ducks/virology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Goose parvovirus (GPV) is a highly pathogenic and lethal virus responsible for Derzsy's disease in goslings and ducklings, significantly influencing the economic viability of waterfowl farming. This necessitates the development of rapid diagnostic techniques for effective disease management. In this study, an optimized clustered regularly interspaced short palindromic repeats (CRISPR)/Acidaminococcus sp. CRISPR associated nuclease 12a (AsCas12a) system was developed for the diagnosis of GPV. The study determined that the optimal conditions for the CRISPR/Cas12a-based fluorescence assay were 20 nM AsCas12a, 5 nM crRNA, and 5 nM single-stranded DNA (ssDNA), whereas the lateral flow assay (LFA) required 20 nM AsCas12a and 4 nM crRNA. Moreover, the fluorescence-based assay and LFA achieved minimum detection limits of 7.8 copies/μL and 78 copies/μL, respectively, representing 1000-fold and 100-fold improvements over conventional PCR methods. Both detection methods exhibited high specificity and demonstrated no cross-reactivity with other prevalent waterfowl pathogens, such as duck plague virus, duck hepatitis viruses, H5 avian influenza virus, waterfowl astrovirus, reovirus, Muscovy duck parvovirus, and novel GPV. The results of the LFA were in complete concordance with laboratory qPCR analyses, thereby affirming their reliability for clinical diagnostics. In conclusion, we have successfully developed a dual-readout GPV detection system utilizing CRISPR/Cas12a technology, which holds significant promise for the early surveillance and containment of GPV outbreaks.},
}
@article {pmid41581881,
year = {2026},
author = {Zhao, AH and Koganti, PP and Qian, M and Garcia, A and O'Day, P and Auchus, RJ and Covey, DF and Selvaraj, V},
title = {Target validation uncouples mitochondrial translocator protein from 19-Atriol-mediated inhibition of steroidogenesis and identifies enzymatic targets.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {3},
pages = {111191},
pmid = {41581881},
issn = {1083-351X},
mesh = {*Receptors, GABA/metabolism/genetics ; Animals ; *Leydig Cells/metabolism/drug effects/cytology ; Mice ; Male ; *Androstenes/pharmacology ; Progesterone/biosynthesis ; *3-Hydroxysteroid Dehydrogenases/metabolism/antagonists &amp; inhibitors/genetics ; Pregnenolone ; Mitochondria/metabolism ; Cell Line ; *Steroids/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {The mitochondrial translocator protein (TSPO) was once proposed to mediate mitochondrial cholesterol import for steroid hormone biosynthesis, but genetic deletion studies in multiple models have refuted this role. Nevertheless, the idea that pharmacological ligands of TSPO can modulate steroid output continues to be invoked. One such compound, 19-Atriol (androst-5-ene-3β,17β,19-triol), was reported to inhibit progesterone synthesis via TSPO binding in MA-10 Leydig cells. To evaluate this proposed mechanism, we used CRISPR/Cas9-generated Tspo-deleted MA-10 cells to study 19-Atriol activity. We found that 19-Atriol inhibited Bt2-cAMP-stimulated steroid output independent of TSPO expression; it acted as a competitive inhibitor of 3β-hydroxysteroid dehydrogenase (3β-HSD), blocking the conversion of pregnenolone to progesterone. Mass spectrometry revealed that 19-Atriol is also a substrate for 3β-HSD, yielding 19-hydroxytestosterone (19-OHT), which itself inhibits 3β-HSD activity. In addition to this effect, both 19-Atriol and 19-OHT decreased cholesterol-to-pregnenolone conversion during stimulation. Partial inhibition of 22R-hydroxycholesterol metabolism by CYP11A1 was observed with 19-Atriol, but not 19-OHT, suggesting direct or indirect effects on this upstream step, potentially involving the steroidogenic acute regulatory protein (STAR). These findings decisively exclude TSPO as a functional mediator of 19-Atriol activity and instead identify direct enzymatic targets within the de novo steroidogenic pathway. By resolving a key mechanistic misattribution, this study underscores the importance of rigorous target validation, particularly for compounds previously assumed to act via TSPO.},
}
@article {pmid41581986,
year = {2026},
author = {Dhariwal, R and Jain, M},
title = {Cell-free systems for low-cost diagnostics.},
journal = {Progress in molecular biology and translational science},
volume = {218},
number = {},
pages = {157-185},
doi = {10.1016/bs.pmbts.2025.08.005},
pmid = {41581986},
issn = {1878-0814},
mesh = {Humans ; Biosensing Techniques ; *Cell-Free System ; Point-of-Care Systems ; Diagnosis ; },
abstract = {Cell-free systems have also become a revolutionary platform for low-cost diagnostics, providing fast, flexible, and scalable solutions to the conventional cell-based assays. Such systems, which utilize the fundamental biochemical machinery of cells without the intricacies of living organisms, have been of great use in point-of-care (POC) diagnostics, particularly in resource-poor environments. This chapter offers a broad overview of the basic principles, design approaches, and technological breakthroughs behind cell-free diagnostic development. It discusses the biochemical underpinnings of cell-free expression, such as ribosomal function, transcriptional control, and energy regeneration, with emphases on the leading platforms including E. coli lysates, wheat germ extracts, and PURE systems. The application of synthetic biology in the form of gene circuits, CRISPR-Cas tools, and RNA aptamers is presented here in the framework of improving the sensitivity and specificity of diagnostics. The chapter further discusses recent innovations in paper-based assays, microfluidic biosensors, and wearable biosensors, which are capable of offering real-time and field-deployable diagnostics. Major challenges in the form of reagent stability, scalability, and regulatory implications are analyzed carefully along with recent trends such as AI-based system design and personalization of diagnostics. In extensive case studies, the chapter highlights the promise of cell-free systems in filling diagnostic gaps, enhancing access to healthcare, and revolutionizing global health. This book strives to offer an encyclopedic sourcebook for researchers, clinicians, and innovators interested in bringing cell-free diagnostics forward.},
}
@article {pmid41582136,
year = {2026},
author = {Guo, S and Zhao, S and Tang, S and Leng, H and Wu, Y and Li, W and Xing, S and Feng, Y and Zhang, Y},
title = {Establishment of a CRISPR/Cas12a/13a-driven dual-detection platform for rapid diagnosis of swine influenza virus and porcine reproductive and respiratory syndrome virus infection.},
journal = {Virology journal},
volume = {23},
number = {1},
pages = {},
pmid = {41582136},
issn = {1743-422X},
support = {2021YFD1800200//National Key Research and Development Program of China/ ; 32170539//National Natural Science Foundation of China/ ; },
mesh = {Animals ; Swine ; *Porcine respiratory and reproductive syndrome virus/isolation &amp; purification/genetics ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Porcine Reproductive and Respiratory Syndrome/diagnosis/virology ; Sensitivity and Specificity ; *Orthomyxoviridae Infections/diagnosis/veterinary/virology ; *Swine Diseases/diagnosis/virology ; Coinfection/diagnosis/virology/veterinary ; },
abstract = {BACKGROUND: Swine influenza virus (SIV) and porcine reproductive and respiratory syndrome virus (PRRSV) are leading pathogens in pigs, whose co-infections exacerbate disease severity. Current diagnostics like RT-PCR lack suitability for rapid, on-site use, while CRISPR-based systems face challenges in convenient multiplex detection.<br><br>RESULTS: We developed an RT-LAMP-CRISPR-Cas12a/13a-LFD dual-detection platform that integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the orthogonal trans-cleavage activities of CRISPR-Cas12a and Cas13a, followed by lateral flow dipstick (LFD) visualization. This assay achieved detection limits of 5 copies/&#xb5;L for SIV and 2 copies/&#xb5;L for PRRSV, and exhibited high specificity against other common swine pathogens. The entire process, including a 20-minute amplification at 40&#xa0;&#xb0;C and 5-minute LFD readout, enables rapid and visual diagnosis. A preliminary validation was conducted using respiratory infection samples, demonstrating high concordance with reference methods and specificity against non-target pathogens.<br><br>CONCLUSIONS: The RT-LAMP-CRISPR-Cas12a/13a-LFD assay provides a sensitive, specific, and potentially field-adaptable tool for the simultaneous detection of SIV and PRRSV. It is ideally suited for early screening and precise control of these pathogens in resource-limited settings.},
}
@article {pmid41582525,
year = {2026},
author = {Ding, S and Li, Y and Wang, F and Liu, Q and Liu, L and Li, J and Wu, Y and Liu, X and Jiang, D and Xu, X and Gao, X and Huang, C},
title = {DNA Flap-Controlled CRISPR/Cas12a Trans-Cleavage Enables Mix-and-Read FEN1 Activity Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {3998-4007},
doi = {10.1021/acs.analchem.5c06447},
pmid = {41582525},
issn = {1520-6882},
mesh = {*Flap Endonucleases/metabolism/analysis ; Humans ; *CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a has emerged as a powerful biosensing tool, owing to its exceptional specificity and trans-cleavage-mediated signal amplification capability, for detecting flap endonuclease 1 (FEN1) activity. However, its reliance on additional preligation or replication steps increases operational complexity and raises the risk of false signals. Herein, we report a DNA flap-controlled CRISPR/Cas12a trans-cleavage (FCT-CRISPR) strategy that enables sensitive, accurate, and mix-and-read detection for FEN1 activity. In FCT-CRISPR, a flap-structured dumbbell DNA probe was ingeniously designed, in which the flap domain serves as a split activator, and the dumbbell scaffold acts as a steric hindrance unit. Upon FEN1 recognition and cleavage, the split activator is liberated from its constrained configuration in the dumbbell scaffold, thereby activating CRISPR/Cas12a trans-cleavage activity. FCT-CRISPR strategy avoids the reliance on exogenous DNA ligation or replication processes, allowing mix-and-read detection of FEN1 activity with a detection limit as low as 0.2 mU and excellent specificity against nontarget enzymes. In addition, the successful detection application in lysates of cancer cells demonstrates the potential of FCT-CRISPR for clinical use. This work establishes a sensitive, accurate, and mix-and-read platform for monitoring FEN1 activity and offers a promising tool for the early diagnosis of FEN1-related diseases.},
}
@article {pmid41584446,
year = {2026},
author = {Yang, F and Ran, Q and Chen, J and Bao, G and Xian, Y and Zhang, C},
title = {Spatiotemporally regulated mitochondrial genome editing via enzyme and NIR-activated CRISPR/Cas9 nanoplatform.},
journal = {Chemical science},
volume = {17},
number = {11},
pages = {5474-5481},
pmid = {41584446},
issn = {2041-6520},
abstract = {Mitochondrial DNA (mtDNA) mutations play critical roles in tumor progression and metabolic reprogramming. Controllable gene editing within tumor cell mitochondria remains a challenge due to the double-membrane barrier and the lack of tumor-selective activation. Herein, we report a dual-responsive CRISPR/Cas delivery platform (UCRP-TPP) that enables spatiotemporally regulated mtDNA editing for targeted tumor therapy. This nanoplatform integrates near infrared light-responsive upconversion nanoparticle (UCNP), an apurinic endonuclease 1 (APE-1)-responsive DNA complex, and a mitochondrial-targeting ligand (TPP), ensuring selective activation and mitochondrial release of Cas9/sgRNA complexes. Upon activation by endogenous APE-1 enzyme and exogenous NIR light, UCRP-TPP induces mtDNA editing by CRISPR/Cas, which leads to mtDNA copy number reduction, mitochondrial membrane depolarization, reactive oxygen species generation, and tumor cell apoptosis. In vivo studies further confirm the robust antitumor efficacy of the UCRP-TPP-based nanoplatform. This work presents a versatile and controllable mitochondrial gene-editing strategy.},
}
@article {pmid41586305,
year = {2025},
author = {Pérez-Rodríguez, M and Serrano-Pertierra, E and Blanco-López, MC},
title = {Advances in biosensor technologies for the detection of antimicrobial resistance in Staphylococcus aureus.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1741845},
pmid = {41586305},
issn = {2235-2988},
mesh = {*Biosensing Techniques/methods ; Humans ; *Staphylococcal Infections/diagnosis/microbiology ; *Methicillin-Resistant Staphylococcus aureus/drug effects/genetics/isolation &amp; purification ; *Staphylococcus aureus/drug effects/genetics/isolation &amp; purification ; CRISPR-Cas Systems ; *Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests/methods ; },
abstract = {The rise of methicillin-resistant Staphylococcus aureus (MRSA) underscores the urgent need for rapid, sensitive, and portable diagnostics. In this paper, we have critically reviewed recent advances in biosensor technologies, integrating nanomaterials, aptamers, CRISPR/Cas systems, and microfluidic lab-on-a-chip platforms, that enable sub-hour and ultrasensitive detection of S. aureus and its resistance genes. These innovations offer powerful alternatives to conventional culture and PCR assays, forming the way for real-time, point-of-care antimicrobial resistance testing. Remaining challenges include matrix interference, lack of standardization, and limited clinical validation, yet continued integration with artificial intelligence and digital systems promises transformative diagnostic capabilities.},
}
@article {pmid41586479,
year = {2026},
author = {Huang, C and Yang, Y and Yin, H and Chang, H and Gong, R and Jiang, Z and Zhang, XE and Chen, Y},
title = {Ultrasensitive CRISPR Platform Enables Rapid and Extraction-Free Detection of Viral Nucleic Acid.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4028-4035},
doi = {10.1021/acs.analchem.5c06518},
pmid = {41586479},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *RNA, Viral/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; *COVID-19/diagnosis/virology ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *COVID-19 Nucleic Acid Testing/methods ; Nasopharynx/virology ; },
abstract = {CRISPR-based molecular diagnostics offers great potential for the rapid identification of pathogens. However, existing one-pot detection systems remain constrained by their restricted versatility and operational complexity. Herein, we report a novel strategy termed interspaced phosphorothioate primer-mediated one-pot detection (iPSOT), which substitutes conventional phosphodiester primers with interspaced phosphorothioate (iPS) primers. The iPS primers demonstrate strong compatibility with AapCas12b during loop-mediated isothermal amplification, enabling robust fluorescence signal generation within 15 min. The iPSOT system achieves highly specific nucleic acid detection and reliably identifies low-copy ribonucleic acid targets at concentrations of as low as 0.5 aM (0.3 copies/μL). Clinical validation further demonstrated that iPSOT enables the direct detection of SARS-CoV-2 from nasopharyngeal swabs without RNA extraction. This method reduces reagent cost and shortens assay time, achieving sample-to-result under 20 min. Overall, iPSOT enhances both sensitivity and specificity in one-pot detection and offers a promising platform for rapid, reliable point-of-care testing and large-scale pathogen surveillance.},
}
@article {pmid41586841,
year = {2026},
author = {Graça, M and Virgolini, N and Correia, R and Escandell, J and Roldão, A},
title = {An improved CRISPR-Cas9 protein-based method for knocking out insect Sf9 cell genes.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {42},
pmid = {41586841},
issn = {1432-0614},
mesh = {Animals ; *CRISPR-Cas Systems ; Sf9 Cells ; *Gene Knockout Techniques/methods ; *Gene Editing/methods ; Apoptosis ; Baculoviridae/genetics ; Spodoptera/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Insect cells are one of the uprising expression systems in the biopharmaceutical industry to produce vaccines and gene therapy vectors, but cell line development has been limited by the lack of established genetic engineering tools and genomic characterization. CRISPR-Cas9 has arisen as a powerful tool for gene editing but has seen little application in insect cells. In this work, a gene editing pipeline for the delivery of a ribonucleoprotein (RNP) complex comprised of a guide RNA and the enzyme Cas9 to insect Sf9 cells was implemented and then applied to knockout caspase initiator Sf-Dronc, aiming at alleviating cell apoptosis during an infection process. The resulting engineered cell lines were characterized as per their phenotype and production of three different product modalities. Utilizing the established workflow, a knockout rate of 68% was achieved with the implemented protocol (vs. the 12% presumed efficiency of a previously reported system) when targeting the fdl gene. When applied to Sf-Dronc, mutants containing deletions in several alleles of the host genome were identified and confirmed by next-generation sequencing. Generated clones exhibited higher apoptosis resistance and delayed onset of cell viability drop following infection with baculovirus. While Sf-Dronc deletion was shown to have negligible impact on the production of rAAV and PfRipr5, production of iVLPS showed an > twofold increase over wild-type Sf9. Overall, this study showcases the successful implementation of an efficient CRISPR-Cas9 pipeline, further leveraging the usage of genetic engineering in insect Sf9 cells towards the development of enhanced cell hosts for biopharmaceutical production. KEY POINTS: • Implementation of an efficient CRISPR-Cas9 RNP complex delivery strategy to insect cells. • Establishment of the genome editing pipeline demonstrated through Sf-Dronc knockout, resulting in increased apoptosis resistance and delayed loss of viability upon baculovirus infection. • Sf-Dronc deletion led to over a twofold increase in the production of influenza VLPs compared to wild-type Sf9 cells.},
}
@article {pmid41587008,
year = {2026},
author = {Cai, M and Song, K and Yao, C and Wang, S and Wang, R and Wang, Q and Chen, H and Wang, H},
title = {Global spread and evolution of KPC-2 and NDM-1-producing Gram-negative bacteria.},
journal = {Science China. Life sciences},
volume = {},
number = {},
pages = {},
pmid = {41587008},
issn = {1869-1889},
abstract = {The co-occurrence of KPC and NDM carbapenemases in Gram-negative bacteria presents a serious and expanding global health threat. This study characterized 338 KPC-2/NDM-1 dual-positive isolates from 23 countries, including 41 clinical strains sequenced through hybrid second- and third-generation platforms from China's national surveillance network. These isolates spanned six genera, 58 species, and 138 sequence types, reflecting substantial taxonomic and geographic diversity. Molecular analysis identified IncFII(p14) plasmids as the principal vectors for cross-genus dissemination of KPC-2, while IncX3, IncN, and IncFIB(pB171)/IncFII(Yp) plasmids were dominant carriers of NDM-1 among the studied strains. Codon usage analysis indicated stronger bias in KPC-2 plasmids (effective codon number: 39.17, optimal codons: 17) compared to NDM-1 plasmids (effective codon number: 41.25, optimal codons: 12), indicating differential evolutionary pressures. Dual-positive strains exhibited significantly higher virulence scores and broader resistance profiles than reference strains (P<0.001). Notably, only 14.6% of isolates harbored Type I-E CRISPR-Cas systems, all of which encoded the anti-CRISPR protein AcrIE10. Furthermore, Type II methyltransferase numbers were significantly enriched in dual-positive strains (P<0.005), suggesting a potential role in modulating host defense evasion. We propose that in Klebsiella spp., KPC-2 plasmids are typically acquired prior to NDM-1 plasmids and can form hybrid plasmids. In non-Klebsiella genera, dual resistance is primarily driven by independent acquisition of high-risk plasmids such as IncFII(p14) and IncX3, without a fixed temporal order. These findings highlight the convergence of global plasmid-mediated resistance, host-pathogen immune interplay, and pan-resistance evolution. Targeting high-risk plasmid lineages and host defense-modulating elements may be key to forecasting resistance emergence and guiding early interventions against dual-carbapenemase-producing pathogens.},
}
@article {pmid41587498,
year = {2026},
author = {Marpaung, DSS and Yap Sinaga, AO},
title = {Toehold-mediated strand displacement in CRISPR/Cas12a reactions: Advances in programmable and universal biosensing strategies.},
journal = {Talanta},
volume = {302},
number = {},
pages = {129442},
doi = {10.1016/j.talanta.2026.129442},
pmid = {41587498},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a-based biosensors have emerged as powerful tools for nucleic acid detection due to their programmability, high sensitivity, and versatility. However, challenges such as PAM dependence, limited mismatch discrimination, and difficulty in detecting non-nucleic acid analytes constrain their universality. Toehold-mediated strand displacement (TMSD) offers a programmable mechanism to overcome these limitations by dynamically regulating hybridization kinetics and molecular interactions. This review systematically summarizes recent advances integrating TMSD into CRISPR/Cas12a systems, including crRNA release, crRNA-DNA activation, activator generation, and reporter signal modulation. By coupling TMSD's precise strand exchange capabilities with Cas12a's trans-cleavage activity, these hybrid biosensors achieve improved specificity, tunable kinetics, and multi-analyte adaptability. The review further discusses design principles, thermodynamic foundations, and application examples across biomedical, environmental, and food diagnostics. Collectively, TMSD-assisted CRISPR/Cas12a biosensing provides a universal, programmable framework for next-generation molecular diagnostics with enhanced control, sensitivity, and functional diversity.},
}
@article {pmid41588051,
year = {2026},
author = {Molina, MC and Quiroga, C},
title = {Functional characterization of a type I-F1 CRISPR-cas system from the clinical isolate Shewanella xiamenensis Sh95 reveals constitutive activity and plasmid-curing capability.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {5409},
pmid = {41588051},
issn = {2045-2322},
support = {ANPCyT 2020-03222//Agencia Nacional de Promoci&#xf3;n de la Investigaci&#xf3;n, el Desarrollo Tecnol&#xf3;gico y la Innovaci&#xf3;n/ ; IP-PUE 0085//Consejo Nacional de Investigaciones Cient&#xed;ficas y T&#xe9;cnicas/ ; PIDAE-UBA 2025 EX-2024-04115022-UBA-DME#REC//Universidad de Buenos Aires/ ; },
abstract = {UNLABELLED: CRISPR-Cas are prokaryotic adaptive immunity defense systems that provide protection through RNA-guided recruited nucleases effectors.Despite the vast diversity in which CRISPR-Cas systems naturally exist, the single effectors from Class 2 were the most studied as genetic engineering tools. Later, the endogenous reprogramming of type I CRISPR-Cas systems showed promising results in diverse bacteria. However, the features and functions of the subtype I-F1 from Shewanella spp. members remain poorly characterized. Here, we report the analysis of the genetic context and activity of a type I-F1 CRISPR-Cas system within the species from the clinical isolate Shewanella xiamenensis Sh95 (SxCRISPR-Cas3). We show that this system transcribes constitutively and contains an internal promoter within cas3f open reading frame. Using endogenous reprogramming assays with synthetic mini CRISPR arrays, we demonstrate that the system is functionally active producing target plasmid interference and plasmid curing. Together, these results represent a simple methodology for obtaining colonies of S. xiamenensis Sh95 cured from the target plasmid and lay the foundation to the future exploration of SxCRISPR-Cas3 as a programmable interference tool.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-34486-2.},
}
@article {pmid41588195,
year = {2026},
author = {Taveneau, C and Chai, HX and D'Silva, J and Bamert, RS and Chen, H and Hayes, BK and Calvert, RW and Purcell, J and Curwen, DJ and Munder, F and Martin, LL and Barr, JJ and Rosenbluh, J and Fareh, M and Grinter, R and Knott, GJ},
title = {De novo design of potent CRISPR-Cas13 inhibitors.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41588195},
issn = {1552-4469},
abstract = {CRISPR-Cas systems are transformative tools for gene editing that can be tuned or controlled by anti-CRISPRs (Acrs)-phage-derived inhibitors that regulate CRISPR-Cas activity. However, Acrs that can inhibit biotechnologically relevant CRISPR systems are relatively rare and challenging to discover. To overcome this limitation, we describe a highly successful and rapid approach that leverages de novo protein design to develop new-to-nature proteins for controlling CRISPR-Cas activity. Here, using Leptotrichia buccalis CRISPR-Cas13a as a representative example, we demonstrate that Acrs designed using artificial intelligence (AIcrs) are capable of highly potent and specific inhibition of CRISPR-Cas13a nuclease activity. We present a comprehensive workflow for design validation and demonstrate AIcr functionality in controlling CRISPR-Cas13 activity in bacterial and human cells. The ability to design bespoke inhibitors of Cas effectors will contribute to the ongoing development of CRISPR-Cas tools in diverse applications across research, medicine, agriculture and microbiology.},
}
@article {pmid41588257,
year = {2026},
author = {Kim, C and Tagmount, A and Zhu, Z and Wilson, F and Li, D and Ostrov, DA and Brad Barbazuk, W and Bacher, R and Vulpe, CD},
title = {Identification of functional genetic components modulating toxicity response to PFOS using genome-wide CRISPR screens in HepG2/C3A cells.},
journal = {Archives of toxicology},
volume = {100},
number = {4},
pages = {1391-1410},
pmid = {41588257},
issn = {1432-0738},
support = {R01ES033625/ES/NIEHS NIH HHS/United States ; R01ES033625/ES/NIEHS NIH HHS/United States ; },
mesh = {Humans ; *Fluorocarbons/toxicity ; *Alkanesulfonic Acids/toxicity ; Hep G2 Cells ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Molecular Docking Simulation ; Genome-Wide Association Study ; },
abstract = {Perfluorooctane sulfonate (PFOS) poses significant health and environmental risks due to its persistence and widespread use and has been linked to various adverse outcomes, such as liver toxicity. Although the molecular responses and toxicity effects of PFOS exposure have been extensively studied, considerable uncertainty remains regarding the causal mechanisms leading to PFOS-associated adverse effects. To help bridge this gap, we conducted CRISPR screens in HepG2/C3A human liver cells exposed to IC25 (170 µM) of PFOS to identify genes and pathways influencing PFOS-induced cytotoxicity. Using a genome-wide CRISPR knockout library targeting 18,819 genes, we identified 340 candidate genes that modulate PFOS-induced cytotoxicity when genetically disrupted (189 gene disruptions increased sensitivity and 151 gene disruptions increased resistance). From these candidate genes, we individually disrupted two candidate genes, SLC6A9 which encodes the glycine transporter GlyT1, and CPSF2, and confirmed increased resistance to PFOS exposure. Further, molecular docking analysis predicts that PFOS directly binds to GlyT1 and functional inhibition of GlyT1 also increases resistance to PFOS exposure. Gene-Disease outcome association analysis using the Comparative Toxicogenomics Database (CTD) indicated an enrichment of candidate genes associated with cancer-related and liver disease phenotypes. KEGG and STRING enrichment analyses found over representation of several biological pathways including DNA damage response and cell cycle. Lastly, cross-species conservation analysis using the top two validated gene targets found that their pathways were highly conserved in several environmentally relevant species. These findings provide new mechanistic and functional insights into PFOS-induced cytotoxicity, highlight potential molecular targets for toxicity mitigation, and establish a foundation for cross-species toxicogenomic modeling of PFOS health effects.},
}
@article {pmid41590269,
year = {2025},
author = {Liang, Z and Zhang, J and Zhang, S},
title = {Engineering a CRISPR-Mediated Dual Signal Amplification-Based Biosensor for miRNA Determination.},
journal = {Biosensors},
volume = {16},
number = {1},
pages = {},
pmid = {41590269},
issn = {2079-6374},
support = {2023A1515110638//Guangdong Basic and Applied Basic Research Foundation/ ; 2025A1515011683//Guangdong Basic and Applied Basic Research Foundation/ ; 2025A04J4037//Guangzhou Science and Technology Planning Project/ ; 2022GDASZH-2022010110//GDAS' Project of Science and Technology Development/ ; },
mesh = {*MicroRNAs/analysis ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; Electrochemical Techniques ; },
abstract = {MicroRNAs, pivotal regulators of gene expression and physiology, serve as reliable biomarkers for early cancer diagnosis and therapy. As one of the earliest discovered miRNAs in the human genome, miRNA-21 provides critical information for early cancer diagnosis, drug therapy, and prognosis. In this work, we harness CRISPR as a bridge to integrate target-induced self-priming hairpin isothermal amplification (SIAM) with terminal transferase (TdT) polymerization labeling, constructing a facile, straightforward electrochemical biosensor for sensitive miRNA-21 detection. Unlike conventional single-strand template-based exponential amplification (EXPAR), the SIAM hairpin undergoes target triggered intramolecular conformational change, initiating extension and strand displacement reactions that suppress nonspecific dimer formation and lower background current. Notably, the assay requires only a single probe, enabling unidirectional signal amplification while nonspecific reactions caused by system complexity. The generated SIAM products activate the Cas12a/crRNA complex to trans-cleave PO4[3-] modified single-stranded DNAs (ssDNAs); the resulting 3' hydroxyl ssDNAs are subsequently labeled by TdT, with the assistance of SA-HRP catalyzing hydrogen peroxide, achieving robust signal amplification. Under optimized conditions, the cathodic current exhibits a logarithmic relationship with miRNA concentrations from 20 fM to 5.0 × 10[8] fM, with a detection limit of 9.2 fM. The biosensor successfully quantified miRNA-21 in commercial serum samples and biological lysates, demonstrating its potential for cancer diagnostics and therapy.},
}
@article {pmid41590284,
year = {2026},
author = {Zhang, Z and Fu, Q and Wen, T and Zheng, Y and Ma, Y and Liu, S and Liu, G},
title = {Integrated Colorimetric CRISPR/Cas12a Detection of Double-Stranded DNA on Microfluidic Paper-Based Analytical Devices.},
journal = {Biosensors},
volume = {16},
number = {1},
pages = {},
pmid = {41590284},
issn = {2079-6374},
support = {22174121, 22211530067, T2250710180//National Natural Science Foundation of China/ ; },
mesh = {*Colorimetry ; Paper ; CRISPR-Cas Systems ; *Biosensing Techniques ; Humans ; Human papillomavirus 16 ; Lab-On-A-Chip Devices ; Gold/chemistry ; *DNA/analysis ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; },
abstract = {Early detection of high-risk human papillomavirus (HPV), particularly HPV16 E7, is critical for cervical cancer prevention. Here, we report a novel, portable, and instrument-free biosensing platform that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection on a microfluidic paper-based analytical device (μPAD) for colorimetric, visual readout of double-stranded DNA (dsDNA). The μPAD features seven functional zones, including lyophilized RPA and CRISPR reagents, and immobilized streptavidin and anti-FAM antibodies for signal generation. Upon target recognition, Cas12a's trans-cleavage activity releases biotinylated-FAM-labeled reporters that form a sandwich complex with gold nanoparticle (AuNP)-conjugated anti-FAM antibodies, producing a visible red signal at the test zone. The gray value of the colorimetric signal correlates linearly with target concentration, enabling the quantitative detection of HPV16 E7 dsDNA down to 100 pM within 60 min. The assay demonstrated high accuracy and reproducibility in spiked samples. By combining isothermal amplification, CRISPR specificity, and paper-based microfluidics, this platform offers a rapid, low-cost, and user-friendly solution for point-of-care HPV screening in resource-limited settings. This work advances the integration of CRISPR diagnostics with μPAD, paving the way for scalable point-of-care molecular diagnostics beyond HPV.},
}
@article {pmid41590404,
year = {2026},
author = {Li, W and Wang, M and Wang, S},
title = {A label-free and universal CRISPR/Cas12a platform for the detection of hazardous substances in food.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {6},
pages = {1216-1223},
doi = {10.1039/d5ay01887k},
pmid = {41590404},
issn = {1759-9679},
mesh = {*CRISPR-Cas Systems/genetics ; *Food Contamination/analysis ; *Hazardous Substances/analysis ; *Food Analysis/methods ; Limit of Detection ; },
abstract = {The present study proposes a label-free and universal CRISPR/Cas12a-based platform for the detection of hazardous substances in food. A label-free reporter, termed the G4 reporter, was carefully engineered. In the absence of the target analyte, the catalytic hairpin assembly (CHA) remains inactive, thereby preventing activation of the CRISPR/Cas12a system and preserving the cleavage of the G4 reporter, which emits a label-free fluorescence signal. Conversely, in the presence of the target analyte, the CHA process is triggered, activating the CRISPR/Cas12a system, which cleaves the G4 reporter into fragments, leading to a decrease in the label-free fluorescence signal. This detection strategy follows a negative response model, wherein quantification is based on the net fluorescence difference between positive and negative controls. The developed label-free signal output modality for the CRISPR/Cas12a system offers advantages of simplicity and cost-effectiveness. Moreover, the proposed method incorporates a CHA process to facilitate signal transduction and activate the subsequent CRISPR system. The system upon integration of CHA with CRISPR functions in a dual-amplification mode, resulting in enhanced signal amplification efficiency and superior sensitivity compared to the standalone CRISPR system. Thus, the platform achieves highly sensitive detection of hazardous substances, with limits of detection (LODs) of 3.0 fg mL[-1] for aflatoxin B1 (AFB1) and 0.02 pg mL[-1] for acetamiprid (ACE). Moreover, trace amounts of AFB1 and ACE were successfully identified in real food samples. By altering the sequences of the detection components, this platform can be readily adapted for the detection of other hazardous substances in food matrices. Therefore, this work introduces a novel, label-free, universal, and sensitive CRISPR/Cas12a-based detection platform, demonstrating considerable potential for applications in food safety surveillance and public health protection.},
}
@article {pmid41590485,
year = {2026},
author = {Gupta, DR and Kasfy, SH and Ali, J and Hia, FT and Hoque, MN and Rahman, M and Islam, T},
title = {Validation and Improvement of a Rapid, CRISPR-Cas-Free RPA-PCRD Strip Assay for On-Site Genomic Surveillance and Quarantine of Wheat Blast.},
journal = {Journal of fungi (Basel, Switzerland)},
volume = {12},
number = {1},
pages = {},
pmid = {41590485},
issn = {2309-608X},
support = {Grant Code: V0156.01//Bill and Melinda Gates Foundation and the Foreign, Commonwealth &amp; Development Office (FCDO), UK/ ; },
abstract = {As an emerging threat to global food security, wheat blast necessitates the development of a rapid and field-deployable detection system to facilitate early diagnosis, enable effective management, and prevent its further spread to new regions. In this study, we aimed to validate and improve a Recombinase Polymerase Amplification coupled with PCRD lateral flow detection (RPA-PCRD strip assay) kit for the rapid and specific identification of Magnaporthe oryzae pathotype Triticum (MoT) in field samples. The assay demonstrated exceptional sensitivity, detecting as low as 10 pg/µL of target DNA, and exhibited no cross-reactivity with M. oryzae Oryzae (MoO) isolates and other major fungal phytopathogens under the genera of Fusarium, Bipolaris, Colletotrichum, and Botrydiplodia. The method successfully detected MoT in wheat leaves as early as 4 days post-infection (DPI), and in infected spikes, seeds, and alternate hosts. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire RPA-PCRD strip assay enabled the detection of MoT within 30 min with no specialized equipment and high technical skills at ambient temperature (37-39 °C). When applied to field samples, it successfully detected MoT in naturally infected diseased wheat plants from seven different fields in a wheat blast hotspot district, Meherpur, Bangladesh. Training 52 diverse stakeholders validated the kit's field readiness, with 88% of trainees endorsing its user-friendly design. This method offers a practical, low-cost, and portable point-of-care diagnostic tool suitable for on-site genomic surveillance, integrated management, seed health testing, and quarantine screening of wheat blast in resource-limited settings. Furthermore, the RPA-PCRD platform serves as an early warning modular diagnostic template that can be readily adapted to detect a wide array of phytopathogens by integrating target-specific genomic primers.},
}
@article {pmid41590698,
year = {2025},
author = {Antonacci, A and Masi, A and Vedi, V and Colella, S and Musella, F and Fiorentino, G and Scognamiglio, V},
title = {CRISPR-Cas Technology Turns Chlamydomonas reinhardtii into a Flagship for Algal Biotechnology.},
journal = {Marine drugs},
volume = {24},
number = {1},
pages = {},
pmid = {41590698},
issn = {1660-3397},
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Biotechnology/methods ; Gene Editing/methods ; *Microalgae/genetics/metabolism ; Biofuels ; Metabolic Engineering/methods ; },
abstract = {Microalgae represent some of the most promising eukaryotic platforms in biotechnology due to their rapid growth, simple cultivation requirements, reliance on sunlight as a primary energy source, and ability to synthesize high-value bioactive compounds. These characteristics have made microalgae attractive candidates in various fields, including biofuel production, carbon capture, and pharmaceutical development. However, several technical limitations have limited their large-scale use as sustainable biofactories. A paradigm shift is currently occurring thanks to the genetic manipulation of microalgae, driven by CRISPR-Cas technology. Significant progress has been made in the model species Chlamydomonas reinhardtii, particularly in the targeted and efficient insertion of foreign DNA. Despite this progress, key challenges remain, and further optimization of CRISPR-Cas methodologies is needed to fully unleash the genetic potential of this organism. This review provides an overview of the convergence of CRISPR-Cas technologies in microalgae research, highlighting their impact on genetic studies, metabolic engineering, and industrial applications. It summarizes recent advances in microalgal genome editing through CRISPR systems, outlines current technical challenges, and highlights future directions for improving the implementation of this innovative technology in microalgal biotechnology.},
}
@article {pmid41591278,
year = {2026},
author = {Li, L and Wang, Y and Liu, L and Lou, Y and Lin, K and Li, T and Yu, C and Han, Y and Wei, H and Wang, D and Wang, S and Rong, Z},
title = {Miniaturized Single-Step Duplex CRISPR Diagnostic Platform for At-Home Molecular Testing of HPV16 and HPV18.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1117-1128},
doi = {10.1021/acssensors.5c03020},
pmid = {41591278},
issn = {2379-3694},
mesh = {*Human papillomavirus 16/genetics/isolation &amp; purification ; *Human papillomavirus 18/genetics/isolation &amp; purification ; Humans ; Female ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Uterine Cervical Neoplasms/diagnosis/virology ; Nucleic Acid Amplification Techniques ; Miniaturization ; Limit of Detection ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The early screening, precise diagnosis, and effective treatment of invasive cervical cancer necessitate at-home molecular testing of human papillomaviruses (HPVs). However, current HPV DNA tests cannot meet the need for an affordable, rapid, and accurate diagnosis using a streamlined workflow. Here, we present a miniaturized single-step duplex CRISPR diagnostic platform, termed SCOPEv2 (Streamlined CRISPR On Pod Evaluation platform, version 2), for rapid and highly sensitive at-home molecular testing of high-risk HPV16 and HPV18 for population screening of cervical cancer. Dual-target recombinase polymerase amplification (RPA) was initially incorporated with Cas12a/Cas13a cleavage reactions in a single-step reaction system. A miniaturized and low-cost dual-color wireless analysis device was further developed to execute the analysis workflow. SCOPEv2 can detect HPV16 and HPV18 with limits of detection of 2.5 copies/μL (5 copies/reaction) and 5 copies/μL (10 copies/reaction) in 30 min, respectively. The analysis results for 128 clinical cervicovaginal swab samples revealed 94.7% sensitivity and 100% specificity. SCOPEv2 demonstrates an easy-to-use workflow, low cost, high analytical performance, and superior clinical feasibility, which enable accurate and simultaneous point-of-care testing of HPV16 and HPV18.},
}
@article {pmid41591594,
year = {2026},
author = {Singhal, P and Saini, S and Saini, O and Bishnoi, A and E R, R and Meena, BR and Singh, J and Yogendra, K},
title = {Molecular gatekeepers: eukaryotic translation factors decoding plant-virus dynamics for resistance engineering.},
journal = {Stress biology},
volume = {6},
number = {1},
pages = {9},
pmid = {41591594},
issn = {2731-0450},
abstract = {Plant viruses are among the most significant biotic stressors, posing a severe threat to crop productivity and global food security. Their success largely depends on the exploitation of host eukaryotic translation factors (eTFs), including initiation factors (eIFs) and elongation factors (eEFs), which act as molecular gatekeepers of the viral life cycle. Key members such as eIF4E, eIF(iso)4E, eIF4G, eEF1A, and eEF1B have been identified as susceptibility factors that mediate viral translation, replication, and systemic movement. Viruses have co-evolved specialized proteins and RNA elements, including VPg and IRES structures, to hijack these host factors and circumvent plant defense barriers. This review synthesizes current understanding of the mechanistic roles of eTFs in virus-host dynamics and highlights strategies to mitigate viral stress. Approaches such as natural allele mining, induced mutagenesis, TILLING/EcoTILLING, RNA interference, and precise genome editing with CRISPR/Cas systems are explored as practical tools for reducing susceptibility. Targeted manipulation of eTFs offers a promising avenue to reprogram plants for resistance while maintaining essential cellular functions. By integrating molecular biology with applied strategies, we propose an eTF-centered framework for resistance breeding within a broader stress biology perspective. Future research combining functional genomics, synthetic biology, and breeding innovation will be pivotal in delivering broad-spectrum, durable, and environmentally sustainable resistance to plant viral stress.},
}
@article {pmid41591771,
year = {2026},
author = {Chen, D and Zhu, B and Zhou, Y and Fang, Z and Zhu, Z and Chen, C and Shen, T},
title = {Dual-mode CRISPR/Cas12a-mediated alkaline phosphatase detection (CAD) biosensor.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {6},
pages = {1239-1247},
doi = {10.1039/d5ay01195g},
pmid = {41591771},
issn = {1759-9679},
mesh = {*Alkaline Phosphatase/analysis/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Immunoassay/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Alkaline phosphatase (ALP), a crucial biomarker for hepatobiliary disorders, bone diseases, and cancer progression, requires ultrasensitive detection methods to meet clinical diagnostic requirements. Current methodologies predominantly depend on single-readout mechanisms that fail to address the growing requirements of sensitivity, operational simplicity, and adaptability to resource-limited settings. Herein, we present a CRISPR/Cas12a-mediated ALP detection (CAD) isothermal amplification system that overcomes these challenges through a novel dual-signal (fluorescence and lateral flow immunoassay (LFIA)) readout mechanism. The system features a rationally engineered hairpin DNA probe (HPP) that initiates Klenow (exo-)-driven polymerase elongation upon ALP recognition, subsequently activating Cas12a's trans-cleavage activity for exponential signal amplification. With fluorescence readout, this cascade amplification strategy achieves unprecedented sensitivity with a detection limit of 0.1 U L[-1] and a wide linear range (0.1-10 U L[-1]), outperforming conventional colorimetric methods by one order of magnitude while maintaining exceptional specificity against biological interferents. Furthermore, the LFIA adaptation of the readout bridges the gap between laboratory-based detection and point-of-care applications. This user-friendly adaptation enables instrument-free visual detection with a clear cut-off value of about 7 U L[-1], offering the potential to effectively differentiate pathological samples from normal physiological levels with an appropriate dilution factor for clinical samples. Our dual-mode biosensing strategy not only enables high-precision quantitative analysis in clinical settings but also facilitates rapid qualitative detection under resource-limited conditions, thus offering significant potential for early disease diagnosis and long-term therapeutic monitoring.},
}
@article {pmid41592109,
year = {2026},
author = {Saglam, M and Tsakirpaloglou, N and Bridgeland, A and Miller, R and Thomson, MJ and Septiningsih, EM},
title = {Carbon nanotube and carbon dot mediated plasmid DNA delivery in cowpea leaves.},
journal = {PloS one},
volume = {21},
number = {1},
pages = {e0340716},
pmid = {41592109},
issn = {1932-6203},
mesh = {*Nanotubes, Carbon/chemistry ; *Plant Leaves/genetics/metabolism ; *Vigna/genetics/metabolism ; *Plasmids/genetics ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; Glucuronidase/genetics/metabolism ; Plants, Genetically Modified/genetics ; Oxidoreductases/genetics ; *Quantum Dots/chemistry ; Gene Editing ; Carbon/chemistry ; },
abstract = {CRISPR-Cas9 technology has been widely used as a key molecular biology tool for crop improvement. However, the advance of this technology has been hindered by host species- or genotype-dependent tissue culture protocols and poor transformation efficiencies. Recent research has shown that plasmid DNA delivered by single-walled carbon nanotubes (SWCNTs) and carbon dots (CDs) can diffuse through plant cell walls, enabling the transient expression of genetic material in plant tissues. However, such an experiment has not been performed in legumes, most of which are considered recalcitrant species for transformation. In this study, we aim to investigate the capability of a SWCNT or CD-based plasmid delivery system in expressing a target gene in cowpea (Vigna unguiculata) leaves via infiltration using the β-glucuronidase (GUS) reporter gene. Further, we aim to see the potential of SWCNTs and CDs for a CRISPR-Cas9 gene construct delivery system, with phytoene desaturase (PDS) as the target gene. Our results showed that SWCNTs and CDs can deliver the GUS reporter gene construct in the surrounding area near the site of the infiltration, which results in the temporary expression of GUS by observing the blue color in this area. Likewise, infiltration of the CRISPR-Cas9 vectors targeting the PDS gene for the knockout resulted in multiplex editing and large deletions within the target gene. Overall, our findings pave the way for overcoming conventional DNA delivery challenges. However, further research is needed to explore optimal germline targets for plant tissues to avoid chimerism and to allow for more efficient CRISPR-Cas9 editing resulting in heritable mutations.},
}
@article {pmid41592354,
year = {2026},
author = {Du, T and Ding, F and Ma, X and Luo, Y and Zhu, D and Wang, L and Su, S},
title = {Ultrasensitive detection of monkeypox virus: harnessing synergistic CRISPR-driven signal amplification on a DNA tetrahedron-mediated sensing interface.},
journal = {Biosensors &amp; bioelectronics},
volume = {298},
number = {},
pages = {118441},
doi = {10.1016/j.bios.2026.118441},
pmid = {41592354},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; *Monkeypox virus/isolation &amp; purification/genetics ; *Mpox, Monkeypox/diagnosis/virology ; Nanostructures/chemistry ; *DNA, Viral/genetics ; Hydrogen Peroxide/chemistry ; DNA/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and ultrasensitive detection of emerging infectious diseases is critical for public health security. Herein, an electrochemical biosensor was developed for ultrasensitive detection of monkeypox virus (MPXV) by integrating CRISPR/Cas12a-driven signal amplification strategy with tetrahedral DNA nanostructure (TDN)-based sensing interface. The added MPXV DNA can efficiently activate the cleavage activity of Cas12a protein, thereby mediating the CRISPR-driven hybridization chain reaction (HCR) on TDN sensing interface. The horseradish peroxidase (HRP)-labeled HCR product can catalyze 3,3',5,5'-tetramethylbenzidine (TMB) and hydrogen peroxide (H2O2) reaction to generate an amplified electrochemical signal. Based on the signal change, the CRISPR-driven electrochemical biosensor exhibited better detection performance comparable to those of pre-amplification CRISPR-based biosensors for MPXV detection, including wide linear range, an ultralow detection limit, exceptional selectivity against non-target viruses (CPXV, ETCV, VZV, HSV), high reproducibility and accepted stability. Integrated with a smartphone-based portable device, the designed point-of-care testing (POCT) electrochemical biosensor can accurately detect MPXV in 10 % human saliva. This work provides a promising sensing platform for rapid, accurate and on-site detection of infectious diseases.},
}
@article {pmid41592905,
year = {2026},
author = {Shabbir, R and Javed, T and Sun, SR and Wang, ZQ and Zhang, W and Gao, SJ and Wang, QN},
title = {Functional genomics in sugarcane breeding: key challenges and strategies.},
journal = {Critical reviews in biotechnology},
volume = {46},
number = {3},
pages = {419-439},
doi = {10.1080/07388551.2026.2614075},
pmid = {41592905},
issn = {1549-7801},
mesh = {*Saccharum/genetics ; *Plant Breeding ; *Genomics/methods ; *Genome, Plant ; Gene Editing ; Genome-Wide Association Study ; CRISPR-Cas Systems ; },
abstract = {Sugarcane, a leading source of sugar and bio-energy around the globe stands at the cross-road of genome complexity and agricultural innovation, offering the immense potential to fuel a sustainable future. Functional genomics with its precise identification and manipulation of genes could enable researchers unlock this potential and accelerate the breeding efforts. However, the polyploid genome of sugarcane with: high heterozygosity, high-repetitive DNA content, multiple copies of homo(eo)logous gene, epistatic interaction of alleles, etc., challenges the gene annotation, genome sequencing, genome editing, and phenotypic characterization. Similarly long breeding cycle, low transformation efficiency, time-consuming, and labor-intensive transformation methods further complicates the genome editing. Recent advances of functional genomics are transforming this scenario, such as current availability of reference genome "R570," has provided a significant insight of genome architect and function. Genome wide association studies (GWAS)/genome selection (GS) are enhancing trait-mapping and prediction of breeding values to accelerate the breeding cycles. The current era of smart breeding with integrative bio-informatics, advance genome editing tools, i.e., CRISPR/Cas-systems (Cas-proteins, Cas-RNPs, d-Cas-RNPs, and CRISPRa/i), and high-throughput phenomics offers a significant approach to: overcome transformation bottlenecks, explore complex trait architect and address polyploidy challenges. Therefore, this review summarizes the key challenges and focuses on elaborating recent advances and suggests optimized strategies for future improvement in functional genomics of sugarcane breeding.},
}
@article {pmid41593344,
year = {2026},
author = {Munir, S and Wan, S and Gao, X and Lai, M and Mu, Z and Wang, H and Liu, Z and Li, F and Xia, L and Tan, Y},
title = {Elucidating the roles of essential genes in autotrophic metabolism and cell morphology of Clostridium ljungdahlii by CRISPRi.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {44},
pmid = {41593344},
issn = {1432-0614},
mesh = {*Clostridium/genetics/metabolism/cytology/growth &amp; development ; *Autotrophic Processes/genetics ; *Genes, Essential ; Ethanol/metabolism ; Fermentation ; Gene Expression Regulation, Bacterial ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Understanding the function of essential genes in Clostridium ljungdahlii is critical for unraveling its autotrophic metabolism and optimizing its potential as a platform for syngas fermentation. However, study on essential genes of this species remains insufficient. Here, we employed an inducible CRISPR interference (CRISPRi) system to investigate the roles of key metabolic and cell division genes in C. ljungdahlii. Targeted repression of genes encoding pyruvate:ferredoxin oxidoreductase (PFOR1, PFOR2), acetaldehyde:ferredoxin oxidoreductase (AOR1, AOR2), and glyceraldehyde phosphate hydrogenase type I (GAP-I) revealed their essential contributions to autotrophic growth, as knockdown strains exhibited impaired growth and reduced ethanol production. Furthermore, downregulation of the cell division gene ftsZ resulted in elongated cell morphology, highlighting its critical role in cell shape regulation. These findings provide new insights into the functional importance of essential genes in C. ljungdahlii and demonstrate how targeted gene repression can advance our understanding of autotrophic metabolism and cellular processes.},
}
@article {pmid41593699,
year = {2026},
author = {Zhu, J and Li, Y and Yu, C and Huang, W and Chen, J and Liu, X and Qin, R and Li, J and Xu, R and Wei, P},
title = {Engineering hypercompact IscB nucleases for efficient and versatile genome editing in rice.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {49},
pmid = {41593699},
issn = {1474-760X},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Endonucleases/genetics/metabolism ; },
abstract = {BACKGROUND: IscB (Insertion sequences Cas9-like OrfB) represents a novel class of RNA-guided nucleases, approximately one-third the size of Cas9 proteins. Despite the limited natural efficiency in eukaryotic cells, recent advances have led to the engineering of several IscBs for mammalian genome editing.<br><br>RESULTS: In this study, we screen and identify high-activity IscB variants for rice. A version of pIscB-v3, combining enOgeuIscB and &#x3c9;RNA-v13, demonstrated superior mutagenesis efficiency compared to other systems. The average editing efficiency of pIscB-v3 is 17.61% from ten endogenous targets, and we obtain edited lines in up to 83.33% of T0 generation with 33.33% of homozygous and bi-allelic mutations. Further analysis reveals that pIscB-v3 exhibits high editing specificity and relaxed target-adjacent motif (TAM) compatibility in rice. Beyond gene knockout systems, we develop cytosine base editors (CBEs) and adenine base editors (ABEs) from pIscB-v3. We find that the ssDNA-targeting SCP1.201 family deaminase Sdd7 outperformed human APOBEC3A in IscB-CBEs for C-to-T conversions in rice. The Sdd7-nIscB achieves precise edits in 22.92% of lines on average, with a maximum frequency of 47.92%. Additionally, TadA8e-nIscB exhibits limited activity. However, fusing an extra copy of TadA-8e to either terminus of TadA8e-nIsc significantly enhances A-to-G conversions.<br><br>CONCLUSIONS: Collectively, our results demonstrate the robust capabilities of IscB to develop an efficient and versatile miniature plant genome editing toolkit to substantially facilitate crop breeding.},
}
@article {pmid41594550,
year = {2025},
author = {Wang, R and Guo, C and Zhang, J and Wang, Z and Jin, W and Liu, W},
title = {Evaluation of the Unintended Effects of fad2-1-Gene-Edited Soybean Line AE15 Seeds.},
journal = {Biomolecules},
volume = {16},
number = {1},
pages = {},
pmid = {41594550},
issn = {2218-273X},
support = {2023YFF1001603//the National Key Research and Development Program/ ; ZDYF2025GXJS151//the Hainan Provincial Sanya Yazhou Bay Science and Technology Innovation Joint Project/ ; },
mesh = {*Glycine max/genetics/metabolism ; *Seeds/genetics/metabolism ; *Gene Editing ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Proteomics ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; },
abstract = {A data-independent acquisition (DIA)-based proteomic analysis was performed to evaluate the unintended effects of fad2-1-gene-edited soybean line AE15 seeds. A total of 561, 269, and 227 differentially expressed proteins (DEPs) were identified in seeds from three consecutive generations of AE15 soybean, respectively, and were primarily enriched in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to carbon metabolism, protein processing in the endoplasmic reticulum, and proteasome function. Furthermore, eight commonly differentially expressed proteins (co-DEPs) were detected across all three generations of AE15 soybean seeds, among which two-beta-amylase and endoplasmic reticulum (ER) lumen protein-retaining receptor-exhibited consistently upregulated expression. In the wild-type soybean control groups, 1063, 989, and 671 DEPs were identified across the three comparisons (ZhH302E3/ZhH10, ZhH10/ZhH42, and ZhH42/ZhH302E3), among which 71 co-DEPs were observed. These findings indicate that the protein expression profile alterations resulting from fad2-1 gene editing are considerably less pronounced compared to those caused by natural genetic variation among soybean seeds.},
}
@article {pmid41594570,
year = {2025},
author = {Anschuetz, A and Robinson, L and Mondesir, M and Melis, V and Platt, B and Harrington, CR and Riedel, G and Schwab, K},
title = {Effect of the Icelandic Mutation APP[A673T] in the Murine APP Gene on Phenotype of Line 66 Tau Mice.},
journal = {Biomolecules},
volume = {16},
number = {1},
pages = {},
pmid = {41594570},
issn = {2218-273X},
support = {PAR1577 and PAR2074//TauRx Therapeutics Ltd Singapore/ ; },
mesh = {Animals ; *tau Proteins/genetics/metabolism ; Mice ; Mice, Transgenic ; Male ; Female ; *Amyloid beta-Protein Precursor/genetics/metabolism ; *Alzheimer Disease/genetics/pathology/metabolism ; Phenotype ; Iceland ; *Mutation ; Humans ; Mice, Inbred C57BL ; Brain/metabolism/pathology ; Disease Models, Animal ; },
abstract = {The Icelandic mutation in the amyloid precursor protein (APP), APP[A673T], has been identified in Icelandic and Scandinavian populations and is associated with a significantly lower risk of developing Alzheimer's disease (AD). The introduction of the human APP[A673T] form led to a reduction in amyloid β-protein (Aβ) production and tau pathology, but the effect of mouse APP[A673T] on tau and Aβ pathology is not well studied. We have crossed line 66 (L66) tau transgenic mice that overexpress the P301S aggregation-prone form of tau with C57Bl6/J mice expressing a single-point mutation edited into the murine APP gene via CRISPR-Cas gene editing, known as mAPP[A673T]. We have performed ELISA, histopathological, and behavioural analyses of heterozygous male/female L66 and L66 xmAPP[A673T] crosses at the age of 6 months to investigate the effect of the murine A673T mutation on tau brain pathology and behavioural deficits in these mice. Using immunohistochemistry, we found only a moderate, yet significant, reduction in mAb 7/51-reactive tau for female L66 x mAPP[A673T] compared to L66 mice. Quantification of tau in soluble/insoluble brain homogenate fractions by ELISA confirmed the lack of overt differences between genotypes, as did our extensive behavioural phenotyping using six different paradigms assessing motor function, olfaction, depression/apathy-like behaviour, as well as exploration and sociability. Therefore, the mAPP[A673T] mutation has a moderate impact on tau pathology but does not appear to impact motor and neuropsychiatric behaviour in L66 tau transgenic mice.},
}
@article {pmid41595237,
year = {2026},
author = {Karnik, M and Tulimilli, SV and Anantharaju, PG and Bettadapura, ADS and Natraj, SM and Mohideen, HS and Dovat, S and Sharma, A and Madhunapantula, SV},
title = {An Overview of the Mechanisms of HPV-Induced Cervical Cancer: The Role of Kinase Targets in Pathogenesis and Drug Resistance.},
journal = {Cancers},
volume = {18},
number = {2},
pages = {},
pmid = {41595237},
issn = {2072-6694},
support = {No. JSSAHER/REG/RES/URG/54/2023-24//Intramural grant sanctioned by JSS AHER/ ; No. JSSAHER/REG/RES/URG/54/2025-26//JSS AHER/ ; },
abstract = {Despite a thorough understanding of the structure of human papillomavirus (HPV) and its genotypic variations (high-risk and low-risk variants), the mechanisms underlying HPV-induced cervical cancer (CC) pathogenesis and the molecular signatures of drug resistance remain to be fully understood. Accumulating evidence has shown the involvement of kinase targets in the induction of drug resistance in high-risk (HR) HPV-CC. Molecularly, the genome of high-risk HPV is reported to control the expression of host kinases. In particular, Aurora kinases A, B, and C (ARKA, ARKB, and ARKC), phosphotidylinositol-trisphosphate kinase (PI3K)-Akt, and Glycogen synthase kinase3-α/β (GSK3 α/β) promote the transformation of infected cells, and also enhance the resistance of cells to various chemotherapeutic agents such as nelfinavir and cisplatin. However, the precise mechanisms through which HPV activates these kinases are yet to be fully elucidated. Furthermore, there is still ambiguity surrounding whether targeting HPV-induced kinases along with HPV-targeted therapies (such as phytopharmaceuticals and PROTAC/CRISPR-CAS-based systems) synergistically inhibit cervical tumor growth. Given the critical role of kinases in the pathogenesis and treatment of CC, a comprehensive review of current evidence is warranted. This review aims to provide key insights into the mechanisms of HPV-induced CC development, the involvement of kinases in drug resistance induction, and the rationale for combination therapies to improve clinical outcomes.},
}
@article {pmid41595497,
year = {2026},
author = {Guan, S and Han, Y and Zhang, J and Du, Y and Chen, Z and Miao, C and Li, J},
title = {Multiplex Gene Editing and Effect Analysis of Yield, Fragrance, and Blast Resistance Genes in Rice.},
journal = {Genes},
volume = {17},
number = {1},
pages = {},
pmid = {41595497},
issn = {2073-4425},
mesh = {*Oryza/genetics/growth &amp; development/microbiology ; *Gene Editing/methods ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Diseases/genetics/microbiology ; Plant Proteins/genetics ; Genes, Plant ; Plant Breeding ; },
abstract = {BACKGROUND: The coordinated improvement of yield, quality and resistance is a primary goal in rice breeding. Gene editing technology is a novel method for precise multiplex gene improvement.<br><br>METHODS: In this study, we constructed a multiplex CRISPR/Cas9 vector targeting yield-related genes (GS3, OsPIL15, Gn1a), fragrance gene (OsBADH2) and rice blast resistance gene (Pi21) to pyramid traits for enhanced yield, quality, and disease resistance in rice. A tRNA-assisted CRISPR/Cas9 multiplex gene editing vector, M601-OsPIL15/GS3/Gn1a/OsBADH2/Pi21-gRNA, was constructed. Genetic transformation was performed using the Agrobacterium-mediated method with the japonica rice variety Xin Dao 53 as the recipient. Mutation editing efficiency was detected in T0 transgenic plants. Grain length, grain number per panicle, thousand-grain weight, 2-acetyl-1-pyrroline (2-AP) content, and rice blast resistance of homozygous lines were measured in the T3 generations.<br><br>RESULTS: Effectively edited plants were obtained in the T0 generation. The simultaneous editing efficiency for all five genes reached 9.38%. The individual gene editing efficiencies for Pi21, GS3, OsBADH2, Gn1a, and OsPIL15 were 78%, 63%, 56%, 54%, and 13%, respectively. Five five-gene homozygous edited lines with two genotypes were selected in the T2 generation. In the T3 generation, compared with the wild-type (WT), the edited homozygous lines showed increased grain number per panicle (14.60-25.61%), increased grain length (7.39-11.16%), increased grain length-width ratio (8.37-13.02%), increased thousand-grain weight (3.79-9.15%), a 42-64 folds increase in the fragrant substance 2-AP content, and significantly enhanced rice blast resistance. Meanwhile, there were no significant changes in other agronomic traits.<br><br>CONCLUSIONS: CRISPR/Cas9-mediated multiplex gene editing technology enabled the simultaneous editing of genes related to rice yield, quality, and disease resistance. This provides an effective approach for obtaining new japonica rice germplasm with blast resistance, long grains, and fragrance.},
}
@article {pmid41596278,
year = {2026},
author = {Bernacka, KU and Michalski, K and Wojciechowski, M and Sowa, S},
title = {Application of SNV Detection Methods for Market Control of Food Products from New Genomic Techniques.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
pmid = {41596278},
issn = {1422-0067},
support = {101137025//Horizon Europe- European Union/ ; DHR.bz.070.2.2024//Polish Ministry of Agriculture and Rural Development/ ; },
mesh = {*Plants, Genetically Modified/genetics ; *Genomics/methods ; *Polymorphism, Single Nucleotide ; *Food, Genetically Modified ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {The detection of single-nucleotide variants (SNVs) is an important challenge in modern genomics, with broad applications in medicine, diagnostics, and agricultural biotechnology. Current detection approaches include PCR-based techniques with high-affinity probes, ligase-based strategies, and sequencing approaches, each with varying degrees of sensitivity, specificity, and practicality. Despite advances in SNV analysis in the medical field, their implementation in the official control and monitoring of genetically modified organisms (GMOs) remains limited. This challenge has gained priority with the advent of new genomic techniques (NGTs), such as CRISPR-Cas nucleases, which allow precise genome editing, including subtle changes at the nucleotide level without introducing foreign DNA. Therefore, traditional methods of GMO detection targeting transgene sequences may not be sufficient to monitor such GMOs. In the European Union, GMO legislation requires distinguishing between conventionally bred and genetically modified plants. The planned introduction of new regulatory categories of NGT plants (NGT1 and NGT2) with different surveillance requirements emphasizes the need for robust, sensitive, and cost-effective SNV detection methods suitable for distinguishing between GMOs, particularly in the context of food and feed safety, traceability, and compliance.},
}
@article {pmid41596437,
year = {2026},
author = {Luo, H and Zou, H and Lin, S and Liu, J and Zhou, G and Gao, L and Huang, J and Li, J and Gao, J and Ma, C},
title = {Multiplex Editing of OsMads26, OsBsr-d1, OsELF3-2 and OsERF922 with CRISPR/Cas9 Confers Enhanced Resistance to Pathogens and Abiotic Stresses and Boosts Grain Yield in Rice (Oryza sativa).},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
pmid = {41596437},
issn = {1422-0067},
mesh = {*Oryza/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Proteins/genetics ; *Stress, Physiological/genetics ; Plant Diseases/microbiology/genetics ; Plants, Genetically Modified/genetics ; *Edible Grain/genetics/growth &amp; development ; Gene Expression Regulation, Plant ; Droughts ; },
abstract = {Rice (Oryza sativa) is one of the world's major staple foods. However, stable rice production is constrained by various biotic and abiotic and stresses. Breeding and cultivation of rice varieties with resistance to multiple pathogens and environmental stresses is the most effective strategy to mitigate the adverse effect of pathogen attacks and abiotic stresses. Recently, researchers have focused on the exploitation of CRISPR/Cas9 technology to manipulate some negative defense-regulator genes to generate rice varieties with broad-spectrum resistance against rice pathogens. In this study, four negative regulator genes of rice blast, OsMads26, OsBsr-1, OsELF3-2 and OsERF922, were selected as CRISPR/Cas9 targets. By simultaneously knocking out all four genes via CRISPR/Cas9 technology, we created three mads26/bsr-1/elf3-2/erf922 quadruple knockout mutants. Our results demonstrated that all quadruple mutants exhibited much higher resistance not only to rice blast and bacterial blight but also to drought and salt stresses than the wildtype. Interestingly, grain yield of all three quadruple mutants was also drastically increased by 17.35% to 21.95%. Therefore, this study provides a novel strategy to rapidly improve rice varieties with broad-spectrum resistance to pathogens, elevated tolerance to abiotic stresses and enhanced yield potential.},
}
@article {pmid41596458,
year = {2026},
author = {Ionas, K and Vukosavljev, M and Bulić, E and Radanović, A and Jocić, S and Kondić-Špika, A and Miladinović, D},
title = {Beyond the Bottleneck: Predicting Regeneration Potential in Sunflower Through Integrated Morphological and Statistical Profiling.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
pmid = {41596458},
issn = {1422-0067},
support = {101081974//European Commission/ ; },
mesh = {*Helianthus/genetics/physiology/growth &amp; development ; *Regeneration/genetics ; Genotype ; Plant Shoots/growth &amp; development/genetics ; Plant Roots/growth &amp; development/genetics ; Plant Breeding ; Gene Expression Regulation, Plant ; Sucrose/pharmacology ; Cluster Analysis ; },
abstract = {This study presents the first integrated analysis of genotype-medium interactions and temporal morphogenesis profiling in sunflower regeneration. It aims to characterize genotype-specific responses, identify predictive morphological markers, and develop a scalable framework for breeding and transformation. Eighteen sunflower genotypes were evaluated to assess organogenic performance. The model genotype Ha-26-PR was used for a complementary experiment, testing varying sucrose concentrations to examine their influence on morphogenic outcomes. Hierarchical Cluster Analysis (HCA), guided by the Elbow method, identified four optimal clusters (K = 4). These aligned with three biologically meaningful categories: High Regenerators (Cluster 1), Moderate/Specific Regenerators (Clusters 2 and 3), and Non-Regenerators (Cluster 4). On S1 medium, NO-SU-12 and AS-1-PR showed superior shoot regeneration, while on R4 medium, HA-26-PR-SU and NO-SU-12 performed best. Genotypes such as NO-SU-12 and AS-1-PR consistently excelled across both media, whereas AB-OR-8 and FE-7 remained non-regenerators. Medium R4 supported superior regeneration, primarily through root formation, while S1 failed to induce roots in any genotype, highlighting the importance of hormonal composition. Although sucrose promoted callus induction, it did not trigger organogenesis. Callus was consistently present across media and time points, but its correlations with shoot and root formation were weak and temporally unstable, limiting its predictive value. Root formation at 14 days (Root 14D) emerged as a robust early predictor of organogenic success. This integration of morphological, temporal, and statistical analyses offers a genotype-tailored regeneration framework with direct applications in molecular breeding and CRISPR/Cas-based genome editing.},
}
@article {pmid41596567,
year = {2026},
author = {Kosmas, CE and Rallidis, LS and Hoursalas, I and Papakonstantinou, EJ and Kostara, CE},
title = {Angiopoietin-like Protein 3 (ANGPTL3) Targeting in the Management of Dyslipidemias.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
pmid = {41596567},
issn = {1422-0067},
mesh = {Humans ; *Angiopoietin-Like Protein 3/antagonists &amp; inhibitors/metabolism/genetics ; *Angiopoietin-like Proteins/antagonists &amp; inhibitors/metabolism/genetics ; *Dyslipidemias/drug therapy/metabolism/genetics ; Animals ; Antibodies, Monoclonal/therapeutic use/pharmacology ; Oligonucleotides, Antisense/therapeutic use ; RNA, Small Interfering/therapeutic use ; },
abstract = {Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality, despite advances in pharmacological prevention and treatment. The burden of CVD necessitates implementing the treatment of risk factors including dyslipidemia. Pharmaceutical advancements and in depth understanding of pathophysiology have enabled innovative therapies targeting pathways underlying lipoprotein metabolism disorders. Angiopoietin protein-like 3 (ANGPTL3) plays a crucial role in the regulation of lipoprotein metabolism, therefore being a potential therapeutic target. Inhibition of ANGPTL3 has emerged as a new therapeutic strategy to reduce LDL-cholesterol levels independent of the LDL receptor function. Therapeutic approaches for ANGPTL3 inhibition range from monoclonal antibodies to nucleic acid therapeutics including antisense oligonucleotides and small interfering RNAs. In this review, we briefly explain the structure and mechanism of action of ANGPTL3 and discuss the therapeutic approaches for targeting ANGPTL3 in the clinical setting. We also discuss Evinacumab, a monoclonal antibody, its structure, mechanism of action, safety, tolerability, pharmacokinetics, and pharmacodynamics, as well as its clinical trial-derived results. The antisense oligonucleotides modify ANGPTL3 mRNA to inhibit protein production, and small interfering RNAs induce mRNA degradation; results from clinical trials were reviewed in detail. Finally, we discuss promising gene editing approaches including clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems.},
}
@article {pmid41596664,
year = {2026},
author = {Luo, X and Ding, Y and Wang, Z and Liu, J},
title = {MED12 Dictates Epithelial Ovarian Cancer Cell Ferroptosis Sensitivity via YAP-TEAD1 Signaling.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
pmid = {41596664},
issn = {1422-0067},
support = {82102732//National Natural Science Foundation of China/ ; 82272698//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Ferroptosis/drug effects/genetics ; Female ; *Carcinoma, Ovarian Epithelial/metabolism/genetics/pathology ; *Transcription Factors/metabolism/genetics ; *Signal Transduction ; Cell Line, Tumor ; YAP-Signaling Proteins ; TEA Domain Transcription Factors ; *Ovarian Neoplasms/metabolism/genetics/pathology ; *DNA-Binding Proteins/metabolism/genetics ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; *Nuclear Proteins/metabolism/genetics ; Drug Resistance, Neoplasm ; Cysteine-Rich Protein 61/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Epithelial ovarian cancer (EOC) represents the most lethal malignancy arising from the female reproductive tract, largely due to the clinical challenge of chemotherapy resistance. Recent studies indicate that ferroptosis-a distinct form of programmed cell death driven by iron accumulation and lipid peroxidation, could potentially exploit a vulnerability in chemoresistant cancer cells. Here, we identify MED12 as a critical regulator of ferroptosis sensitivity in EOC through modulation of the YAP-TEAD1 signaling pathway. Using CRISPR/Cas9-mediated knockout and rescue experiments in EOC cell lines, we demonstrate that MED12 deficiency significantly enhances sensitivity to ferroptosis inducers (RSL3 and Erastin), as evidenced by reduced IC50 values. Transcriptomic and chromatin accessibility analyses reveal that MED12 loss activates YAP signaling through TEAD1 upregulation, increasing chromatin accessibility at YAP-TEAD1 target loci and elevating the expression of downstream effectors CYR61 and CTGF. Pharmacological inhibition of YAP with verteporfin or siRNA-mediated TEAD1 knockdown reverses ferroptosis sensitivity in MED12-deficient cells, confirming pathway specificity. These findings establish MED12 as a modulator of the YAP-TEAD1-ferroptosis axis and suggest that targeting this pathway could overcome chemoresistance in MED12-deficient EOC. Our work provides a mechanistic foundation for exploiting ferroptosis induction as a therapeutic strategy in ovarian cancer.},
}
@article {pmid41596696,
year = {2026},
author = {Boren, C and Kumar, R and Gollahon, L},
title = {In Silico Design and Characterization of a Rationally Engineered Cas12j2 Gene Editing System for the Treatment of HPV-Associated Cancers.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
pmid = {41596696},
issn = {1422-0067},
support = {NA//Texas Tech University Association of Biologists Grants-in-Aid/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/therapy/virology/genetics ; *Neoplasms/therapy/virology/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Oncogene Proteins, Viral/genetics ; Computer Simulation ; },
abstract = {CRISPR-Cas9 systems have enabled unprecedented advances in genome engineering, particularly in developing treatments for human diseases, like cancer. Despite potential applications, limitations of Cas9 include its relatively large size and strict targeting requirements. Cas12j2, a variant ofCasΦ-2, shows promise for overcoming these limitations. However, its effectiveness in mammalian cells remains relatively unexplored. This study sought to develop an optimized CRISPR-Cas12j2 system for targeted knockout of the E6 oncogene in HPV-associated cancers. A combination of computational tools (ColabFold, CCTop, Cas-OFFinder, HADDOCK2.4, and Amber for Molecular Dynamics) was utilized to investigate the impact of engineered modifications on structural integrity and gRNA binding of Cas12j2 fusion constructs, in potential intracellular conditions. Cas12j2_F2, a Cas12j2 variant designed and evaluated in this study, behaves similarly to the wild-type Cas12j2 structure in terms of RMSD/RMSF profiles, compact Rg values, and minimal electrostatic perturbation. The computationally validated Cas12j2 variant was incorporated into a custom expression vector, co-expressing the engineered construct along with a dual gRNA for packaging into a viral vector for targeted knockout of HPV-associated cancers. This study provides a structural and computational foundation for the rational design of Cas12j2 fusion constructs with enhanced stability and functionality, supporting their potential application for precise genome editing in mammalian cells.},
}
@article {pmid41596715,
year = {2026},
author = {Ansari, RA and Rezaee Danesh, Y and Castello, I and Vitale, A},
title = {Molecular Identification and RNA-Based Management of Fungal Plant Pathogens: From PCR to CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {27},
number = {2},
pages = {},
pmid = {41596715},
issn = {1422-0067},
mesh = {*CRISPR-Cas Systems ; *Plant Diseases/microbiology/prevention &amp; control/genetics ; *Fungi/genetics/pathogenicity ; Polymerase Chain Reaction/methods ; *RNA, Fungal/genetics ; Plants/microbiology ; },
abstract = {Fungal diseases continue to limit global crop production and drive major economic losses. Conventional diagnostic and control approaches depend on time-consuming culture-based methods and broad-spectrum chemicals, which offer limited precision. Advances in molecular identification have changed this landscape. PCR, qPCR, LAMP, sequencing and portable platforms enable rapid and species-level detection directly from plant tissue. These tools feed into RNA-based control strategies, where knowledge of pathogen genomes and sRNA exchange enables targeted suppression of essential fungal genes. Host-induced and spray-induced gene silencing provide selective control without the long-term environmental costs associated with chemical use. CRISPR/Cas9 based tools now refine both diagnostics and resistance development, and bioinformatics improves target gene selection. Rising integration of artificial intelligence indicates a future in which disease detection, prediction and management connect in near real time. The major challenge lies in limited field validation and the narrow range of fungal species with complete molecular datasets, yet coordinated multi-site trials and expansion of annotated genomic resources can enable wider implementation. The combined use of molecular diagnostics and RNA-based strategies marks a shift from disease reaction to disease prevention and moves crop protection towards a precise, sustainable and responsive management system. This review synthesizes the information related to current molecular identification tools and RNA-based management strategies, and evaluates how their integration supports precise and sustainable approaches for fungal disease control under diverse environmental settings.},
}
@article {pmid41596857,
year = {2026},
author = {Tsouggou, N and Korozi, E and Pemaj, V and Drosinos, EH and Kapolos, J and Papadelli, M and Skandamis, PN and Papadimitriou, K},
title = {Advances in Shotgun Metagenomics for Cheese Microbiology: From Microbial Dynamics to Functional Insights.},
journal = {Foods (Basel, Switzerland)},
volume = {15},
number = {2},
pages = {},
pmid = {41596857},
issn = {2304-8158},
abstract = {The cheese microbiome is a complex ecosystem strongly influenced by both technological practices and the processing environment. Moving beyond traditional cultured-based methods, the integration of shotgun metagenomics into cheese microbiology has enabled in-depth resolution of microbial communities at the species and strain levels. The aim of the present study was to review recent applications of shotgun metagenomics in cheese research, underscoring its role in tracking microbial dynamics during production and in discovering genes of technological importance. In addition, the review highlights how shotgun metagenomics enables the identification of key metabolic pathways, including amino acid catabolism, lipid metabolism, and citrate degradation, among others, which are central to flavor formation and ripening. Results of the discussed literature demonstrate how microbial composition, functional traits, and overall quality of cheese are determined by factors such as raw materials, the cheesemaking environment, and artisanal practices. Moreover, it highlights the analytical potentials of shotgun metagenomics, including metagenome-assembled genomes (MAGs) reconstruction, characterization of various genes contributing to flavor-related biosynthetic pathways, bacteriocin production, antimicrobial resistance, and virulence, as well as the identification of phages and CRISPR-Cas systems. These insights obtained are crucial for ensuring product's authenticity, enabling traceability, and improving the assessment of safety and quality. Despite shotgun metagenomics' advantages, there are still analytical restrictions concerning data handling and interpretation, which need to be addressed by importing standardization steps and moving towards integrating multi-omics approaches. Such strategies will lead to more accurate and reproducible results across studies and improved resolution of active ecosystems. Ultimately, shotgun metagenomics has shifted the field from descriptive surveys to a more detailed understanding of the underlying mechanisms shaping the overall quality and safety of cheese, thus bringing innovation in modern dairy microbiology.},
}
@article {pmid41597209,
year = {2026},
author = {Ren, Z and Zhou, J and Yang, D and Guo, Y and Zhang, J and Xu, J and Chen, YE},
title = {Gene Editing Therapies Targeting Lipid Metabolism for Cardiovascular Disease: Tools, Delivery Strategies, and Clinical Progress.},
journal = {Cells},
volume = {15},
number = {2},
pages = {},
pmid = {41597209},
issn = {2073-4409},
support = {R42 GM122181/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *Cardiovascular Diseases/therapy/genetics/metabolism ; *Lipid Metabolism/genetics ; *Genetic Therapy/methods ; Animals ; *Gene Transfer Techniques ; CRISPR-Cas Systems ; },
abstract = {Gene editing technologies have revolutionized therapeutic development, offering potentially curative and preventative strategies for cardiovascular disease (CVD), which remains a leading global cause of morbidity and mortality. This review provides an introduction to the state-of-the-art gene editing tools-including ZFNs, TALENs, CRISPR/Cas9 systems, base editors, and prime editors-and evaluates their application in lipid metabolic pathways central to CVD pathogenesis. Emphasis is placed on targets such as PCSK9, ANGPTL3, CETP, APOC3, ASGR1, LPA, and IDOL, supported by findings from human genetics, preclinical models, and recent first-in-human trials. Emerging delivery vehicles (AAVs, LNPs, lentivirus, virus-like particles) and their translational implications are discussed. The review highlights ongoing clinical trials employing liver-targeted in vivo editing modalities (LivGETx-CVD) and provides insights into challenges in delivery, off-target effects, genotoxicity, and immunogenicity. Collectively, this review captures the rapid progress of LivGETx-CVD from conceptual innovation to clinical application, and positions gene editing as a transformative, single-dose strategy with the potential to redefine prevention and long-term management of dyslipidemia and atherosclerotic cardiovascular disease.},
}
@article {pmid41597233,
year = {2026},
author = {Bao, C and Channell, CI and Tseng, YH and Bailey, J and Sbaiti, N and Demirkol, A and Tsang, SH},
title = {Chronic In Vivo CRISPR-Cas Genome Editing: Challenges, Long-Term Safety, and Outlook.},
journal = {Cells},
volume = {15},
number = {2},
pages = {},
pmid = {41597233},
issn = {2073-4409},
support = {the Foundation Fighting Blindness TA-GT-0321-0802-COLU-TRAP, the Lynette and Richard Jaffe Foundation, NYEE Foundation, the Rosenbaum Family Foundation, the Gebroe Family Foundation, the Research to Prevent Blindness (RPB) Physician-Scientist Award, unres//Jonas Children's Vision Care is supported by the National Institute of Health U01EY030580, U01EY034590 R24EY028758, R24EY027285, R01EY033770, R01EY018213, R01EY024698,/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; Genetic Therapy/methods ; },
abstract = {CRISPR/Cas systems have transformed molecular medicine, yet the field still lacks principled guidance on when transient editing suffices versus when sustained exposure through in vivo viral delivery is necessary and how to keep prolonged exposure safe. Notably, EDIT-101 was designed for a permanent edit in post-mitotic photoreceptors with lifelong Cas9 persistence. This review addresses this gap by defining the biological and therapeutic conditions that drive benefit from extended Cas activity while minimizing risk. We will (i) examine relationships between expression window and efficacy across Cas9/Cas12/Cas13 modalities, (ii) identify genome-wide off-target liabilities alongside orthogonal assays, and (iii) discuss controllable, self-limiting, and recallable editor platforms. By separating durable edits from persistent nuclease exposure, and by providing validated control levers, this work establishes a generalizable framework for safe, higher-efficacy CRISPR medicines. Furthermore, we highlight key studies in cell lines, murine models, non-human primates, and humans that examine the long-term effects of sustained expression of CRISPR/Cas systems and discuss the safety and efficacy of such approaches. Current evidence demonstrates promising therapeutic outcomes with manageable safety profiles, although there is a need for continued monitoring as CRISPR/Cas therapies are increasingly applied in clinical contexts and therapies are developed for broader clinical applications.},
}
@article {pmid41598172,
year = {2025},
author = {Magyar-Tábori, K and Udupa, SM and Hanász, A and Juhász, C and Mendler-Drienyovszki, N},
title = {Rising Demand for Winter Crops Under Climate Change: Breeding for Winter Hardiness in Autumn-Sown Legumes.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {1},
pages = {},
pmid = {41598172},
issn = {2075-1729},
abstract = {Climate change in the Pannonian region is accelerating a shift toward autumn sowing of cool-season grain legumes (pea, faba bean, lentil, chickpea, lupine) to achieve higher yields, greater biomass production, enhanced nitrogen fixation, improved soil cover, and superior resource use efficiency compared with spring sowing. However, successful overwintering depends on the availability of robust winter-hardy cultivars. This review synthesizes recent breeding advances, integrating traditional approaches-such as germplasm screening, hybridization, and field-based selection-with genomics-assisted strategies, including genome-wide association studies (GWAS), quantitative trait locus (QTL) mapping, marker-assisted selection (MAS), and CRISPR/Cas-mediated editing of CBF transcription factors. Key physiological mechanisms-LT50 determination, cold acclimation, osmoprotectant accumulation (sugars, proline), and membrane stability-are assessed using field survival rates, electrolyte leakage assays, and chlorophyll fluorescence measurements. Despite challenges posed by genotype × environment interactions, variable winter severity, and polygenic trait control, the release of cultivars worldwide (e.g., 'NS-Mraz', 'Lavinia F', 'Ghab series', 'Pinklevi', and 'Rézi') and ongoing breeding programs demonstrate substantial progress. Future breeding efforts will increasingly rely on genomic selection (GS), high-throughput phenomics, pangenomics, and G×E modeling to accelerate the development of climate-resilient legume cultivars, ensuring stable and sustainable production under increasingly unpredictable winter conditions.},
}
@article {pmid41598889,
year = {2025},
author = {Wang, Y and Zhang, C and Li, MJ and Iqbal, A and Ahmed, KS and Idrees, A and Habiba, and Yang, BM and Jiang, L},
title = {Exploring the Role of Pheromones and CRISPR/Cas9 in the Behavioral and Olfactory Mechanisms of Spodoptera frugiperda.},
journal = {Insects},
volume = {17},
number = {1},
pages = {},
pmid = {41598889},
issn = {2075-4450},
support = {2024C014-2//Innovation Capacity Building Project of the Jilin Provincial Development and Reform Commission./ ; },
abstract = {Globally, Spodoptera frugiperda is a major threat to many important crops, including maize, rice, and cotton, causing significant economic damage. To control this invasive pest, environmentally friendly pest control techniques, including pheromone detection and identification of potential molecular targets to disrupt S. frugiperda mating communication, are needed. Female moths biosynthesize pheromones and emit them from the pheromone gland, which significantly depends on the intrinsic factors of the moth. Male S. frugiperda have a sophisticated olfactory circuit on their antennae that recognizes pheromone blends via olfactory receptor neurons (ORNs). With its potential to significantly modify the insect genome, CRISPR/Cas9 offers a revolutionary strategy to control this insect pest. The impairing physiological behaviors and disrupting the S. frugiperda volatile-sensing mechanism are the main potential applications of CRISPR/Ca9 explored in this review. Furthermore, the release of mutant S. frugiperda for their long-term persistence must be integral to the adoption of this technology. Looking forward, CRISPR/Cas9-based gene drive systems have the potential to synergistically target pheromone signaling pathways in S. frugiperda by disrupting pheromone receptors and key biosynthesis genes, thereby effectively blocking intraspecific communication and reproductive success. In conclusion, CRISPR/Cas9 provides an environmentally friendly and revolutionary platform for precise, targeted pest management in S. frugiperda.},
}
@article {pmid41598917,
year = {2026},
author = {Pei, X and Xie, J and Liang, C and Utkina, AO},
title = {Next-Generation Precision Breeding in Peanut (Arachis hypogaea L.) for Disease and Pest Resistance: From Multi-Omics to AI-Driven Innovations.},
journal = {Insects},
volume = {17},
number = {1},
pages = {},
pmid = {41598917},
issn = {2075-4450},
abstract = {Peanut (Arachis hypogaea L.) is a globally important oilseed and food legume, yet its productivity is persistently constrained by devastating diseases and insect pests that thrive under changing climates. This review aims to provide a comprehensive synthesis of advances in precision breeding and molecular approaches for enhancing disease and pest resistance in peanut. Traditional control measures ranging from crop rotation and cultural practices to chemical protection have delivered only partial and often unsustainable relief. The narrow genetic base of cultivated peanut and its complex allotetraploid genome further hinder the introgression of durable resistance. Recent advances in precision breeding are redefining the possibilities for resilient peanut improvement. Multi-omics platforms genomics, transcriptomics, proteomics, and metabolomics have accelerated the identification of resistance loci, effector-triggered immune components, and molecular cross-talk between pathogen, pest, and host responses. Genome editing tools such as CRISPR-Cas systems now enable the precise modification of susceptibility genes and defense regulators, overcoming barriers of conventional breeding. Integration of these molecular innovations with phenomics, machine learning, and remote sensing has transformed resistance screening from manual assessment to real-time, data-driven prediction. Such AI-assisted breeding pipelines promise enhanced selection accuracy and faster deployment of multi-stress-tolerant cultivars. This review outlines current progress, technological frontiers, and persisting gaps in leveraging precision breeding for disease and pest resistance in peanut, outlining a roadmap toward climate-resilient, sustainable production systems.},
}
@article {pmid41599009,
year = {2025},
author = {Lv, YR and Liu, YY and Zhang, R and Yang, B and Xue, SY and Ding, YL and Jia, JT and Bayaer, H and Bagen, A and Chen, RB and Tunala, S and Zhao, L and Liu, YH},
title = {Rapid and Simple Detection of Mycobacterium avium subsp. paratuberculosis Using a Lateral Flow Assay Based on CRISPR-Cas12a Combined with Recombinase Polymerase Amplification or Nested PCR.},
journal = {Pathogens (Basel, Switzerland)},
volume = {15},
number = {1},
pages = {},
pmid = {41599009},
issn = {2076-0817},
support = {1.(YF20240007) 2.(BR251303) 3.(YLXKZX-NND-012)//1.Ordos Science &amp; Technology Plan 2.Special Project for the Construction of Scientific Research and Innovation Teams, Class B Team 3.Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; },
mesh = {*CRISPR-Cas Systems ; *Mycobacterium avium subsp. paratuberculosis/genetics/isolation &amp; purification ; *Paratuberculosis/diagnosis/microbiology ; *Polymerase Chain Reaction/methods ; Animals ; Sensitivity and Specificity ; Feces/microbiology ; Recombinases/genetics ; DNA, Bacterial/genetics ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Paratuberculosis (PTB), caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic intestinal disease in ruminants. PTB is difficult to diagnose, control, and eradicate, leading to substantial economic losses. Thus, sensitive and specific detection methods are urgently required. crRNA and primers targeting the MAP ATPase FtsK gene were designed for recombinase polymerase amplification (RPA) and nested PCR. Fecal DNA was amplified using RPA or nested PCR, purified with Tris-saturated phenol-chloroform-isoamyl alcohol, and detected via CRISPR-Cas12a. Moreover, signals were read using a qPCR instrument, fluorescence reader, or lateral flow strips. RPA-CRISPR-Cas12a and nested PCR-CRISPR-Cas12a assays were optimized and validated on 50 clinical samples and 7 MAP cultures. The limits of detection were 1 × 10[-10] μg/μL for RPA-CRISPR-Cas12a and 1 × 10[-14] μg/μL for nested PCR-CRISPR-Cas12a. Efficient cleavage of the ssDNA reporter occurred at DNA concentrations of ≥1 × 10[-4] μg/μL, producing a strong fluorescent signal. All three detection methods showed perfect agreement with reference assays across both sample sets. This study presents the first integration of RPA or nested PCR with CRISPR-Cas12a for MAP detection, enabling rapid, specific, and highly sensitive diagnosis. Flexible detection options allow adaptation to available resources and bacterial loads, supporting practical use in PTB control.},
}
@article {pmid41599202,
year = {2026},
author = {Yoon, B and Kim, JA and Kang, YK},
title = {CRISPR-Cas-Mediated Reprogramming Strategies to Overcome Antimicrobial Resistance.},
journal = {Pharmaceutics},
volume = {18},
number = {1},
pages = {},
pmid = {41599202},
issn = {1999-4923},
support = {grant number RS-2024-00417430//National Research Foundation of Korea (NRF)/ ; grant number RS-2024-00399808//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET)/ ; 2025-RISE-16-001//Regional Innovation System &amp; Education (RISE) program/ ; A26234, CTRQQR-2021\100009//CRUK Convergence Science Centre at The Institute of Cancer Research, London, and Imperial Col-lege London/ ; },
abstract = {Antimicrobial resistance (AMR) is escalating worldwide, posing a serious threat to global public health by driving infections that are no longer treatable with conventional antibiotics. CRISPR-Cas technology offers a programmable and highly specific therapeutic alternative by directly targeting the genetic determinants responsible for resistance. Various CRISPR systems can restore antibiotic susceptibility and induce selective bactericidal effects by eliminating resistance genes, disrupting biofilm formation, and inhibiting virulence pathways. Moreover, CRISPR can suppress horizontal gene transfer (HGT) by removing mobile genetic elements such as plasmids, thereby limiting the ecological spread of AMR across humans, animals, and the environment. Advances in delivery platforms-including conjugative plasmids, phagemids, and nanoparticle-based carriers-are expanding the translational potential of CRISPR-based antimicrobial strategies. Concurrent progress in Cas protein engineering, spatiotemporal activity regulation, and AI-driven optimization is expected to overcome current technical barriers. Collectively, these developments position CRISPR-based antimicrobials as next-generation precision therapeutics capable of treating refractory bacterial infections while simultaneously suppressing the dissemination of antibiotic resistance.},
}
@article {pmid41599334,
year = {2026},
author = {Wang, X and Chen, X and Zhou, Y and Zhao, Y and Shi, C and Li, R and Liu, L and Mu, C and Song, W and Wang, C},
title = {Establishment of CRISPR-Cas9-Mediated Gene Editing in the Swimming Crab Portunus trituberculatus.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {2},
pages = {},
pmid = {41599334},
issn = {1420-3049},
support = {2022YFD2400104//the National Key R&amp;D Program of China/ ; 32341058 and 31972783//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Brachyura/genetics ; Electroporation ; Myostatin/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; INDEL Mutation ; },
abstract = {Portunus trituberculatus is an economically important marine crustacean in East Asia's aquaculture industry. Nevertheless, precise genome modification has not yet been established. In this study, we evaluated the applicability of the CRISPR-Cas9 gene editing system in P. trituberculatus using electroporation for efficient delivery of the Cas9-sgRNA complex into zygotes. We systematically investigated electroporation parameters, including buffer composition, voltage, capacitance, and pulse times. Our results showed that artificial seawater was a superior buffer to phosphate-buffered saline (PBS) and identified an effective electroporation condition of 600 V, 1 μF capacitance, and two pulses, resulting in approximately 72.7% fluorescent zygotes. Under these electroporated conditions, we detected gene indels and putative insertion events at the targeted locus of myostatin (mstn) gene. These results demonstrate the feasibility of Cas9-based genome editing in P. trituberculatus and provide a proof-of-concept for functional genomics studies and future genetic improvement of this species.},
}
@article {pmid41600812,
year = {2025},
author = {Tian, C and Feng, L and Zhou, X and Huang, K and Wang, F and Luo, R and Meng, F and Yang, H and Qiao, C and Wang, X and Shi, J and Chen, Y},
title = {A Portable One-Tube Assay Integrating RT-RPA and CRISPR/Cas12a for Rapid Visual Detection of Eurasian Avian-like H1N1 Swine Influenza Virus in the Field.},
journal = {Viruses},
volume = {18},
number = {1},
pages = {},
pmid = {41600812},
issn = {1999-4915},
support = {2021YFD1800200//Nnational Key Research and Development Program of China/ ; },
mesh = {*Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; Animals ; *CRISPR-Cas Systems ; Swine ; Sensitivity and Specificity ; *Orthomyxoviridae Infections/diagnosis/virology/veterinary ; *Swine Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Humans ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The widespread circulation of Eurasian avian-like H1N1 (EA H1N1) swine influenza virus poses significant zoonotic and pandemic risks worldwide. However, current diagnostic methods are difficult to deploy in the field, as they generally require specialized laboratory infrastructure and trained personnel. Here, we present a novel dual-signal detection platform that combines reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology for rapid, on-site EA H1N1 detection. We established an integrated one-tube assay by designing and optimizing RT-RPA primers targeting a conserved region of the hemagglutinin (HA) gene, together with engineered CRISPR/Cas12a guide RNAs exhibiting high specificity. The platform incorporates two complementary readout modes: real-time fluorescence monitoring and visual colorimetric detection using a smartphone. The assay shows excellent analytical specificity, with no cross-reactivity observed against other swine influenza virus subtypes or common swine pathogens, (including CSFV, PRRSV, PEDV, PCV, TGEV, and RV). The detection limit is 2 copies/μL, and the entire procedure can be completed within 30 mins using simple portable equipment. When evaluated on 86 clinical samples, the assay demonstrated 94.18% concordance with RT-qPCR. Compared with conventional diagnostic methods, this RT-RPA-CRISPR/Cas12a assay offers greater convenience and cost-effectiveness. Its strong potential for field-based rapid testing underscores promising application prospects in swine influenza surveillance and control programs.},
}
@article {pmid41601145,
year = {2026},
author = {Jiao, Y and Liu, Y and Sun, F and Wang, W and Li, H and Gao, Q and Li, Y and Lu, N and Tian, X and Ding, X and Du, J},
title = {Genetically Modified Plant Beneficial Microorganisms: A Sustainable Solution or a New Challenge for Agriculture.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {5},
pages = {4235-4248},
doi = {10.1021/acs.jafc.5c14342},
pmid = {41601145},
issn = {1520-5118},
mesh = {*Plant Diseases/microbiology/prevention &amp; control/immunology ; *Bacteria/genetics/metabolism ; Genetic Engineering ; *Microorganisms, Genetically-Modified/genetics/physiology ; *Plants, Genetically Modified/microbiology/genetics ; *Crops, Agricultural/microbiology/genetics/immunology ; Agriculture ; *Plants/microbiology/genetics/immunology ; },
abstract = {Plant diseases significantly impact crop yield and quality, while conventional pesticide treatments often disrupt beneficial plant microbiota essential for pathogen prevention and immune regulation. Although plant beneficial microorganisms (PBMs) show promise as disease control agents, their effectiveness is constrained by strain-dependent variations, survival challenges, and inconsistent immune responses. Recent advances in genetic engineering, particularly CRISPR-Cas systems combined with complementary technologies like RecE/T, enable precise modifications of PBMs to enhance their protective potential. Enhanced PBMs improve functionality via multiple mechanisms: targeted gene-expression-mediated colonization, specific antimicrobial activity, and immune regulation. Studies demonstrate that genetically modified PBMs can prevent and control plant diseases through competitive exclusion, antibiotic production, barrier reinforcement, and immune modulation. We analyzed the considerations for the environmental release of engineered PBMs to reduce risks. Future research should focus on optimizing PBMs for specific applications while addressing biosafety concerns, thereby unlocking their full potential in safeguarding plant health.},
}
@article {pmid41601654,
year = {2025},
author = {Yin, L and He, W and Wang, Y and Zhang, H and Huang, M and Yan, Y and Li, S and Feng, X and Saenz, F and Zhang, J and Zhu, D and Yang, C and Ma, T and Fu, J and Chen, J},
title = {FACS-based genome-wide CRISPR screening platform identifies modulators of CD47.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1684539},
pmid = {41601654},
issn = {1664-3224},
mesh = {*CD47 Antigen/genetics/metabolism/immunology ; Animals ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Humans ; Flow Cytometry/methods ; Phagocytosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Macrophages/immunology/metabolism ; Female ; Immunity, Innate ; },
abstract = {BACKGROUND: CD47 is a key innate immune checkpoint that enables tumor cells to evade macrophage-mediated clearance.<br><br>METHODS/RESULTS: To systematically identify genetic regulators of CD47 surface expression, we performed FACS-based genome-wide CRISPR screens in three murine cancer cell lines B16 (melanoma), MC38 (colon adenocarcinoma), and EMT6 (breast carcinoma).<br><br>RESULTS: Comparative analysis of cells with high or low CD47 surface expression using DrugZ revealed CD47 itself as the top hit, validating the screens. Notably, DNAJC13 emerged as a consistent and robust regulator of CD47 expression across all three cell lines. Functional validation using DNAJC13-knockout cells confirmed a significant reduction in CD47 surface levels. Furthermore, in co-culture assays with macrophages, DNAJC13-deficient tumor cells exhibited increased susceptibility to phagocytosis, supporting a functional role for DNAJC13 in innate immune evasion. Finally, we verify that DNAJC13-knockout decrease tumor burden when treated with CD47 blockade.<br><br>CONCLUSIONS: Overall, this study highlights a previously unrecognized regulator of CD47 and demonstrates the utility of high-throughput FACS-based CRISPR screening to uncover modulators of key immune checkpoint pathways.},
}
@article {pmid41603018,
year = {2026},
author = {Dolder, RE and Friedman, CE and Loiben, AM and Yang, KC and Glazer, AM},
title = {High-Throughput Methods for Variant Functional Assessment in Cardiac Disease.},
journal = {Circulation. Genomic and precision medicine},
volume = {19},
number = {1},
pages = {e005239},
pmid = {41603018},
issn = {2574-8300},
support = {R35 GM150465/GM/NIGMS NIH HHS/United States ; I01 BX006428/BX/BLRD VA/United States ; F32 HL164108/HL/NHLBI NIH HHS/United States ; R01 HL164675/HL/NHLBI NIH HHS/United States ; R00 HG010904/HG/NHGRI NIH HHS/United States ; R01 HL171174/HL/NHLBI NIH HHS/United States ; K99 HL177347/HL/NHLBI NIH HHS/United States ; 25PRE1360565/AHA/American Heart Association-American Stroke Association/United States ; },
mesh = {Humans ; *High-Throughput Nucleotide Sequencing/methods ; *Heart Diseases/genetics ; *Genetic Variation ; Myocytes, Cardiac/metabolism ; *High-Throughput Screening Assays/methods ; Gene Editing ; Animals ; },
abstract = {In vitro functional modeling of genetic variants has revolutionized our understanding of which variants can cause cardiac disorders, providing insights into their molecular underpinnings. This review provides an overview of high-throughput methods used for the functional assessment of variants implicated in inherited cardiac diseases. Advances in gene-editing technology now enable the efficient generation of cells expressing individual genetic variants or libraries of variants for robust functional studies. We discuss innovative assays that can evaluate dozens or hundreds of variants sequentially. For example, the electrophysiological properties of numerous cardiac ion channel variants in genes linked to inherited arrhythmias can be characterized using automated patch clamping. The mechanical properties of cardiomyocytes expressing candidate cardiomyopathy variants can be assessed using techniques such as atomic force microscopy, traction force microscopy, and impedance-based methods. Multiplexed assays of variant effect are an emerging family of techniques that use gene-specific or general assays, combined with next-generation sequencing, to characterize hundreds or thousands of pooled genetic variants. We examine the key advantages and limitations of each method and outline future goals for the field. Innovative in vitro studies of cardiac genetic variants will enhance our understanding of variant-disease relationships and improve diagnosis, screening, and treatment options for these disorders.},
}
@article {pmid41603277,
year = {2026},
author = {Li, J and Shao, T and Cao, XJ and Wang, YX and Kong, DM},
title = {An allosteric key strand controlled adaptable CRISPR/Cas12a biosensing platform for point-of-care testing of multiple types of targets.},
journal = {Lab on a chip},
volume = {26},
number = {4},
pages = {917-929},
doi = {10.1039/d5lc01029b},
pmid = {41603277},
issn = {1473-0189},
mesh = {*Biosensing Techniques/instrumentation ; *CRISPR-Cas Systems ; *Point-of-Care Testing ; DNA/genetics/chemistry/analysis ; Humans ; Allosteric Regulation ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Currently, the CRISPR/Cas12a based sensor has become a powerful tool for gene editing and molecular diagnostics. However, most CRISPR/Cas12a sensors are primarily limited to the detection of a single target type, due to their strict dependence on the specific recognition of the PAM sequence within a precisely designed double-stranded DNA (dsDNA) and crRNA for cleavage activity regulation. Herein, we designed an allosteric key strand (KS) controlled CRISPR/Cas12a biosensor via toehold-based strand displacement reaction (TSDR). By simply reconfiguring KS into different conformations with functional nucleic acid structures, this sensor could selectively respond to various target molecules from nucleic acids to non-nucleic acid molecules without changing the sequence of crRNA and targeted PAM-dsDNA. The trans-cleavage activity of CRISPR/Cas12a could be triggered through leveraging proximity-based TSDR in response to target binding. The proposed sensor achieved sensitive and specific detection of various targets, including nucleic acids (HPV-16), small molecules (kanamycin), and enzymes (uracil-DNA glycosylase). Furthermore, by integrating lateral flow assay technology, this CRISPR/Cas12a-based system enabled point-of-care testing (POCT) for the detection of multiple target types. This approach can overcome the sequence-specific limitations, thereby improving the versatility of CRISPR/Cas12a sensors for extending more target types detection. We anticipate this innovative technology will serve as a flexible and accessible sensing platform, facilitating rapid diagnosis in the field of POCT and enabling its broader application across diverse biotechnological domains.},
}
@article {pmid41603430,
year = {2026},
author = {Li, W and Yang, Y and Xu, W and Zhu, Y and Li, Y and Cao, L and Lyu, S and Liu, J and Xie, Y and Li, X and Hu, X and Huang, L},
title = {An Extraction-free One-Pot Assay for Rapid Field Discrimination of African Swine Fever Virus Variants by a Single-Step RPA-CRISPR/Cas12a Strategy.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1201-1213},
doi = {10.1021/acssensors.5c03287},
pmid = {41603430},
issn = {2379-3694},
mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Animals ; Swine ; *African Swine Fever/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; },
abstract = {African swine fever virus (ASFV) causes devastating outbreaks in swine populations worldwide. The co-existence of wild-type and emerging gene-deleted variants (e.g., ASFV-ΔI177L) necessitates rapid on-site diagnostic tools for strain identification, which is critical for timely disease control and tailored farm management. Here, we developed a field-deployable, extraction-free one-pot assay (CORDSv2) that combines multiplex RPA and CRISPR/Cas12a to target conserved ASFV sequences and specific fluorescent markers (eGFP/mCherry) of gene-deleted variants. The assay achieved ultrasensitive detection (LOD: 6 copies/μL) and demonstrated 97.9% accuracy with 96 field samples. To streamline field operations, we integrated an extraction-free protocol (for serum/saliva) with freeze-dried reagent microspheres, enabling stable storage and direct use with minimal manual handling. When paired with a portable fluorometer (hippoCORDS), the system completes the entire sample-to-answer workflow within 1 h with a single step: addition of lysate to the microspheres. This robust, portable system addresses the urgent need for simple, on-site ASFV variant surveillance and is adaptable for other high-threat pathogens.},
}
@article {pmid41603729,
year = {2026},
author = {Huang, Y and Xu, H and Zhang, T and Liao, Y and Hu, J and Huang, Z and Hu, H and Li, P and Fan, L and Xie, J},
title = {Mycobacterial non-homologous end joining is required for antiphage defense.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603729},
issn = {1362-4962},
support = {82472325//National Natural Science Foundation/ ; 82072246//National Natural Science Foundation/ ; CSTB2024NSCQ-MSX0703//Natural Science Foundation of Chongqing/ ; //Chongqing Public Health Key Specialty (Discipline) Construction Project/ ; CSTB2024NSCQ-MSX0703//Natural Science Foundation of Chongqing/ ; },
mesh = {*DNA End-Joining Repair ; *Mycobacterium smegmatis/virology/genetics ; DNA Breaks, Double-Stranded ; Oxidative Stress ; Homologous Recombination ; },
abstract = {In the ongoing arms race with phages, bacteria have evolved diverse defense systems, such as CRISPR-Cas and restriction-modification systems. The DNA double-strand break repair system represents a core mechanism for maintaining genomic integrity and is vital for cell survival. However, it remains unknown whether and how these repair systems contribute to phage resistance. This study systematically investigates the role of the non-homologous end joining (NHEJ) during phage infection in Mycobacterium smegmatis. We found that NHEJ deficiency compromises host resistance to phage SWU1, as evidenced by increased plaque counts and reduced bacterial survival. Mechanistically, phages exploit host NHEJ for genomic repair; however, the error-prone nature of NHEJ leads to imperfect repair at phage cos sites, thereby blocking replication. The host modulates the balance between NHEJ and homologous recombination (HR) to control repair fidelity: NHEJ loss shifts the balance toward high-fidelity HR, which in turn promotes phage survival. Furthermore, NHEJ deficiency exacerbates infection-induced oxidative stress, leading to a compromise in bacterial viability. Our findings reveal the multifaceted functions of NHEJ in mycobacterium-phage interactions and provide new insights into how DNA repair systems shape antiphage defense and coevolution.},
}
@article {pmid41603733,
year = {2026},
author = {Kwon, H and Kim, J and Zhou, L and Dean, A},
title = {LDB1 regulates gene expression and chromatin structure in pluripotency and lineage differentiation.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603733},
issn = {1362-4962},
support = {//Intramural Program of the National Institute of Diabetes/ ; //Digestive and Kidney Diseases/ ; 075033/GF/NIH HHS/United States ; },
mesh = {Animals ; *Cell Differentiation/genetics ; Mice ; Kruppel-Like Factor 4 ; *Chromatin/chemistry/metabolism/genetics ; Cell Lineage/genetics ; *DNA-Binding Proteins/genetics/physiology/metabolism ; SOXB1 Transcription Factors/metabolism/genetics ; Embryoid Bodies/metabolism/cytology ; Erythroblasts/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; Kruppel-Like Transcription Factors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Mice, Knockout ; CRISPR-Cas Systems ; *Pluripotent Stem Cells/metabolism/cytology ; Enhancer Elements, Genetic ; Embryonic Stem Cells/metabolism/cytology ; *Gene Expression Regulation ; LIM Domain Proteins ; },
abstract = {Chromatin organization is a pivotal factor in stem cell pluripotency and differentiation. However, the role of enhancer looping protein LIM domain-binding 1 (LDB1) in stem cells remains to be fully explored. We generated Ldb1(-/-) embryonic stem cells (ESCs) using CRISPR/Cas9 editing and observed a reduction in key stem cell factors SOX2 and KLF4 upon LDB1 loss. Differential gene expression, including of the Lin28-mediated self-renewal pathway genes, was observed between wild-type and Ldb1(-/-) ESC. LDB1 occupied super enhancers, including those of pluripotency genes, in ESC together with pluripotency factors, and LDB1 loss resulted in loss of Sox2 interactions with the SCR enhancer. Embryoid bodies (EBs) derived from Ldb1(-/-) ESC displayed reduced expression of lineage-specific markers. Ldb1(-/-) ESC had impaired ability to undergo terminal differentiation to erythroblasts, and gene dysregulation was very pronounced in Ldb1(-/-) erythroblasts. Conditional LDB1-deficient mice displayed reduced hematopoietic stem cell markers on bone marrow cells and dysregulation of the Lin28 pathway. Thus, LDB1 function is critical for ESC and EB development and becomes progressively more important for normal gene expression during differentiation to erythroblasts.},
}
@article {pmid41603736,
year = {2026},
author = {Fuglsang, A and Rout, SS and Koutna, EB and Sofos, N and Gallego, AR and Montoya, G},
title = {Conformational dynamics of CRISPR-Cas type I-F-HNH inform nickase engineering in a cascade scaffold.},
journal = {Nucleic acids research},
volume = {54},
number = {3},
pages = {},
pmid = {41603736},
issn = {1362-4962},
support = {NNF14CC0001//Novo Nordisk Foundation Center for Protein Research (CPR)/ ; NNF14CC0001//Novo Nordisk Foundation/ ; NNF24SA0098829//Novo Nordisk Foundation/ ; NNF0024386//Novo Nordisk Foundation/ ; NNF17SA0030214//Novo Nordisk Foundation/ ; NNF18OC0055061//Novo Nordisk Foundation/ ; NNF24SA0098829//Novo Nordisk Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; DNA Cleavage ; *Deoxyribonuclease I/genetics/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; Catalytic Domain ; Protein Engineering ; Humans ; Cryoelectron Microscopy ; Gene Editing ; Models, Molecular ; DNA, Single-Stranded/metabolism/genetics/chemistry ; DNA/metabolism/chemistry ; Protein Conformation ; },
abstract = {The type I-FHNH CRISPR-Cas system is a non-canonical Class 1 effector complex distinguished by the replacement of the Cas3 recruitment domain with a catalytic HNH domain in Cas8, enabling autonomous DNA cleavage without accessory nucleases. Using cryo-EM, we determined high-resolution structures of the effector complex in three catalytic states-precatalytic, NTS-cleaved, and post-catalytic-revealing a dynamic trajectory of the HNH domain through inward, middle, and outward conformations. Biochemical assays demonstrated that the complex cleaves the nontarget strand (NTS) prior to the target strand (TS), consistent with a sequential cleavage mechanism similar to Cas12 effectors but notably lacking trans-cleavage activity on single-stranded DNA. Structural comparisons confirmed a minimal PAM requirement (5'-CN) and a constrained HNH catalytic site poised for precise strand scission. We engineered a ΔLinker variant of Cas8 that repositions the HNH domain, selectively abolishing TS cleavage and converting the system into a programmable NTS-specific nickase. Importantly, we validated the functionality of both wild-type and mutant complexes in human cells. While the wild-type system induced indels and base substitutions, the ΔLinker variant triggered targeted single-strand nicks without double-stranded breaks. Together, our work establishes type I-FHNH as a compact and precise genome editing platform with in vivo efficacy.},
}
@article {pmid41603755,
year = {2026},
author = {Yuan, X and Yang, F and Chen, X and Xiao, P and Shen, L},
title = {Single-Nucleotide Variation Analysis in Oral Squamous Cell Carcinoma-Related ctDNA by dCas9/sgRNA Recognition-Mediated Proximity Ligation-Triggered Terminal Hairpin Formation and Self-Priming Amplification.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4205-4212},
doi = {10.1021/acs.analchem.5c07087},
pmid = {41603755},
issn = {1520-6882},
mesh = {Humans ; *Circulating Tumor DNA/genetics/blood ; *Mouth Neoplasms/genetics/blood/diagnosis ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; *Carcinoma, Squamous Cell/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Circulating tumor DNA (ctDNA) represents a promising noninvasive biomarker for cancer diagnosis, including oral cancer. However, its clinical translation is currently limited by the lack of precise and reliable detection techniques. In this study, we developed a novel fluorescent biosensor for the detection of single-nucleotide variations in ctDNA, which integrates dual dCas9/sgRNA complexes for target recognition, proximity ligation-initiated terminal hairpin formation and self-priming amplification (PS-THSP), and Cas12a/crRNA-mediated signal output. A key innovation of this design is its multilayered specificity strategy, combining mutation-specific recognition by dual dCas9/sgRNA, proximity-dependent ligation, and Cas12a/crRNA-assisted verification of PS-THSP amplicons. This integrated approach offers a significant advance over existing CRISPR/Cas-based methods that rely primarily on signal amplification. Furthermore, the biosensor achieves high sensitivity through the synergistic coupling of PS-THSP amplification and Cas12a trans-cleavage activity, enabling a broad dynamic range spanning 6 orders of magnitude and a detection limit as low as 0.12 fM within 120 min. When applied to serum samples, the biosensor reliably detected ctDNA with high accuracy, demonstrating its strong potential for clinical cancer diagnostics.},
}
@article {pmid41603863,
year = {2026},
author = {Calderón, L and Schäfer, M and Rončević, M and Rauschmeier, R and Jaritz, M and Schwickert, TA and Sun, Q and Pauli, A and Zuber, J and Busslinger, M},
title = {In vivo CRISPR/Cas9 screens identify new regulators of B cell activation and plasma cell differentiation.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {3},
pages = {},
pmid = {41603863},
issn = {1540-9538},
support = {//Boehringer Ingelheim/ ; 740349/ERC_/European Research Council/International ; LT00427/2013//Human Frontier Science Program/ ; },
mesh = {Animals ; *Cell Differentiation/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Plasma Cells/immunology/cytology ; *B-Lymphocytes/immunology/cytology ; Mice ; *Lymphocyte Activation/genetics/immunology ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Immune responses to pathogens lead to the generation of plasma cells through a complex interplay of B cells with their microenvironment in lymphoid organs. To identify new regulators of B cell activation and plasmablast differentiation in the context of the splenic microenvironment, we established an in vivo system for pooled sgRNA CRISPR/Cas9 screens in immunized mice. To improve the infection efficiency of naïve B cells, we generated Cd23-Cre Rosa26LSL-EcoR/+ mice exhibiting increased expression of the ecotropic lentivirus receptor EcoR on naïve B cells. Upon adoptive B cell transfer and immunization of recipient mice, 379 sgRNAs, targeting genes with high expression in plasma cells, were analyzed for their effects on plasmablast generation. Gene hits, encoding 23 positive and 18 negative regulators of B cell activation, plasmablast differentiation, or homeostasis, were uniquely identified in these in vivo screens. Validated genes encoded proteins involved in cell adhesion, signal transduction, protein folding, iron transport, and enzymatic processes. Hence, our in vivo screening system identified novel regulators controlling B cell-mediated immune responses.},
}
@article {pmid41604096,
year = {2026},
author = {Almufarriji, FM},
title = {Nanocarrier-mediated CRISPR-Cas delivery: a novel approach against antibiotic-resistant superbugs.},
journal = {Saudi pharmaceutical journal : SPJ : the official publication of the Saudi Pharmaceutical Society},
volume = {34},
number = {1},
pages = {5},
pmid = {41604096},
issn = {1319-0164},
abstract = {Antibiotic resistance (ABR) is a leading cause of death and a major public health threat globally. Without appropriate interventions, annual ABR-associated deaths have been projected to reach 10 million by 2050 worldwide. Hence, it is critical to develop novel therapeutic interventions that would be able to tackle ABR by targeting mainly the pathogenic microbes, while lessening harm to beneficial microbes. There is an increasing research interest in CRISPR-Cas (CC) systems owing to their potential in controlling and preventing horizontal gene transfer and spread of antibiotic resistance. In addition, CC systems offer several advantages, including high efficiency, rapid turnaround time, low cost, and easy design, which allow these systems to effectively and precisely target antibiotic-resistant bacteria. CRISPR-based gene therapy offers numerous benefits; however, the major limitation in clinical translation is the safe and effective delivery of CRISPR components to target organs or cells, thus hindering its potential in therapeutic interventions. Nanocarriers (NCs) can help the CC systems to overcome their off-target effects by precisely delivering the systems to the target cells. NCs can also be engineered for target site release, payload protection, and high specificity, which can further ensure delivery of the components of CC in the target cells or regions without harming surrounding tissues. This review summarizes the principles and mechanisms of CC systems, highlights their applications against antibiotic-resistant bacteria, and discusses emerging nanocarrier-based delivery strategies that may enhance the clinical utility of CRISPR-Cas technologies in managing ABR.},
}
@article {pmid41605211,
year = {2026},
author = {Lin, J and Lin, Y and Liu, N and Cao, W and Zhang, J and Wen, S and Zhang, Y and Liao, W and Hong, Z and Lin, Y and Liu, Q and Liu, H and Li, Q and Chen, B and Li, M and Luo, Z and Yang, L and Yang, Y and Zheng, SH and Wang, Y and Chu, H and Hu, Y and Qin, Y and Luo, BX and Tian, S and Chen, Y and Yan, T and Yang, L and Wang, H and Liu, T and Jiang, Y and Lu, Z},
title = {AAVLINK: A potent DNA-recombination method for large cargo delivery in gene therapy.},
journal = {Cell},
volume = {189},
number = {3},
pages = {969-986.e17},
doi = {10.1016/j.cell.2025.12.039},
pmid = {41605211},
issn = {1097-4172},
mesh = {*Dependovirus/genetics ; Animals ; *Genetic Therapy/methods ; Mice ; Genetic Vectors/genetics ; Humans ; *Recombination, Genetic ; *Gene Transfer Techniques ; Nerve Tissue Proteins/genetics/metabolism ; HEK293 Cells ; CRISPR-Cas Systems ; Transduction, Genetic/methods ; },
abstract = {Delivery of therapeutic genes is essential for successful gene therapy. Adeno-associated viruses (AAVs) are a prime vector for carrying gene cargoes. However, the limited packaging capacity of AAVs poses a major challenge for large gene transduction. Here, we devised a strategy termed AAV with translocation linkage (AAVLINK), leveraging Cre/lox-mediated intermolecular DNA recombination to overcome cargo size constraints. This AAVLINK strategy enabled superior gene segmentation flexibility, robust gene reconstitution efficiency, and a marked reduction in truncated protein products. AAVLINK drove expression of intact Shank3 or SCN1A and rescued behavior and seizure phenotypes of mutant mice, respectively. Moreover, we generated AAVLINK2.0 with destabilized Cre to address biosafety concerns. Importantly, we used AAVLINK to build a vector bank for 193 large genetic-disorder-associated genes and 5 CRISPR-based tools with verified gene reconstitution. Altogether, our study establishes a robust method to facilitate delivery of large gene cargoes using AAVs.},
}
@article {pmid41605290,
year = {2026},
author = {Shi, L and Zhang, M and Zheng, R and Kwok, LY and Zhang, W},
title = {Comparative genomics reveals two major lineages of Bifidobacterium adolescentis in the human gut, driven by divergent adaptation in China and the United States.},
journal = {Journal of advanced research},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jare.2026.01.071},
pmid = {41605290},
issn = {2090-1224},
abstract = {INTRODUCTION: Bifidobacterium adolescentis is a key beneficial member of the human gut microbiota, but its genomic diversity and evolutionary drivers across human populations remain poorly characterized.<br><br>OBJECTIVES: Understanding genomic functional heterogeneity and evolutionary patterns in human gut-derived B. adolescentis.<br><br>METHODS: We performed a comparative genomic analysis of 395 B. adolescentis, mainly from China (n&#xa0;=&#xa0;169) and the United States (n&#xa0;=&#xa0;146), with smaller sets from Australia, Italy, and the United Kingdom, to investigate functional heterogeneity and evolutionary mechanisms. Our analysis integrated core and pan-genome architecture, phylogenomics, single nucleotide polymorphism (SNP)-based population structure, carbohydrate-active enzyme profiles, CRISPR-Cas systems, antibiotic resistance genes, and recombination dynamics.<br><br>RESULTS: The pan-genome was open and highly plastic. Phylogenetic reconstruction identified two major clades with strong geographic stratification: Chinese isolates predominantly clustered in Clade B, while U.S. isolates grouped in Clade A. Functional annotation showed regional specialization in carbohydrate-active enzymes, with Chinese isolates enriched in glycosyltransferase families and U.S. isolates in carbohydrate-binding module and carboxylesterase families, likely reflecting dietary adaptations. Genomic islands were hotspots for horizontal gene transfer, harboring region-specific carbohydrate-active enzymes and antibiotic resistance genes such as tet(W/32/O) and ermX, which were frequently located in Chinese isolates. Recombination was found to be the primary driver of genetic diversity, with recombination-to-mutation ratios approaching and exceeding 3.0 in Chinese and U.S. isolates. Linkage disequilibrium decay further supported higher recombination rates in these populations.<br><br>CONCLUSION: B. adolescentis has diverged into two major genomic lineages, primarily associated with isolates from China and the U.S. This divergence reflects adaptation to distinct host-associated ecological factors, such as diet, antibiotic exposure, and lifestyle, and is predominantly driven by extensive homologous recombination rather than point mutations. These findings highlight how regional selective pressures shape the genomic and functional landscape of this key gut symbiont.},
}
@article {pmid41605395,
year = {2026},
author = {Zhang, J and Han, B and Zhang, X and Xie, X and Zhao, F and Zhang, W and Jiang, Y and Zhang, X},
title = {A CRISPR/Cas12a-mediated marker-free fluorescent biosensor constructed based on an automated 3D DNA walker-enabled signal amplification for sensitive detection of aflatoxin B1.},
journal = {International journal of biological macromolecules},
volume = {344},
number = {Pt 2},
pages = {150549},
doi = {10.1016/j.ijbiomac.2026.150549},
pmid = {41605395},
issn = {1879-0003},
mesh = {*Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Food Contamination/analysis ; Silver/chemistry ; Fluorescence ; *DNA/chemistry ; *Endodeoxyribonucleases ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The efficient and sensitive detection of mycotoxins is critical to ensure food safety and maintain public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensor based on an automated 3D DNA walker-enabled 'one-to-many' signal amplification was developed for sensitive detection of aflatoxin B1. Due to the efficient amplification effect of DNA walkers and the strong fluorescence properties of silver nanoclusters, a sensitive output of the amplified signal was produced by precisely regulating the activated trans-cleavage activity of the specific target DNA of Cas12a. In the established fluorescence biosensor, a small amount of aflatoxin B1 promoted the production of a large amount of activator by the established 3D DNA walker, which stimulated the trans-cleavage activity of Cas12a to degrade the single-stranded DNAs for synthesis of silver nanoclusters, leading to a decreased fluorescent signal. The established biosensor was able to achieve the sensitive detection of aflatoxin B1 under optimal conditions, obtaining a detection limit of 45.38 pg/mL in a linear range of 0.05 to 10 ng/mL. In addition, the developed biosensor showed good recoveries in spiked food samples (peanut milk and drinking water) at different concentrations. This work provided new insights for the applications of DNA walker and the development of a marker-free fluorescent biosensing platform based on CRISPR/Cas for the detection of mycotoxins.},
}
@article {pmid41605411,
year = {2026},
author = {Zhang, Q and Yang, Y and Huang, X and Ma, J and Duan, Y and Ma, G and Lei, H},
title = {Prime editing for ocular gene therapy and disease modeling: a narrative review of advances, delivery, and translational readiness.},
journal = {Experimental eye research},
volume = {265},
number = {},
pages = {110891},
doi = {10.1016/j.exer.2026.110891},
pmid = {41605411},
issn = {1096-0007},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems ; Translational Research, Biomedical ; *Eye Diseases/therapy/genetics ; Disease Models, Animal ; Gene Transfer Techniques ; },
abstract = {Prime editing is a versatile "search-and-replace" genome-editing technology that enables precise and flexible genome correction of genetic sequences by reverse-transcribing an RNA template encoded at the 3' end of a prime editing guide RNA (pegRNA). It supports the introduction of nucleotide substitutions, and insertions or/and deletions (indels) in living cells without requiring double-stranded DNA breaks or exogenous donor templates. Since its introduction in 2019, prime editing has advanced rapidly-from the first-generation prime editor (PE1) to PE7 and other next-generation variants-with editing efficiencies increasing from 0.7 to 5.5 % to more than 50 % in vitro. Optimization strategies including engineering of the Cas9 and reverse transcriptase domains, refinement of pegRNA architecture, recruitment of auxiliary proteins, and modulation of DNA repair pathways have substantially enhanced editing efficiency, product purity, and target scope across diverse cell types and tissues. These developments are particularly relevant to ophthalmology, where many blinding disorders arise from point mutations or small indels ideally suited for prime editing-based correction. Recent work in retinal cells and animal models has demonstrated the growing feasibility of prime editing to treat inherited retinal diseases, modulate pathological angiogenesis, and achieve precise gene repair in post-mitotic photoreceptors and retinal pigment epithelial cells. As delivery vectors and newer PE variants improve, prime editing is a plausible next-generation platform for a wide range of ocular diseases.},
}
@article {pmid41605509,
year = {2026},
author = {Li, J and Chen, X and Yang, Y and Xu, Y and Lai, M and Shi, L and Lin, X and Chen, W and Peng, H},
title = {Electron Transfer Mechanism-Mediated Host-Guest Nanoswitch Powered Amplification-Free CRISPR/Cas12a-Electrochemiluminescence Bioassay for Alzheimer's Disease Diagnosis.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4102-4111},
doi = {10.1021/acs.analchem.5c06614},
pmid = {41605509},
issn = {1520-6882},
mesh = {*Alzheimer Disease/diagnosis ; Humans ; *Electrochemical Techniques/methods ; Gold/chemistry ; *Luminescent Measurements/methods ; *Amyloid beta-Peptides/analysis/blood ; beta-Cyclodextrins/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Metal Nanoparticles/chemistry ; Electron Transport ; Methylene Blue/chemistry ; Limit of Detection ; },
abstract = {Developing a high-performance amplification-free electrochemiluminescence (ECL) assay platform that operates at a low trigger potential is a promising strategy for broadening the applications of ECL sensing. In this work, we present a host-guest interaction-mediated split-type CRISPR/Cas12a-ECL assay platform by using the highly sensitive host-guest recognition between the β-cyclodextrin-functionalized gold nanoclusters (β-CD-AuNCs) probe and methylene blue (MB) system as a proof of concept. Efficient ECL quenching of β-CD-AuNCs by MB is achieved via an electron transfer mechanism based on host-guest recognition between them. By integrating the high-specific recognition and cleavage activity of the CRISPR/Cas technology, the high quantum yield, and low trigger potential β-CD-AuNCs-based ECL probes, together with the highly sensitive and selective host-guest recognition-based split-type assay design, a novel "trinity" detection platform has been successfully constructed. Using Amyloid-β oligomers (AβOs), a key biomarker for Alzheimer's disease (AD) diagnosis and therapy, as the analyte, this amplification-free CRISPR/Cas-ECL biosensing platform enables ultrasensitive and accurate detection of AβO without requiring additional signal amplification strategies. The proposed sensing platform exhibits a linear detection range from 1.0 × 10[-8] to 1.0 × 10[-1] μg/mL for AβO detection, with a detection limit as low as 0.2 fg/mL (S/N = 3). This sensitivity approaches single-molecule levels and is 3-4 orders of magnitude lower than that of traditional ELISA. Furthermore, owing to its outstanding performance including high specificity, excellent selectivity, superior sensitivity, and strong anti-interference capability, the platform demonstrates remarkable detection performance in monitoring AβO in clinical AD blood samples, showing a good Pearson's correlation between the method and ELISA results. This work provides a powerful tool for clinical diagnosis and paves the way for therapeutic development, while also offering a rational design strategy for next-generation ECL biosensing platforms.},
}
@article {pmid41605928,
year = {2026},
author = {Ganguly, C and Aribam, SD and Dos Santos, AM and Martin, L and Thomas, LM and Shao, Y and Rajan, R},
title = {Bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41605928},
issn = {2041-1723},
support = {R35GM153297 and R44GM133270//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; //Research Council of the University of Oklahoma Norman Campus/ ; R35 GM153297/GM/NIGMS NIH HHS/United States ; R44 GM096678/GM/NIGMS NIH HHS/United States ; P30 GM145423/GM/NIGMS NIH HHS/United States ; MCB-1716423 and MCB-2424888//National Science Foundation (NSF)/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics/chemistry ; CRISPR-Cas Systems/genetics ; Allosteric Regulation ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/genetics/chemistry ; *Francisella/genetics/enzymology ; *R-Loop Structures ; DNA Cleavage ; Gene Editing ; DNA/metabolism ; },
abstract = {CRISPR-Cas12a, an RNA-based DNA targeting system, is widely used for genome editing and biomarker detection. To mitigate the off-target DNA cleavage of Cas12a, we previously developed a Francisella novicida Cas12a variant (FnoCas12a[KD2P]) by introducing double proline substitutions (K969P/D970P) in a conserved arginine-rich helix called the bridge helix (BH). In this work, we use a combinatorial approach to understand the molecular mechanisms of BH-mediated activation of Cas12a for DNA cleavage. We report five structures of FnoCas12a[KD2P] that are at different states of conformational activation. Comparison of the variant and wild-type (FnoCas12a[WT]) structures, along with activity assays and computational simulations, establishes the loop-to-helical transition and bending of the BH as an allosteric trigger for RNA-DNA hybrid propagation. These changes track with the previously reported coupled remodeling of BH and helix 1 of RuvC motif-II as well as the REC lobe movements needed to accommodate the growing hybrid. The transition of the BH is essential for the loop-to-helical transition of the "lid", which in turn opens the RuvC active site pocket for DNA entry and cleavage. Pairwise 3D structural comparison of the BH and RuvC of Cas12 and Cas9 families provides insight into the diversity of BH's structural organization in these mechanistically similar enzymes.},
}
@article {pmid41606937,
year = {2025},
author = {Jiang, W and Zhu, T and Zhou, S and Pan, L and Qiao, Z and Wang, M and Yang, D},
title = {Recent advances in electrochemical-based CRISPR/Cas biosensing for nucleic acid and non-nucleic acid pathogenic microorganism detection.},
journal = {Food research international (Ottawa, Ont.)},
volume = {221},
number = {Pt 1},
pages = {117213},
doi = {10.1016/j.foodres.2025.117213},
pmid = {41606937},
issn = {1873-7145},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Nucleic Acids/analysis ; *Food Microbiology/methods ; *Bacteria/genetics/isolation &amp; purification ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The widespread presence of pathogenic microorganisms in food and environmental sources poses a persistent threat to public health. Conventional detection methods-including culture, microscopy, and biochemical assays-are limited by low sensitivity, cross-reactivity, and prolonged turnaround times, particularly when microbial loads are low or phenotypic overlap occurs. These limitations underscore the urgent need for diagnostic platforms that combine speed, specificity, and sensitivity. The advent of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) systems has revolutionized microbial diagnostics, driving the emergence of electrochemical CRISPR/Cas (EC-CRISPR/Cas) biosensors. This review surveys four principal electrochemical CRISPR/Cas (EC-CRISPR/Cas) platforms-Cas9, Cas12a, Cas13a, and Cas14a-emphasizing their structural characteristics, biosensing mechanisms, and signal amplification strategies for both nucleic acid and non-nucleic acid pathogen detection. We first outline the molecular architecture and functional mechanisms of each Cas protein in the context of biosensing. EC-CRISPR/Cas detection strategies are classified as nucleic acid-based (either amplification-free or amplification-dependent) or non-nucleic acid-based, the latter primarily relying on aptamer-mediated recognition. We also provide a comparative analysis of signal enhancement techniques and application scenarios across bacterial, viral, fungal, and parasitic pathogens. Importantly, we identify key limitations of current systems-such as poor reusability, signal drift, and challenges in point-of-care deployment-and present emerging solutions including crRNA engineering, nanomaterial integration, and artificial intelligence-guided biosensor design. These innovations hold strong potential to enhance sensitivity, specificity, and real-time performance, offering a foundation for next-generation, scalable EC-CRISPR/Cas diagnostics.},
}
@article {pmid41606942,
year = {2025},
author = {Fraiture, MA and D'aes, J and Gobbo, A and Delvoye, M and Meunier, AC and Frouin, J and Guiderdoni, E and Deforce, D and De Vogelaere, C and De Keersmaecker, SCJ and Vanneste, K and Roosens, NHC},
title = {Genetic fingerprints derived from genome database mining allow accurate identification of genome-edited rice in the food chain via targeted high-throughput sequencing.},
journal = {Food research international (Ottawa, Ont.)},
volume = {221},
number = {Pt 1},
pages = {117218},
doi = {10.1016/j.foodres.2025.117218},
pmid = {41606942},
issn = {1873-7145},
mesh = {*Oryza/genetics ; *High-Throughput Nucleotide Sequencing/methods ; *Plants, Genetically Modified/genetics ; *Genome, Plant ; *Gene Editing/methods ; Polymorphism, Single Nucleotide ; *Food, Genetically Modified ; Databases, Genetic ; *DNA Fingerprinting/methods ; CRISPR-Cas Systems ; },
abstract = {Genome-edited (GE) organisms are currently classified as GMOs according to European legislation, requiring traceability and labelling in the food and feed supply chain. However, unambiguous identification of a specific GE organism with one or more induced single nucleotide variations (SNVs) dispersed across the genome remains challenging. This study explored whole-genome sequencing-based characterization, public genome databases, and machine learning tools to select key genetic elements and create a unique fingerprint for distinguishing a specific GE line. As a case study, a GE Nipponbare rice line containing a single CRISPR-Cas-induced SNV was used. To experimentally assess the detection of this fingerprint, a targeted high-throughput sequencing approach, including multiplex PCR-based enrichment of key genetic elements, was developed and successfully tested. This promising proof-of-concept demonstrates the potential of combining a unique genetic fingerprint with targeted high-throughput sequencing to facilitate the accurate detection of GE organisms, thereby supporting food traceability and regulatory compliance for the development of new GE lines, as well as protecting associated intellectual property.},
}
@article {pmid41607132,
year = {2026},
author = {Yu, K and Li, H and Hu, Y and Yu, Y and Deng, S and Yang, Y and Guo, M and Li, M and Zhe, M and He, H and Fan, C},
title = {Integrating transcriptomics and high-throughput gene editing uncovers shoot apical meristem regulators in Brassica napus.},
journal = {Plant physiology},
volume = {200},
number = {2},
pages = {},
doi = {10.1093/plphys/kiag032},
pmid = {41607132},
issn = {1532-2548},
support = {2022YFD1200400//National Key Research and Development Program of China/ ; 2023YFF1000700//National Key Research and Development Program of China/ ; 31671279//National Natural Science Foundation of China/ ; 2024BBA001//Agricultural Gene Editing Platform Technology and Breeding Research &amp; Development/ ; },
mesh = {*Brassica napus/genetics/growth &amp; development ; *Meristem/genetics/growth &amp; development ; *Gene Editing/methods ; *Transcriptome ; Gene Expression Profiling ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Plant Shoots/genetics/growth &amp; development ; Plant Proteins/genetics/metabolism ; Mutation ; },
abstract = {The shoot apical meristem (SAM) determines plant architecture, but the key components of its regulatory network remain elusive in rapeseed (Brassica napus L.). Here, we integrated transcriptomic profiling of 3 multilocular silique mutants (Bnaclv1, Bnaclv2, and Bnaclv3) across key SAM development stages (IM, stage6, and stage8) with large-scale CRISPR/Cas9 functional screening to identify regulators of SAM maintenance. Differential gene expression and GO enrichment highlighted genes significantly associated with meristem development processes. Weighted gene co-expression network analysis of stage-specific transcriptomes identified 42 candidate genes potentially related to SAM development. To enable systematic functional screening, we established a high-throughput multiplex CRISPR/Cas9 pipeline, simultaneously targeting 198 sites across 42 candidate genes through optimized sgRNA design and pooled transformation. We successfully obtained mutants for 25 genes with homozygous mutants for 9 genes. Phenotypic analysis demonstrated that mutants of BnaSCPL family genes (SCPL29, SCPL44, and SCPL45) exhibited a multi-stem phenotype and disrupted SAM organization. Mechanistic studies revealed that BnaSCPL mutations disrupt the canonical CLV3/WUS feedback loop, uncovering their roles in SAM homeostasis. Additionally, knockout of BnaLFY homologs caused permanent vegetative state and sterility, demonstrating their conserved role in floral meristem identity in Brassica napus. Collectively, our study not only elucidates the critical function of BnaSCPLs in SAM maintenance but also establishes a regulatory framework for understanding meristem phase transitions in B. napus, providing potential targets for crop architecture improvement.},
}
@article {pmid41608243,
year = {2026},
author = {Mitrofanov, A and Beisel, CL and Baumdicker, F and Alkhnbashi, OS and Backofen, R},
title = {Comprehensive analysis of CRISPR array repeat mutations reveals subtype-specific patterns and links to spacer dynamics.},
journal = {microLife},
volume = {7},
number = {},
pages = {uqaf050},
pmid = {41608243},
issn = {2633-6693},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated protein (Cas) systems are adaptive immune mechanisms in bacteria and archaea that protect against invading genetic elements by integrating short fragments of foreign DNA into CRISPR arrays. These arrays consist of repetitive sequences interspersed with unique spacers, guiding Cas proteins to recognize and degrade matching nucleic acids. The integrity of these repeat sequences is crucial for the proper function of CRISPR-Cas systems, yet their mutational dynamics remain poorly understood. In this study, we analyzed 56 343 CRISPR arrays across 25 628 diverse prokaryotic genomes to assess the mutation patterns in CRISPR array repeat sequences within and across different CRISPR subtypes. Our findings reveal, as expected to some extent, that mutation frequency is substantially higher in terminal repeat sequences compared to internal repeats consistently across system types. However, the mutation patterns exhibit an unexpected amount of variation among different CRISPR subtypes, suggesting that selective pressures and functional constraints shape repeat sequence evolution in distinct ways. Understanding these mutation dynamics provides insights into the stability and adaptability of CRISPR arrays across diverse bacterial and archaeal lineages. Additionally, we elucidate a novel relationship between repeat mutations and spacer dynamics, demonstrating that hotspots for terminal repeat mutations coincide with regions exhibiting higher spacer conservation. This observation corroborates recent findings indicating that spacer deletions occur at a frequency 374 times greater than that of mutations and are significantly influenced by repeat misalignment. Our findings suggest that repeat mutations might play a pivotal role in spacer retention or loss, or vice versa, thereby highlighting an evolutionary trade-off between the stability and adaptability of CRISPR arrays.},
}
@article {pmid41608579,
year = {2026},
author = {Sanchez-Pupo, RE and Kelly, JJ and Shalaby, N and Xia, Y and Martinez-Santiesteban, FM and Lau, J and Verriet, IE and Fox, MS and Hicks, JW and Thiessen, JD and Ronald, JA},
title = {Imaging CRISPR-edited CAR-T cell therapies with optical and positron emission tomography reporters.},
journal = {Theranostics},
volume = {16},
number = {7},
pages = {3227-3245},
pmid = {41608579},
issn = {1838-7640},
mesh = {*Positron-Emission Tomography/methods ; Animals ; Humans ; *Receptors, Chimeric Antigen/genetics/metabolism ; *Immunotherapy, Adoptive/methods ; Mice ; Gene Editing/methods ; *T-Lymphocytes/immunology/metabolism/transplantation ; CRISPR-Cas Systems ; Genes, Reporter ; Cell Line, Tumor ; Symporters/genetics ; Female ; Dependovirus/genetics ; Optical Imaging/methods ; },
abstract = {Rationale: Chimeric antigen receptor (CAR) T cell therapies have shown remarkable success in treating hematological cancers and are increasingly demonstrating potential for solid tumors. CRISPR-based genome editing offers a promising approach to enhance CAR-T cell potency and safety, yet challenges such as inefficient tumor homing and toxicities in normal tissues, limit broader adoption. Advanced imaging technologies, including bioluminescence imaging (BLI) and positron emission tomography (PET), provide real-time visualization of CAR-T cell behavior in vivo. Here, we developed Trackable Reporter Adaptable CRISPR-Edited CAR (tRACE-CAR) T cells, a modular system for site-specific integration of CARs and imaging reporters. Methods: The luciferase reporter AkaLuciferase (AkaLuc) or the human sodium iodide symporter (NIS) was cloned downstream of the CAR in adeno-associated virus (AAV) donors for imaging. CAR-reporter cassettes were inserted into the T-cell receptor α constant locus of primary human T cells via CRISPR editing and AAV transduction. Editing efficiency was assessed by flow cytometry. In vitro cytotoxicity was evaluated across multiple effector-to-target ratios. In vivo, BLI and PET imaging were used for tracking CAR-T cells in tumor-bearing immunodeficient mice. Results: T cell receptor (TCR) knockout efficiency exceeded 85% and CAR expression reached 70-80%. Reporter-engineered CAR-T cells exhibited significant cytotoxicity and outperformed naïve T cells. In vivo, AkaLuc BLI and [18]F-tetrafluoroborate PET enabled non-invasive tracking of viable CAR-T cells. Administration route (intravenous, peritumoral, or intraperitoneal) significantly influenced CAR-T cell distribution and therapeutic effectiveness. Conclusion: tRACE-CAR enables precise optical and PET tracking of CRISPR-edited CAR-T cells in models of leukemia and ovarian cancer, allowing dynamic, non-invasive monitoring of cell distribution in both tumors and off-target tissues. This imaging-enabled platform could lead to more personalized, effective CRISPR-edited CAR cell therapies.},
}
@article {pmid41608958,
year = {2026},
author = {Zhang, Y and Walker, RSK and Sunna, A and Barber, TJ and Li, M},
title = {Droplet Digital CRISPR for Nucleic Acid Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {14},
pages = {e17470},
pmid = {41608958},
issn = {2198-3844},
support = {DP200102269//Australian Research Council/ ; DP240100795//Australian Research Council/ ; GNT2017679//National Health and Medical Research Council/ ; },
mesh = {*Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acids/analysis/genetics ; *Microfluidics/methods ; },
abstract = {Droplet digital (dd) clustered regularly interspaced short palindromic repeats (CRISPR) integrates the high sequence specificity of CRISPR-based nucleic acid detection with the absolute quantification capability of digital droplet microfluidics, offering high sensitivity, precision, and scalability. By partitioning samples into thousands to millions of picoliter microdroplets, ddCRISPR enables single-molecule resolution and minimizes background interference. This review summarizes the principles of droplet generation, manipulation, and detection in ddCRISPR platforms, as well as recent advances in amplification-based and amplification-free detection strategies. Representative applications are highlighted for viral, bacterial, and other DNA/RNA biomarker detection. Current challenges, including workflow automation, droplet stability, multiplexing, and assay portability, are discussed alongside future perspectives such as artificial intelligence (AI)-assisted analysis, point-of-care integration, and high-throughput multiplexed detection. These insights aim to guide the translation of ddCRISPR technologies from laboratory research to robust, scalable, and accessible diagnostic solutions.},
}
@article {pmid41609044,
year = {2026},
author = {Anschuetz, A and Listyono, R and Vorley, T and Platt, B and Harrington, CR and Riedel, G and Schwab, K},
title = {The Icelandic Mutation in the Murine APP Gene, mAPP[A673T], on Amyloid-β Plaque Burden in the 5×FAD Alzheimer Model.},
journal = {Journal of integrative neuroscience},
volume = {25},
number = {1},
pages = {48581},
doi = {10.31083/JIN48581},
pmid = {41609044},
issn = {0219-6352},
support = {PAR1577//TauRx Therapeutics Ltd., Singapore/ ; PAR2074//TauRx Therapeutics Ltd., Singapore/ ; },
mesh = {Animals ; *Alzheimer Disease/genetics/pathology/metabolism ; *Amyloid beta-Protein Precursor/genetics/metabolism ; Disease Models, Animal ; *Plaque, Amyloid/pathology/genetics/metabolism ; Mice, Transgenic ; Male ; *Amyloid beta-Peptides/metabolism ; Mice, Inbred C57BL ; Female ; Presenilin-1/genetics ; Mice ; Humans ; Mutation ; *Brain/pathology/metabolism ; Iceland ; },
abstract = {BACKGROUND: The protective Icelandic mutation in the amyloid precursor protein (APP) gene, APP[A673T], identified in Icelandic and other Nordic populations is associated with a significantly lower risk of developing Alzheimer's disease (AD). Conflicting results have been reported for the human APP[A673T] mutation in various knock-in models of AD, but the effect of the mouse APP[A673T] form in 5× familial AD (5×FAD) mice has never been investigated.<br><br>METHODS: We crossed C57Bl6/J mice expressing a single point mutation edited into the murine APP gene via Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated (CRISPR-Cas) gene editing, termed mAPP[A673T], with 5&#xd7;FAD mice that overexpress human APP carrying the Swedish (K670N/M671L), Florida (I716V), and London (V717I) mutations as well as human presenilin-1 (PS1) with two mutations (M146L and L286V); the resulting mice were termed 5&#xd7;FAD &#xd7; mAPP[A673T] mice. We investigated amyloid beta-protein (A&#x3b2;) pathology in 5&#xd7;FAD &#xd7; mAPP[A673T], 5&#xd7;FAD and their respective controls, mAPP[A673T], and C57Bl6/J wild type mice, at 6-months of age using immunohistochemistry, immunoblotting, and enzyme-linked immunosorbent assay (ELISA).<br><br>RESULTS: We found a moderate yet significant reduction in A&#x3b2; plaque size in male 5&#xd7;FAD &#xd7; mAPP[A673T] compared with 5&#xd7;FAD mice. No differences were observed for soluble/insoluble A&#x3b2;40 and A&#x3b2;42 levels per se, but lower plaque count/area was found in 5&#xd7;FAD &#xd7; mAPP[A673T] mice when A&#x3b2;42/A&#x3b2;40 ratios were low, suggesting a genotype-dependent sensitivity to A&#x3b2; aggregation and accumulation.<br><br>CONCLUSIONS: The mAPP[A673T] mutation has the potential to modify A&#x3b2; pathology in 5&#xd7;FAD mice at the age of 6 months.},
}
@article {pmid41610326,
year = {2026},
author = {Guo, W and Cheng, Y and Yin, H and Li, S and Wan, Y and Li, C and Jiang, C and Zhou, J and Yuan, X and Wang, J},
title = {CRISPR-AD: Combinational Detection of Blood Protein and miRNA with Digital CRISPR-Based Assay Enable to Improve the Diagnostic Performance of Alzheimer's Disease.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {3849-3859},
doi = {10.1021/acs.analchem.5c06080},
pmid = {41610326},
issn = {1520-6882},
mesh = {Humans ; *MicroRNAs/blood/genetics ; *Alzheimer Disease/diagnosis/blood/genetics ; Biomarkers/blood ; *tau Proteins/blood ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Aged ; Female ; Male ; Cognitive Dysfunction/blood/diagnosis ; Smartphone ; },
abstract = {Blood-based biomarkers present a noninvasive approach for detecting and assessing Alzheimer's disease (AD) pathophysiology, always by using sophisticated instrumentation for accurate detection. Here, we introduce CRISPR-AD, a CRISPR/Cas-based digital assay designed for the combined detection of protein and microRNA in blood. This method achieves a limit of detection (LOD) as low as 60 fg/mL for phosphorylated tau217 (p-tau217) and 0.5 fM for microRNA-34a-5p (miRNA34a), enabling successful detection in both AD patients and healthy individuals. We find that the combined use of these biomarkers improves the ability to distinguish between AD patients and healthy participants, particularly in individuals with mild cognitive impairment (MCI). Additionally, we have developed a portable device that integrates a smartphone as an imaging system for point-of-care testing (POCT), offering the potential for early stage AD screening. This study represents the first effort to evaluate the combined detection of blood protein and microRNA biomarkers for AD, underscoring the potential of multiple biomarker combinations for more accurate AD diagnosis.},
}
@article {pmid41610742,
year = {2026},
author = {Jiao, R and Ni, Q and Zhao, R and Zhu, X and Wang, L and Jiao, J and Jiang, H and Wu, Q and Yao, S and Yao, L and Liu, K and Qin, P},
title = {Dual CRISPR/Cas-driven amplification-free surface-enhanced Raman scattering biosensor combined with a smartphone for simultaneous detection of total and live target bacteria.},
journal = {Biosensors &amp; bioelectronics},
volume = {299},
number = {},
pages = {118446},
doi = {10.1016/j.bios.2026.118446},
pmid = {41610742},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Spectrum Analysis, Raman/instrumentation ; *CRISPR-Cas Systems/genetics ; *Smartphone ; Limit of Detection ; *Bacteria/isolation &amp; purification/genetics ; DNA, Bacterial/genetics ; Staphylococcus aureus/isolation &amp; purification ; },
abstract = {Simultaneous detection of total and live counts of target bacteria is significant but challenging. To address this challenge, here we proposed a dual CRISPR/Cas-driven amplification-free surface-enhanced Raman scattering (SERS) biosensor, termed cc-SERS. The biosensor was constructed based on the property that DNA remains stable while some RNA degrades rapidly after bacterial death. Briefly, the target DNA and RNA from live bacteria could activate both CRISPR/Cas12a and CRISPR/Cas13a, while dead bacteria could only activate CRISPR/Cas12a through the target DNA. In the absence of the target bacteria, neither CRISPR/Cas12a nor CRISPR/Cas13a could be activated. Therefore, the characteristic Raman signal at 1079 cm[-1] generated by the target DNA-activated CRISPR/Cas12a indicated the presence of the target bacteria (the sum of dead and live), while the characteristic Raman signal at 593 cm[-1] produced by the target RNA-activated CRISPR/Cas13a indicated the presence of the live target bacteria. With this unique signaling pattern, the biosensor is capable of detecting both total and live target bacteria in a single tube with a detection limit of ∼10 CFU/mL. The introduction of a rapid pre-processing procedure and a smartphone-assisted portable Raman spectrometer enabled the entire process to be completed in the field within 45 min. Thanks to the excellent programmability of CRISPR/Cas systems, the biosensor has been successfully applied to the detection of Staphylococcus aureus and Cronobacter sakazakii, respectively. As a proof-of-concept, this work opens a promising avenue for the simultaneous detection of total and live target bacteria.},
}
@article {pmid41610849,
year = {2026},
author = {Samelson, AJ and Ariqat, N and McKetney, J and Rohanitazangi, G and Parra Bravo, C and Bose, RS and Travaglini, KJ and Lam, VL and Goodness, D and Ta, T and Dixon, G and Marzette, E and Jin, J and Tian, R and Tse, E and Abskharon, R and Pan, HS and Carroll, EC and Lawrence, RE and Gestwicki, JE and Rexach, JE and Eisenberg, DS and Kanaan, NM and Southworth, DR and Gross, JD and Gan, L and Swaney, DL and Kampmann, M},
title = {CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1517-1534.e19},
pmid = {41610849},
issn = {1097-4172},
support = {F32 AG076281/AG/NIA NIH HHS/United States ; U54 NS123746/NS/NINDS NIH HHS/United States ; R01 AG075802/AG/NIA NIH HHS/United States ; K99 AG080116/AG/NIA NIH HHS/United States ; R01 AG085357/AG/NIA NIH HHS/United States ; U19 AG060909/AG/NIA NIH HHS/United States ; R01 AG070895/AG/NIA NIH HHS/United States ; R00 AG080116/AG/NIA NIH HHS/United States ; R01 AG082141/AG/NIA NIH HHS/United States ; U54 NS100717/NS/NINDS NIH HHS/United States ; F32 AG063487/AG/NIA NIH HHS/United States ; R01 AG062359/AG/NIA NIH HHS/United States ; P30 CA082103/CA/NCI NIH HHS/United States ; R01 AG060477/AG/NIA NIH HHS/United States ; U24 AG072458/AG/NIA NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *tau Proteins/metabolism/genetics ; *Neurons/metabolism/cytology ; *Proteostasis ; Tauopathies/metabolism/pathology/genetics ; Ubiquitin-Protein Ligases/metabolism ; *CRISPR-Cas Systems/genetics ; Ubiquitination ; Mitochondria/metabolism ; Proteasome Endopeptidase Complex/metabolism ; },
abstract = {Aggregation of the protein tau defines tauopathies, the most common age-related neurodegenerative diseases, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation, dysfunction, and death. However, molecular mechanisms underlying cell-type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi screen in induced pluripotent stem cell (iPSC)-derived neurons. The screen uncovered both known and unexpected pathways, including UFMylation and GPI anchor biosynthesis, which control tau oligomer levels. We discovered that the E3 ubiquitin ligase CRL5[SOCS4] controls tau levels in human neurons, ubiquitinates tau, and is correlated with resilience to tauopathies in human disease. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, generating disease-relevant tau proteolytic fragments and changing tau aggregation in vitro. These results systematically reveal principles of tau proteostasis in human neurons and suggest potential therapeutic targets for tauopathies.},
}
@article {pmid41610854,
year = {2026},
author = {Daly, J and Piatnitca, L and Al-Seragi, M and Krishnamoorthy, V and Wisnovsky, S},
title = {CRISPR activation screens map the genomic landscape of cancer glycome remodeling.},
journal = {Cell genomics},
volume = {6},
number = {4},
pages = {101139},
pmid = {41610854},
issn = {2666-979X},
mesh = {Humans ; Glycosylation ; *Polysaccharides/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Sialic Acid Binding Immunoglobulin-like Lectins/metabolism/genetics ; *Neoplasms/genetics/metabolism ; Sulfotransferases/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Glycomics/methods ; Gene Expression Regulation, Neoplastic ; },
abstract = {Many cancers upregulate the expression of sialic acid-containing glycans. These oligosaccharides engage inhibitory sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on immune cells, allowing cancer cells to evade immune surveillance. The genetic mechanisms underlying this process remain poorly defined. In this study, we performed gain-of-function CRISPR activation (CRISPRa) screens to define genetic pathways that regulate expression of Siglec-binding glycans. We show that Siglec ligand expression is controlled through genetic competition between genes that catalyze α2-3 sialylation and GlcNAcylation of galactose residues. Cancer glycome remodeling is also aided by the overexpression of "professional ligands" that facilitate Siglec-glycan binding. Notably, we also find that expression of the CD24 gene is genetically dispensable for cell surface binding of the inhibitory receptor Siglec-10. Finally, we identify the sulfotransferase enzyme GAL3ST4 as a potential driver of immune evasion in glioma cells. Our study provides a unique genomic atlas of cancer-associated glycosylation and identifies immediately actionable targets for cancer immunotherapy.},
}
@article {pmid41611149,
year = {2026},
author = {Setia, A and Patil, D and Randhave, NV and Vaishali, and Verma, N and Rani, K and Kale, A and Wagh, B and Kumar, V and Malik, AK and Sharon Mano Pappu, J and Deshmukh, K and Muthu, MS},
title = {CRISPR/Cas9 genome editing-based preclinical theranostics, biomarkers and drug delivery systems for cancer applications.},
journal = {International journal of biological macromolecules},
volume = {345},
number = {},
pages = {150582},
doi = {10.1016/j.ijbiomac.2026.150582},
pmid = {41611149},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy/diagnosis/drug therapy ; *Drug Delivery Systems/methods ; *Biomarkers, Tumor/genetics ; *Theranostic Nanomedicine/methods ; Animals ; Genetic Therapy/methods ; },
abstract = {Cancer development is driven by diverse genetic aberrations, underscoring the importance of innovative approaches like gene therapy for effective treatment. The CRISPR/Cas9 gene-editing system has all the makings of a game-changing technique for future disease treatment, owing to its pinpoint accuracy and efficiency in deleting disease-causing genes or correcting damaging base mutations. A number of efficient Cas9 variants and derivatives were recently designed to tackle the intricate genomic modifications that accompany illnesses. In addition, CRISPR/Cas9 based systems are increasingly explored for biomarker sensing and cancer diagnostics. Early identification, real-time monitoring, and therapy stratification are made possible by CRISPR-driven biosensors, which can detect circulating tumor DNA, microRNAs, or exosomal RNA with high specificity and sensitivity. Furthermore, a variety of stimuli-responsive delivery strategies, including chemical and peptide-assisted systems, light-activated mechanisms, glutathione-sensitive carriers, and pH-responsive platforms, have been explored to improve intracellular release efficiency thereby enhancing the precision of CRISPR/Cas9-mediated gene editing in cancer therapy. The CRISPR/Cas9-enabled theranostic platforms employ engineered nanocarriers to simultaneously deliver gene-editing tools and imaging agents, thereby facilitating synchronized treatment monitoring and improved therapeutic precision. This review emphasizes the transformative potential of CRISPR/Cas9-integrated theranostics, which combine targeted gene editing with advanced imaging for enhanced therapeutic monitoring and efficacy in cancer treatment.},
}
@article {pmid41611402,
year = {2026},
author = {Hou, J and Wang, Y and Yuan, W and Gong, Y and Yu, Y and Qin, X and Li, H and Zhang, Y and Shi, H and Chen, Y and Zhang, X},
title = {Thermosensitive hydrogel-enhanced RPA-CRISPR/Cas12a biosensor for ultrasensitive detection of methylated loci in breast cancer ctDNA.},
journal = {Analytica chimica acta},
volume = {1388},
number = {},
pages = {345101},
doi = {10.1016/j.aca.2026.345101},
pmid = {41611402},
issn = {1873-4324},
mesh = {Humans ; *Biosensing Techniques/methods ; *Breast Neoplasms/genetics/blood/diagnosis ; *CRISPR-Cas Systems ; *DNA Methylation ; Female ; *Circulating Tumor DNA/genetics/blood ; *Hydrogels/chemistry ; *Temperature ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Methylation differences exist between breast cancer tissues and normal tissues. The release of methylated circulating tumor DNA (ctDNA) by tumor cells provides a foundation for breast cancer liquid biopsy using methylated ctDNA. However, detection of low-abundance methylated loci in ctDNA remains a significant challenge to date. Existing one-tube detection systems cannot avoid target depletion caused by CRISPR/Cas12a cleavage, leading to reduced sensitivity.<br><br>RESULTS: This study developed a thermosensitive hydrogel-based one-tube RPA-CRISPR/Cas12a detection system, combined with methylation-sensitive restriction endonucleases (MSRE), for the detection of specific methylated loci in breast cancer ctDNA. This method achieves spatial separation while maintaining connectivity of reaction phases in a single tube for the first time, and the thermosensitive hydrogel does not exert inhibitory effects on either system. The system can specifically recognize methylated target molecules with a limit of detection (LOD) as low as 1&#xa0;&#xd7;&#xa0;10[-8]&#xa0;ng/&#x3bc;L (&#x2248;70 copies/&#x3bc;L), outperforming the current glycerol-enhanced one-tube reaction system. It is capable of distinguishing methylated fractions as low as 0.05&#xa0;%, with a sensitivity twice that of the gold standard methylation-specific quantitative PCR (Methylight). Detection of genomic DNA (gDNA) from tumor tissues and paired plasma ctDNA of 15 clinical patients using this method showed both sensitivity and specificity reaching 100&#xa0;%.<br><br>SIGNIFICANCE: This novel, highly sensitive, efficient, and portable detection method innovatively resolves the target depletion issue caused by CRISPR/Cas12a cleavage in traditional RPA-CRISPR/Cas12a systems via thermosensitive hydrogel-mediated single-tube phase separation technology. It provides a new technical pathway for the accurate detection of low-abundance methylated circulating tumor DNA (ctDNA), will significantly enhance the level of breast cancer liquid biopsy, and offer strong support for the diagnosis and differential diagnosis of breast cancer.},
}
@article {pmid41611404,
year = {2026},
author = {Park, S and Koo, B and Kim, MG and Lee, EY and Lee, HJ and Roh, Y and Lee, M and Bae, CE and Lee, SW and Kang, YA and Shin, Y},
title = {CADEM: Species-level detection of mycobacterial cfDNA via CRISPR for pulmonary disease diagnosis.},
journal = {Analytica chimica acta},
volume = {1388},
number = {},
pages = {345085},
doi = {10.1016/j.aca.2026.345085},
pmid = {41611404},
issn = {1873-4324},
mesh = {Humans ; *CRISPR-Cas Systems ; *Cell-Free Nucleic Acids/genetics/blood/analysis ; *Lung Diseases/diagnosis/microbiology ; *DNA, Bacterial/genetics/blood ; Lab-On-A-Chip Devices ; Mycobacterium avium Complex/genetics/isolation &amp; purification ; Microfluidic Analytical Techniques ; },
abstract = {BACKGROUND: Pulmonary infections caused by Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) present significant clinical challenges due to overlapping symptoms and different treatments. In particular, accurate identification of NTM species such as Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MABC) is essential, as these species show drug susceptibility profiles that differ markedly from MTB. However, conventional culture-based diagnostics are time-consuming, and current molecular assays often lack resolution and rely heavily on sputum specimens. To address these limitations, liquid biopsy using bacterial-derived circulating cell-free DNA (cfDNA) offers a minimally invasive alternative, and CRISPR/Cas12a technology provides the sensitivity required to detect its low levels.<br><br>RESULTS: We developed CADEM (CRISPR-Assisted Detection via Enrichment of Mycobacterium-derived cfDNA using Microfluidic technology), a streamlined diagnostic system that integrates microfluidic cfDNA enrichment, targeted amplification, and CRISPR/Cas12a-based detection. The microfluidic platform enables high-yield recovery of cfDNA from large-volume clinical samples without the need for cell lysis. Optimized Cas12a-crRNA complexes enable highly sensitive and specific detection of MAC-, MABC-, and MTB -specific amplicons, achieving 10- to 100-fold greater sensitivity than end-point PCR and probe-based real-time PCR. In a validation set of 20 clinical specimens (7 positives and 13 healthy controls), CADEM identified all MAC, MABC, and MTB cases with full accuracy and no false positives. The CRISPR detection step produced a clear fluorescence readout within 20&#xa0;min and, together with enrichment and amplification, delivered species-level results within a 2-h workflow.<br><br>SIGNIFICANCE: CADEM offers an accurate and streamlined molecular approach for distinguishing MAC, MABC, and MTB at the species-level to support appropriate diagnosis and treatment. By combining microfluidic cfDNA enrichment with CRISPR-based detection, CADEM enables efficient analysis from liquid biopsy samples for pulmonary disease diagnosis. The system is also compatible with isothermal amplification, supporting future adaptation for point-of-care testing in resource-limited settings.},
}
@article {pmid41611486,
year = {2026},
author = {Lu, Q and Teng, W and Liang, Y and Wang, Y},
title = {[Advances in genome editing and lodging resistance in sugarcane].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {33-52},
doi = {10.13345/j.cjb.250555},
pmid = {41611486},
issn = {1872-2075},
support = {XDA0450000//the Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Saccharum/genetics ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Gene Editing/methods ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Zinc Finger Nucleases/genetics ; },
abstract = {Sugarcane (Saccharum spp.) is an important cash crop that provides about 90% of sugar and 40% of bioethanol in China. Due to its large genome and complicated genetic background, conventional breeding is difficult to achieve efficient genetic improvement of sugarcane. Genome editing is a disruptive technology in life sciences, enabling precise and efficient modification of target genes. From zinc-finger nucleases (ZFNs) to transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system and the derived base editing and prime editing, these technologies have greatly advanced genetic research and upgraded biological breeding. With the decoding of the sugarcane genome, genome editing has provided a new technical means for the genetic improvement of polyploid sugarcane. This article provides a comprehensive review of the trajectory of genome editing in plants, the optimization of the CRISPR/Cas system, the genetic transformation status of sugarcane, the development of sugarcane genomics, and the application of genome editing in sugarcane. It focuses on exploring the application prospects of genome editing in breeding lodging-resistant sugarcane varieties. This review aims to provide valuable references for promoting the use of genome editing in sugarcane breeding.},
}
@article {pmid41611487,
year = {2026},
author = {Huang, X and Zeng, G and Shen, X},
title = {[Research progress and prospects in CRISPR-Cas9 gene editing in sorghum].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {53-61},
doi = {10.13345/j.cjb.250419},
pmid = {41611487},
issn = {1872-2075},
support = {ZLGC201902-15//the Innovation-driven Assistance Project of China Association for Science and Technology/ ; },
mesh = {*Sorghum/genetics/growth &amp; development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Plant Breeding/methods ; },
abstract = {Sorghum is an important grain and cash crop in China, and the promotion and application of this crop have long been constrained by a shortage of genetic resources. Issues such as narrow genetic background and outdated breeding techniques have severely hindered the development and dissemination of new sorghum varieties. Although gene editing has demonstrated significant potential in the genetic improvement of crops since its inception, the application of this technology in sorghum remains lagging. This paper provides a systematic review of the latest breakthroughs in CRISPR-Cas9 in sorghum. Focusing on abiotic stress, growth and development, and quality, we explore the innovative applications of this technology in expanding genetic diversity, improving stress tolerance, optimizing plant architecture and yield potential, and enhancing quality characteristics. Additionally, we analyze the main technical challenges including low genetic transformation efficiency and insufficient adaptability of editing tools facing the gene editing in sorghum. Finally, we make an outlook on the future prospects of next-generation gene editing technologies in the genetic improvement of sorghum. This paper can provide important theoretical references for sorghum molecular breeding, and hold significant practical significance for safeguarding China's food security and enhancing the competitiveness of the sorghum industry.},
}
@article {pmid41611506,
year = {2026},
author = {Ji, Y and Bian, X and Feng, Z and Li, F and Hu, Z and Hu, Y and Liu, P and Ma, B and Chen, X},
title = {[Map-based cloning and functional analysis of the golden panicle and brown rice gene gpr1].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {1},
pages = {356-366},
doi = {10.13345/j.cjb.250438},
pmid = {41611506},
issn = {1872-2075},
support = {32272096//the National Natural Science Foundation of China/ ; LZ24C130003//the Natural Science Foundation of Zhejiang Province/ ; 2023R404039//the Zhejiang Provincial Undergraduate Science and Technology Innovation Activity Plan ("Xinmiao" Talents Program)/ ; 2024-2-017 and 2023-2-014//the Science and Technology Key Projects of Jinhua City/ ; },
mesh = {*Oryza/genetics/metabolism ; Cloning, Molecular ; *Plant Proteins/genetics ; Chromosome Mapping ; *Genes, Plant ; CRISPR-Cas Systems ; Pigmentation/genetics ; Flavonoids/biosynthesis ; },
abstract = {The colors of panicle hull and brown rice are crucial agronomic traits in rice, which are widely used in breeding and genetic research. Here, a rice variety named 'LAL SAR' characterized by golden panicle and brown rice was identified from Nepal. Genetic analysis revealed that these phenotypes were controlled by a single recessive nuclear gene, which was designated as gpr1 (golden panicle and brown rice 1). Using the strategy of map-based cloning, we mapped gpr1 into a 47 kb interval on the rice chromosome 3 and identified its candidate gene as LOC_Os03g60509. This gene encodes a chalcone isomerase, a key enzyme in the flavonoid biosynthetic pathway. Through PCR sequencing and RT-PCR analysis, a long-fragment insertion was found in the promoter of gpr1 in 'LAL SAR', which completely suppressed the expression of this gene. Using CRISPR/Cas9 technology, we successfully knocked out GPR1 (the dominant allele of gpr1) from a Oryza sativa subsp. japonica variety 'Zhonghua 11'. The knockout mutant plants exhibited golden panicles and brown rice, with significantly increased naringenin chalcone content in the hull. Our results indicate that GPR1 participates in the flavonoid biosynthesis, providing a critical theoretical foundation and a gene resource for rice quality improvement and genetic enhancement.},
}
@article {pmid41611887,
year = {2026},
author = {Gordon, A and Yoon, SJ and Bicks, LK and Martín, JM and Pintacuda, G and Arteaga, S and Wamsley, B and Guo, Q and Elahi, L and Dolmetsch, RE and Bernstein, JA and O'Hara, R and Hallmayer, JF and Lage, K and Pasca, SP and Geschwind, DH},
title = {Developmental convergence and divergence in human stem cell models of autism.},
journal = {Nature},
volume = {651},
number = {8106},
pages = {707-719},
pmid = {41611887},
issn = {1476-4687},
support = {RM1 MH132651/MH/NIMH NIH HHS/United States ; R01 MH109912/MH/NIMH NIH HHS/United States ; R01 MH110927/MH/NIMH NIH HHS/United States ; U01 MH115745/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/pathology/cytology ; Organoids/metabolism/pathology/cytology ; *Autism Spectrum Disorder/genetics/pathology ; Mutation/genetics ; Cell Differentiation/genetics ; Neural Stem Cells/metabolism/cytology/pathology ; Protein Interaction Maps/genetics ; *Models, Biological ; CRISPR-Cas Systems/genetics ; Female ; Transcriptome/genetics ; Male ; Cell Line ; },
abstract = {Two decades of genetic studies in autism spectrum disorder (ASD) have identified more than 100 genes harbouring rare risk mutations[1-13]. Despite this substantial heterogeneity, transcriptomic and epigenetic analyses have identified convergent patterns of dysregulation across the ASD postmortem brain[14,15-17]. To identify shared and distinct mechanisms of ASD-linked mutations, we assembled a large patient collection of human induced pluripotent stem (hiPS) cells, consisting of 70 hiPS cell lines after stringent quality control representing 8 ASD-associated mutations, idiopathic ASD, and 20 lines from non-affected control individuals. Here we used these hiPS cell lines to generate human cortical organoids, profiling by RNA sequencing at four distinct time points up to 100 days after in vitro differentiation. Early time points harboured the largest mutation-specific changes, but different mutations converged on shared transcriptional changes as development progressed. We identified a shared RNA and protein interaction network, which was enriched in ASD risk genes and predicted to drive the observed downstream changes in gene expression. CRISPR-Cas9 screening of these candidate transcriptional regulators in induced human neural progenitors validated their downstream convergent molecular effects. These data illustrate how risk associated with genetically defined forms of ASD can propagate by means of transcriptional regulation to affect convergently dysregulated pathways, providing new insight into the convergent impact of ASD genetic risk on human neurodevelopment.},
}
@article {pmid41612423,
year = {2026},
author = {Nizovtseva, I and Rezaykin, A and Korenskaia, A and Zakhartsev, M and Chigireva, A and Starodumov, I and Chernushkin, D},
title = {Identification and comparative genomic analysis of prophage sequences and CRISPR‒Cas immunity in Methylococcus genomes: insights into industrial methane bioconversion.},
journal = {Biotechnology for biofuels and bioproducts},
volume = {19},
number = {1},
pages = {},
pmid = {41612423},
issn = {2731-3654},
support = {24-24-00454//Russian Science Support Foundation/ ; },
abstract = {BACKGROUND: Methylococcus species utilize methane as the sole carbon and energy source, converting it into biomass and other metabolic end products. Owing to this metabolic capacity, they hold particular promise in industrial C1 biotechnology, especially for the production of protein-rich feed. However, the industrial cultivation of Methylococcus-based consortia on methane is inherently nonsterile, exposing the process to potential biological risks that may compromise the stability, duration and productivity of cultivation. One of the most critical threats is bacteriophage infection, whose triggers for rapid phage-mediated lysis and resulting economic losses remain incompletely understood. Elucidating these processes is paramount for devising strategies to mitigate or prevent detrimental outcomes.<br><br>RESULTS: In this investigation, nine publicly accessible genomes of Methylococcus species were examined, culminating in the identification of eleven prophage sequences distributed variably among the genomes. Sequence annotations revealed that nine prophages are potentially functional and intact, whereas the rest carry incomplete gene sets indicative of nonviability. Phylogenetic analyses corroborated the substantial diversity of prophages, which formed distinct clusters related to &#x3b3;-proteobacteria phages. Furthermore, comparative genomic analyses demonstrated a high degree of structural conservation despite the presence of rearrangements. The annotation of the CRISPR&#x2012;Cas systems provided insights into additional dimensions of phage&#x2012;bacteria interactions. Examination of prophage integration sites did not reveal any disruption of metabolic gene structures, thus suggesting minimal risk of deleterious phenotypic outcomes.<br><br>CONCLUSIONS: These findings considerably advance the current understanding of the genetic diversity and biological properties of prophages infecting Methylococcus species, underscoring the importance of holistic approaches for the detection and analysis of these elements. Our findings underscore the need for routine prophage monitoring in industrial methanotrophic consortia, with the pipeline established here serving as a foundational framework for future refinement and industrial adaptation.},
}
@article {pmid41612768,
year = {2026},
author = {Wang, Y and Zhang, X and Lin, Y and Zhang, X and Yang, Z and Wan, Y and Negahdary, M},
title = {Single-Tube Dual-Gene Detection of Methicillin-Resistant Staphylococcus aureus via Selective Trans-Cleavage Preferences of Cas9 and Cas12a.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {3772-3781},
doi = {10.1021/acs.analchem.5c05789},
pmid = {41612768},
issn = {1520-6882},
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification ; Animals ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Penicillin-Binding Proteins/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Micrococcal Nuclease/genetics ; },
abstract = {Rapid and accurate detection of methicillin-resistant Staphylococcus aureus (MRSA) is essential for guiding clinical treatment and preventing infections. Current dual-gene detection methods based on CRISPR-Cas systems often require additional transcription steps, which increase the complexity of the assay and extend the turnaround time. Here, we report a single-tube dual-gene detection platform that leverages the orthogonal trans-cleavage preferences of Cas9 and Cas12a. By exploiting the loss of RNA cleavage activity in Cas12a when guided by split crRNA, and the inability of Cas9 to cleave DNA hairpin probes, we established a single-tube assay capable of simultaneously detecting the MRSA resistance gene (mecA) and the S. aureus-specific nuclease gene (nuc). The platform achieved attogram-level sensitivity and single-cell detection with high specificity against non-MRSA strains. Validation in an in vivo tilapia infection model demonstrated complete concordance with qPCR, reaching 100% positive and negative percent agreements. This work presents a streamlined, accurate, and practical approach for dual-gene pathogen detection, expanding the potential of Cas protein orthogonality for multiplex diagnostics.},
}
@article {pmid41613398,
year = {2025},
author = {Mehmood, MA and Iqbal, MM and Ashfaq, M and Chen, S and Wang, J},
title = {Advanced molecular tools for surveillance and management of tobamoviruses.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1718133},
pmid = {41613398},
issn = {1664-462X},
abstract = {Tobamoviruses are a group of plant viruses that can cause yield losses of up to 70% and reduce fruit quality by 30-50%. Historically, tobamoviruses were dominated by tobacco mosaic virus (TMV) and tomato mosaic virus (ToMV). However, the landscape is rapidly shifting with the emergence of economically significant viruses such as tomato mottle mosaic virus (ToMMV) and tomato brown rugose fruit virus (ToBRFV). Both can circumvent the previously durable Tm-2[2] resistance in tomato and spread across multiple continents. This shift coincides with dramatic leaps in diagnostic tools, which have enhanced surveillance capabilities. Sensitive detection of tobamoviruses in the field with minimal sample preparation can be achieved using latest technologies such as isothermal amplification, CRISPR/Cas-hybrid assays or next-generation sequencing. Virus-host interactions underscore that viral proteins, including replicase components, are potent suppressors of RNA silencing (VSRs). Small RNA profiling and network analyses of viral movement proteins reveal complex mechanisms of immune evasion and resistance breakdown. These findings are largely based on dominant NB-LRR genes such as L, Tm-1, and Tm-2[2] . However, evidence indicates that ToBRFV can bypass this resistance via mutation in the movement protein, so supplementary methods should be considered. This review covers latest approaches, such as genome editing with CRISPR, targeting susceptibility genes, RNA interference (RNAi), and multi-omics approaches (transcriptomics, proteomics, metabolomics, ionomics), that can facilitate real-time surveillance and breeding for enhanced resilience. Moreover, the use of bio-formulations and nano-formulations as eco-friendly alternatives against tobamoviruses is discussed in detail. Climate change further complicates disease dynamics by undermining temperature-sensitive resistance, altering virus prevalence, and exacerbating yield losses. The rapid emergence of new tobamoviruses, which threatens the economy, necessitates a comprehensive approach. The integration of molecular diagnostics using CRISPR, omics technologies, designed protective systems, and climate-augmented disease prediction offers a detailed blueprint for the sustainable control of tobamoviruses and crop protection.},
}
@article {pmid41613863,
year = {2025},
author = {Safrygina, AA and Orlov, YL},
title = {Genetically engineered approaches to the treatment of cystic fibrosis.},
journal = {Biophysical reviews},
volume = {17},
number = {5},
pages = {1333-1358},
pmid = {41613863},
issn = {1867-2450},
abstract = {Failure of functions of CFTR (cystic fibrosis transmembrane conduction regulator) gene, which encodes a protein of a selective ion channel, is causing cystic fibrosis. Cystic fibrosis is a severe systemic monogenic disease with an autosomal recessive type of inheritance, which significantly reduces the duration and quality of life of patients. It is one of the most common hereditary diseases. Studying of molecular functions of CFTR protein in different types of cells, its structural and functional network interactions are critically important for the development of a new and more effective pathogenetic therapy. We are reviewing papers on the structure of the CFTR protein and its pathogenic genetic variants, as well as methods of pathogenetic therapy of cystic fibrosis by CFTR modulators and gene engineering. Recent gene engineering approaches to keep CFTR functions are discussed, such as gene-replacement therapy and genome editing, as well as viral and non-viral delivery systems and strategies of genomic editors.},
}
@article {pmid41614578,
year = {2026},
author = {Barragán-Borrero, V and de Santana Lopes, A and Rodrigues Batista, ED and Höfer, M and Elias, R and Chakraborty, A and Ponce-Mañe, A and Descombes, C and Diezma-Navas, L and Petraki, L and Huber, M and Xu, S and Marí-Ordóñez, A},
title = {Strain, procedures, and tools for reproducible genetic transformation and genome editing of the emerging plant model Spirodela polyrhiza.},
journal = {The New phytologist},
volume = {250},
number = {2},
pages = {735-756},
pmid = {41614578},
issn = {1469-8137},
support = {512079118//Emmy Noether Programme/ ; //&#xd6;sterreichischen Akademie der Wissenschaften/ ; 14002787004056//Erasmus+/ ; //Johannes Gutenberg-Universit&#xe4;t Mainz/ ; 407023052//Deutsche Forschungsgemeinschaft/ ; 427577435//Deutsche Forschungsgemeinschaft/ ; 438887884//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Araceae/genetics ; *Transformation, Genetic ; *Gene Editing/methods ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Reproducibility of Results ; Agrobacterium ; Transgenes ; },
abstract = {Duckweeds (Lemnaceae) have excellent potential for fundamental and applied research due to ease of cultivation, small size, and continuous fast clonal growth. However, their usage as model organisms and platforms for biotechnological applications is often limited by the lack of universal genetic manipulation methods necessary for transgene expression, gene editing, and other methods to modify gene expression. To identify suitable strains for genetic manipulation of the giant duckweed, Spirodela polyrhiza, we screened several genotypes for callus induction and regeneration and established genetic transformation. We identified SP162 to be amenable to Agrobacterium-mediated transformation via tissue culture. The procedure is robust and reproducible across laboratories, allowing stable expression of different reporter genes and selectable markers, enabling CRISPR/Cas9-mediated genome editing. In addition, due to a weak small RNA-based silencing response, S. polyrhiza sustains prolonged periods of transgene activity in transient expression assays. To promote duckweed research and encourage the adoption of S. polyrhiza, we have made SP162 (ID#: 5676) and its genome publicly available and provide here detailed procedures for its cultivation and transformation. Furthermore, we created a web server to explore its genome, retrieve gene sequences, and implement orthologous gene search and a gRNA design function for diverse CRISPR/Cas-based applications (https://agxu.uni-mainz.de/SP162/).},
}
@article {pmid41614935,
year = {2026},
author = {El Semary, NAH and Fadiel, A and Eichenbaum, KD and Alhusayni, SA},
title = {Prokaryotic Molecular Defense Mechanisms and Their Potential Applications in Cancer Biology: A Special Consideration for Cyanobacterial Systems.},
journal = {Current issues in molecular biology},
volume = {48},
number = {1},
pages = {},
pmid = {41614935},
issn = {1467-3045},
support = {KFU253867//Vice Presidency of Higher studies and Scientific research, Deanship of Scientific research, King Faisal University, AlHufuf, Al-Ahsa, Kingdom of Saudi Arabia, postcode: 31982/ ; },
abstract = {Cyanobacteria harbor sophisticated molecular defense systems that have evolved over billions of years to protect against viral invasion and foreign genetic elements. These ancient photosynthetic organisms possess a diverse array of restriction-modification (R-M) systems and CRISPR-Cas arrays that present challenges for genetic engineering, but also offer unique opportunities for cancer-targeted biotechnological applications. These systems exist in prokaryotes mainly as defense mechanisms but they are currently used in molecular applications as gene editing tools. Moreover, latest developments in nucleases such as zinc finger nucleases (ZFNs), TALENs (transcription-activator-like effector nucleases) are discussed. A comprehensive genomic analysis of 126 cyanobacterial species found 89% encode multiple R-M systems, averaging 3.2 systems per genome, creating formidable barriers to transformation but also providing molecular machinery that could be harnessed for precise recognition and targeting of cancer cells. This review critically examines the dual nature of these defense systems, their ecological functions, and the emerging strategies to translate their molecular precision into advanced anticancer therapeutics. Hence, the review main objectives are to explore the recent understanding of these mechanisms and to exploit the knowledge gained in opening new avenues for cancer-focused targeted interventions, while acknowledging the significant challenges to translate these systems from laboratory curiosities to practical applications.},
}
@article {pmid41615337,
year = {2026},
author = {Liu, X and Wang, X and He, S and Li, M and Lu, C and Xing, C},
title = {An oxidative cleavage-based HCR-CRISPR/Cas12a biosensor for highly sensitive detection of hypochlorous acid.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1317-1324},
doi = {10.1039/d5an01299f},
pmid = {41615337},
issn = {1364-5528},
mesh = {*Biosensing Techniques/methods ; *Hypochlorous Acid/analysis ; *CRISPR-Cas Systems ; Oxidation-Reduction ; Limit of Detection ; DNA/chemistry/genetics ; Nucleic Acid Hybridization ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; *Water Pollutants, Chemical/analysis ; Bacterial Proteins ; },
abstract = {Hypochlorous acid (HClO) is a critical disinfectant in public health and water treatment, yet its excessive presence in environmental matrices poses significant risks to human health (e.g., respiratory irritation, tissue damage) and aquatic ecosystems (e.g., biodiversity loss). Herein, we developed an oxidative cleavage-based hybridization chain reaction (HCR)-CRISPR/Cas12a biosensor for highly sensitive detection of HClO. The sensor utilizes a dual-lock switch mechanism: HClO selectively cleaves a phosphorothioated (PS) DNA hairpin (Lock 1), releasing an initiator strand to trigger the HCR and generate PAM-containing long dsDNA. Simultaneously, the locked crRNA (L-crRNA), which is caged by a PS-modified silent chain (Lock 2), can be released when the PS-modified silent chain is degraded under HClO incubation, allowing the activated crRNA to dynamically regulate Cas12a-crRNA complex formation. Then, HCR-dsDNA activates Cas12a's trans-cleavage activity, enabling fluorescence signal readout via reporter DNA cleavage. The dual-lock strategy minimizes nonspecific activation in CRISPR/Cas12a systems, significantly enhancing sensitivity and specificity. Our work establishes a robust platform for environmental pollutant monitoring, with applications in water safety assessment and food quality control.},
}
@article {pmid41616513,
year = {2026},
author = {Xu, L and Jin, J and Lyu, W and Liang, X and Wang, Q and Zhang, J and Luo, Y and Chen, J and Lu, H and Li, X and Shen, F},
title = {Point-of-care profiling of H. pylori virulence and antibiotic resistance from endoscopic biopsies using an integrated restriction enzyme-CRISPR microfluidic platform.},
journal = {Biosensors &amp; bioelectronics},
volume = {299},
number = {},
pages = {118438},
doi = {10.1016/j.bios.2026.118438},
pmid = {41616513},
issn = {1873-4235},
mesh = {*Helicobacter pylori/genetics/pathogenicity/drug effects/isolation &amp; purification ; Humans ; *Helicobacter Infections/microbiology/diagnosis/drug therapy ; *Drug Resistance, Bacterial/genetics ; Point-of-Care Systems ; Anti-Bacterial Agents/pharmacology ; Virulence/genetics ; CRISPR-Cas Systems ; Lab-On-A-Chip Devices ; Biopsy ; Biosensing Techniques ; Clarithromycin/pharmacology ; Levofloxacin/pharmacology ; Polymorphism, Single Nucleotide ; Gastric Mucosa/microbiology/pathology ; },
abstract = {Endoscopically obtained gastric mucosal biopsies contain abundant molecular information that could inform Helicobacter pylori (H. pylori) eradication therapy. However, the lack of point-of-care tools for processing solid tissue limits rapid, on-site genotyping, leading to empirical therapy and higher eradication failure rates. Here, we present the SlipChip-based On-site and User-friendly Testing (SCOUT) together with the restriction enzyme-assisted CRISPR/Cas12a (RCut) method for point-of-care genotyping from raw endoscopic gastric biopsies. This fully automated platform enables analysis of key virulence genes and resistance-associated single-nucleotide variants (SNVs) at mutant allele frequencies down to 0.1 %. Results were available within 1 h, enabling actionable molecular profiling during the same endoscopic encounter. In 159 clinical validations, SCOUT showed concordant virulence profiling with quantitative PCR (κ = 0.978) and accurately detected clarithromycin and levofloxacin resistance mutations with up to 100 % sensitivity and specificity. These results demonstrate that SCOUT bridges the gap between diagnostic sampling and informed eradication strategies, providing a scalable framework for point-of-care molecular genotyping.},
}
@article {pmid41616988,
year = {2026},
author = {Yao, B and Öktem, M and Yang, G and Wang, Q and Daniels, MA and Dokter, I and Lefferts, JW and Gonçalves, MAFV and Doevendans, PA and van Mil, A and Sluijter, JPG and Schiffelers, R and Mastrobattista, E and Lei, Z},
title = {LAH5-mediated delivery of prime editor ribonucleoprotein complexes for genome editing.},
journal = {International journal of pharmaceutics},
volume = {692},
number = {},
pages = {126622},
doi = {10.1016/j.ijpharm.2026.126622},
pmid = {41616988},
issn = {1873-3476},
mesh = {Humans ; *Gene Editing/methods ; *Ribonucleoproteins/administration &amp; dosage/genetics ; HEK293 Cells ; *Cell-Penetrating Peptides/administration &amp; dosage/chemistry ; Induced Pluripotent Stem Cells/metabolism ; Myocytes, Cardiac/metabolism ; CRISPR-Cas Systems ; },
abstract = {Prime editing (PE) is a precise gene-editing technology with potential for treating genetic disorders, but efficient delivery systems remain a challenge. Viral vectors offer high efficiency but pose safety concerns related with their immunogenicity, while non-viral methods struggle with stability and scalability. Cell-penetrating peptides (CPPs) present a promising alternative due to their low immunogenicity. In this study, we explored LAH5, a histidine-rich CPP, for delivering PE ribonucleoproteins (RNPs) into PLN R14del mutant cell lines. We purified engineered SpGPEmax protein, evaluating its intracellular uptake and editing frequency in HEK293T.PLN R14del reporter cells and human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our results demonstrate that LAH5 effectively delivers intracellularly SpGPEmax RNP components, resulting in correction of the R14del mutation, thereby offering a viable non-viral strategy for direct cellular precise genome editing.},
}
@article {pmid41617656,
year = {2026},
author = {He, M and Chen, J and Chao, M and Yin, F and Pan, H and Wang, Q and Cheng, W and Wang, Z and Xiang, Y},
title = {Novel One-Pot Detection of Tumor mRNA Vaccine by the Target Serving as crRNA of the Split Cas12a System and Synergistically Triggering an Entropy-Driven Reaction.},
journal = {Analytical chemistry},
volume = {98},
number = {5},
pages = {4134-4142},
doi = {10.1021/acs.analchem.5c06932},
pmid = {41617656},
issn = {1520-6882},
mesh = {*Entropy ; *Cancer Vaccines/analysis/genetics ; *RNA, Messenger/genetics/analysis ; Humans ; *mRNA Vaccines/analysis ; *Endodeoxyribonucleases/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {mRNA vaccines, as a new form of vaccine, are gradually revealing their potential in the field of tumor treatment. With the development of mRNA vaccines, monitoring their levels has become particularly important. In this work, a novel and innovative strategy was proposed for the first time, which directly used the target as crRNA and simultaneously triggered an entropy-driven split Cas12a system for triple signal amplification for the high-sensitivity detection of mRNA vaccines. This study broke the traditional full-length crRNA and innovatively designed a split crRNA structure, directly using the target mRNA as the variable sequence of the crRNA. This design avoided the need for additional introduction of variable sequences, thereby eliminating nonspecific signal leakage and multicomponent interference from the source. At the same time, the target molecule served as the initiating chain for the EDC reaction, allowing the split Cas12a system to be coupled with the entire entropy-driven reaction process, achieving efficient collaboration and triple signal amplification and significantly improving the detection sensitivity and specificity. Moreover, the entire detection process required only one reaction step, which was simple, fast, and efficient. The aim was to provide strategies for screening and pharmacokinetic studies of mRNA vaccines.},
}
@article {pmid41617991,
year = {2026},
author = {Nagahata, N and Kato, K and Yamada, S and Kannan, S and Okazaki, S and Isayama, Y and Hiraizumi, M and Yamashita, K and Koonin, EV and Zhang, F and Nishimasu, H},
title = {Structural visualization of the molecular evolution of CRISPR-Cas9.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {2},
pages = {304-317},
pmid = {41617991},
issn = {1545-9985},
mesh = {Cryoelectron Microscopy ; *CRISPR-Cas Systems/genetics ; *Evolution, Molecular ; RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; Models, Molecular ; *CRISPR-Associated Protein 9/chemistry/genetics/ultrastructure/metabolism ; DNA/metabolism/chemistry ; },
abstract = {RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR-Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element-guided activity) systems, respectively. Sequence and structural comparisons indicate that IscB (~500 residues) evolved into Cas9 (~700-1,600 residues) through protein expansion coupled with guide RNA miniaturization. However, the specific sequence of events in this evolutionary transition remains unknown. Here, we report cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases-two IscBs and two Cas9s-each in complex with its cognate guide RNA and target DNA. Comparisons of these four complex structures to previously reported IscB and Cas9 structures indicate that evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the acquisition of the zinc-finger-containing REC3 domain, followed by bridge helix extension and REC1 domain acquisition. These structural changes led to expansion of the REC lobe, increasing the target DNA cleavage specificity. Additionally, the structural conservation of the RNA scaffolds indicates that the dual CRISPR RNA (crRNA) and trans-activating crRNA guides of CRISPR-Cas9 evolved from the single ωRNA guides of OMEGA systems. Our findings provide insights into the succession of structural changes involved in the exaptation of transposon-associated RNA-guided nucleases for the role of effector nucleases in adaptive immune systems.},
}
@article {pmid41618915,
year = {2026},
author = {Liu, Q and Huang, Y and Zhou, R and Ding, H and Nie, Q and Gong, X and Zuo, T and Wang, S and Liang, Y and Li, Y},
title = {A Minimal and Portable CRISPR Platform Based on Bifidobacterial Cas9 Enables Genome Editing in E. coli Nissle 1917.},
journal = {ACS synthetic biology},
volume = {15},
number = {4},
pages = {1544-1556},
doi = {10.1021/acssynbio.5c00911},
pmid = {41618915},
issn = {2161-5063},
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Bifidobacterium/genetics ; Plasmids/genetics ; Metabolic Engineering/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; Genome, Bacterial ; },
abstract = {Genetic manipulation of core gut probiotics remains challenging due to endogenous cellular barriers and a scarcity of efficient molecular tools, limiting progress in live biotherapeutic development. Here, we characterized the native type II-C CRISPR-Cas system in Bifidobacterium longum subsp. longum GNB (B. longum GNB). Through integrated bioinformatic analysis and high-throughput protospacer adjacent motif (PAM) screening, we identified a novel 5'-NNRMAT-3' (where R = A/G, M = A/C) motif recognized by its compact Cas9 nuclease (BLCas9). The stringent PAM dependency of BLCas9 was unequivocally confirmed by in vitro cleavage assays. Leveraging this endogenous mechanism, we developed a dual-plasmid editing platform for robust and multiplex genome engineering in the probiotic strain Escherichia coli Nissle 1917 (EcN). Application of this system notably enhanced extracellular γ-aminobutyric acid (GABA) production in EcN through targeted metabolic engineering. Our work provides the first molecular dissection of a type II-C system in Bifidobacterium longum and establishes a generalizable framework for the discovery and application of compact programmable nucleases, suggesting a viable strategy for modulating host physiology via the gut-brain axis.},
}
@article {pmid41620000,
year = {2026},
author = {Shah, W and Hussain, M and Serwat, A and Bilal, M and Raza, Y and Mansoor, A and Faraz, A},
title = {CRISPR/Cas9 and reproductive failure: applications, ethical challenges, and future perspectives in human germline genome editing.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {584},
number = {},
pages = {120875},
doi = {10.1016/j.cca.2026.120875},
pmid = {41620000},
issn = {1873-3492},
mesh = {Humans ; *Gene Editing/ethics ; *CRISPR-Cas Systems/genetics ; *Germ Cells/metabolism ; Animals ; *Infertility/genetics/therapy ; },
abstract = {Reproductive failure affects millions of couples worldwide and frequently arises from genetic defects that impair gametogenesis, fertilization, or early embryonic development. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome-editing technology has emerged as a powerful experimental platform for dissecting infertility-associated genes and, in principle, correcting pathogenic variants in germline cells or preimplantation embryos. This review critically examines current applications of CRISPR/Cas9 in reproductive biology, including disease modeling in animal systems, editing of spermatogonial stem cells (SSCs), manipulation of oocytes and zygotes, and proof-of-concept studies in human embryos. Particular emphasis is placed on the major technical barriers that currently preclude clinical translation, including off-target mutagenesis, embryo mosaicism, and the low efficiency of homology-directed repair relative to non-homologous end joining. Limitations related to delivery strategies, DNA damage responses, chromosomal rearrangements, and the genetic heterogeneity of infertility are also evaluated. Comparative discussion highlights how germline editing differs fundamentally from somatic CRISPR therapies that have already reached clinical application in hematologic disorders. The review further analyzes ethical and regulatory challenges associated with heritable genome modification, including long-term safety, consent across generations, international governance disparities, and the continued reliance on assisted reproductive technologies combined with preimplantation genetic testing as safer clinical alternatives. Collectively, current evidence indicates that CRISPR/Cas9 remains primarily a research tool for elucidating reproductive biology rather than an imminent therapeutic option for human infertility. Continued technological refinement, rigorous preclinical validation, and globally harmonized oversight will be essential before germline applications can be ethically or clinically justified.},
}
@article {pmid41620031,
year = {2026},
author = {Jiang, Y and Yang, Z and Yang, J and Li, Y and Liu, J and Zhao, L and Ge, J},
title = {Development of a rapid and portable detection method for canine distemper virus based on CRISPR-Cas13a.},
journal = {Journal of virological methods},
volume = {342},
number = {},
pages = {115355},
doi = {10.1016/j.jviromet.2026.115355},
pmid = {41620031},
issn = {1879-0984},
mesh = {Animals ; *Distemper Virus, Canine/isolation &amp; purification/genetics ; Sensitivity and Specificity ; Dogs ; *CRISPR-Cas Systems ; *Distemper/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; RNA, Viral/genetics ; },
abstract = {Canine distemper virus (CDV) is a pathogenic microorganism that severely affects the respiratory, digestive, and nervous systems, causing multi-systemic symptoms. It infects nearly all terrestrial carnivores worldwide, particularly the Canidae and Mustelidae families, posing a serious threat to global socio-economic and public health security. Given the importance of etiological treatment and early diagnosis, developing novel detection methods with improved accuracy, rapidity, and user-friendliness is necessary for effective prevention and control of CDV infection. In this study, we established a novel testing method using recombinase-aid amplification (RAA) coupled with CRISPR-Cas13a and optimized the working concentration of CRISPR RNA (crRNA) and Cas13a for the lateral flow detection (LFD) of CDV. The RAA-CRISPR-Cas13a-LFD for CDV did not cross-react against other prevalent canine pathogens and the sensitivity can detect as little as 10[2] copies/μL of CDV cDNA plasmids. Additionally, combined with HUDSON this RAA-CRISPR-Cas13a-LFD method could be used to detect clinical samples within 1.5 h, with performance comparable to that of RT-PCR. The results for the RAA-CRISPR-Cas13a detection could be visualized using either fluorescence or lateral flow strips for in field-deployable viral diagnosis. Overall, our developed method showed good potential in point-of-care testing (POCT) to control and reduce the losses by CDV infection.},
}
@article {pmid41620285,
year = {2026},
author = {Sohn, M and Pantsulaia, G and Brody, J},
title = {Mouse and human T cell Cas9-RNP/nucleofection-mediated gene-editing.},
journal = {Methods in cell biology},
volume = {201},
number = {},
pages = {91-107},
doi = {10.1016/bs.mcb.2025.03.020},
pmid = {41620285},
issn = {0091-679X},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; *CD8-Positive T-Lymphocytes/metabolism ; *Ribonucleoproteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics/metabolism ; *Transfection/methods ; },
abstract = {CRISPR/Cas9 technology has revolutionized genome editing, providing a precise and expeditious means of genetic modification. This breakthrough has enhanced our understanding of gene function, including T cell immunology. Efficient gene editing in primary T cells not only offers a robust investigative tool for dissecting gene function but also holds promise for advancing T cell-based immunotherapies, including next-generation chimeric antigen receptor T cells. In this article, we introduce a highly efficient gene editing protocol for unstimulated human CD8 T cells and unstimulated and stimulated murine CD8 T cells, utilizing transient nucleofection of ribonucleoprotein complexes composed of synthesized modified single guide RNAs (sgRNAs) and purified Cas9 protein. This protocol, initially devised for primary CD8 T cells, can be readily adapted to other primary cell cultures through optimization of nucleofection conditions as well. In essence, our method provides a practical and powerful approach for achieving precise and swift gene knockout in primary CD8 T cells.},
}
@article {pmid41620519,
year = {2026},
author = {Popović, J and Hahut, A and Torres, GE and Vincent, A and Soto-Echevarria, N and Wray, B and Bartom, ET and Paunesku, T and Goodman, CR and Woloschak, GE},
title = {Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {6797},
pmid = {41620519},
issn = {2045-2322},
support = {CA221848-07/NH/NIH HHS/United States ; CA221848-07/NH/NIH HHS/United States ; },
mesh = {Humans ; *ErbB Receptors/genetics/metabolism/chemistry ; *CRISPR-Cas Systems ; *Epidermal Growth Factor/metabolism ; *Gene Editing ; Protein Binding ; Cell Line, Tumor ; Protein Domains ; Uterine Cervical Neoplasms/genetics/metabolism ; Mutation ; Amino Acid Substitution ; Female ; Binding Sites ; },
abstract = {Elevated Epidermal Growth Factor Receptor (EGFR) expression is observed in most cervical cancers, and it is frequently associated with poor clinical outcomes. The limited efficacy of existing EGFR-targeted therapies in cervical cancer highlights the need for a deeper understanding of EGFR role in this cancer type. To investigate EGFR separately from its interaction with Epidermal Growth Factor (EGF), we removed the key amino acids from the ligand bindings site. We used CRISPR/Cas9 genome editing to generate a panel of EGFR mutant cell lines and then sequenced and characterized them in detail. Studying the phenotypes of mutant cell clones, we show that a pair of amino acid substitutions L14R and Y45M within Domain I of EGFR protein completely disrupts EGF binding and changes EGFR subcellular distribution. A single substitution Y45M significantly reduced EGF binding but did not lead to subcellular redistribution of EGFR. Upon editing, EGFR mRNA and protein expression were decreased in mutant clones compared to wild type cells. Genome wide profiling of different CRISPR/Cas9 clones confirmed correct editing of EGFR with no off target CRISPR/Cas9 generated mutations. At the same time, spontaneous mutations that could impact cell phenotypes were detected in mutant clones. Disruption of ligand binding domain of EGFR by sequential knock in CRISPR/Cas9 genome editing altered subcellular localization and phosphorylation of EGFR in cervical cancer cells. The results presented here provide insights that may accelerate the development of CRISPR/Cas9-based therapies for EGFR-dependent cancers and reinforce the importance of thorough evaluation of CRISPR/Cas9-generated phenotypes.},
}
@article {pmid41620608,
year = {2026},
author = {Pahlevan Kakhki, M and Rangani, F and Ewing, E and Starvaggi Cucuzza, C and Zheleznyakova, G and Kalomoiri, M and Kenny, L and Raghavan, A and Rao Prakash, C and van den Hoeven, G and Venkata S Badam, T and Covacu, R and Andreou, I and Needhamsen, M and Kular, L and Jagodic, M},
title = {Comprehensive profiling of CRISPR/dCas9 epigenome editors indicates a complex link between on and off target effects.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {41620608},
issn = {1474-760X},
mesh = {*CRISPR-Cas Systems ; DNA Methylation ; *Gene Editing/methods ; Humans ; *Epigenome ; DNA Methyltransferase 3A ; DNA (Cytosine-5-)-Methyltransferases/metabolism/genetics ; Promoter Regions, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epigenesis, Genetic ; },
abstract = {BACKGROUND: CRISPR/dCas9-based epigenome editing systems, including DNA methylation epimodifiers, have greatly advanced molecular functional studies, revolutionizing their precision and applicability. Despite their promise, challenges such as the magnitude and stability of the on-target editing and unwanted off-target effects underscore the need for improved tool characterization and design.<br><br>RESULTS: We systematically compare specific targeting and genome-wide off-target effects of available and novel dCas9-based DNA methylation editing tools over time. We demonstrate that multimerization of the catalytic domain of DNA methyltransferase 3A enhances editing potency but also induces widespread, early methylation deposition at low-to-medium methylated promoter-related regions with specific gRNAs and also with non-targeting gRNAs. A small fraction of the methylation changes associated with transcriptional dysregulation and mapped predominantly to bivalent chromatin associating both with transcriptional repression and activation. Additionally, specific non-targeting control gRNAs cause pervasive and long-lasting methylation-independent transcriptional alterations particularly in genes linked to RNA and energy metabolism. CRISPRoff emerges as the most efficient tool for stable promoter targeting, with fewer and less stable off-target effects compared to other epimodifiers but with persistent transcriptome alterations.<br><br>CONCLUSIONS: Our findings highlight the delicate balance between potency and specificity of epigenome editing and provide critical insights into the design and application of future tools to improve their precision and minimize unintended consequences.},
}
@article {pmid41621146,
year = {2026},
author = {Kundu, A and Jerala, R},
title = {Small RNAs, big potential: Engineering microRNA-based synthetic gene circuits.},
journal = {Current opinion in chemical biology},
volume = {90},
number = {},
pages = {102652},
doi = {10.1016/j.cbpa.2026.102652},
pmid = {41621146},
issn = {1879-0402},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Gene Regulatory Networks ; *Synthetic Biology/methods ; Animals ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression. Their dysregulation is closely associated with various diseases, positioning them as biomarkers of cellular state. Synthetic biology has leveraged these properties to engineer miR-based genetic circuits capable of sensing and interpreting endogenous miR levels. Early miR-OFF systems relied on reporter gene repression but were limited by ambiguous signal interpretation. Subsequent advances introduced miR-ON architectures, logic-based classifiers integrating multiple miRs, and layered regulatory strategies combining transcriptional, translational, and cleavage-based modules to enhance sensitivity and specificity. Recent innovations include CRISPR-associated miR-responsive systems and incoherent feed-forward loop (iFFL) architectures that stabilize gene expression amid cellular variability, shifting applications from passive sensing to therapeutic intervention. Despite challenges such as leakage, cellular resource resources, and delivery, progress in orthogonal miR toolkits, computational modeling, and RNA-based delivery platforms is rapidly driving miR-based circuits toward diagnostic and therapeutic applications.},
}
@article {pmid41621837,
year = {2026},
author = {Patraskaki, M and Seyedkatouli, N and Schlicker, L and Warmoes, MO and Cordero-Maldonado, ML and Heins-Marroquin, U and Linster, CL},
title = {Unveiling Immune System Perturbations in Early Development Through Zebrafish Models of NADHX Repair Deficiency.},
journal = {Journal of inherited metabolic disease},
volume = {49},
number = {2},
pages = {e70149},
pmid = {41621837},
issn = {1573-2665},
support = {PRIDE17/12244779/PARK-QC//Fonds National de la Recherche Luxembourg/ ; PRIDE19/14063202/ACTIVE//Fonds National de la Recherche Luxembourg/ ; C22/BM/17198760//Fonds National de la Recherche Luxembourg/ ; //Fondation Juniclair/ ; },
mesh = {Animals ; Zebrafish ; Disease Models, Animal ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; NAD/metabolism ; Phenotype ; *Immune System/metabolism ; *Racemases and Epimerases/genetics/deficiency ; },
abstract = {The vital cofactors NADH and NADPH are prone to hydration, forming hydroxylated redox-inactive derivatives (NADHX and NADPHX) in cells. These damaged metabolites are repaired by two highly conserved enzymes, an NAD(P)HX dehydratase (NAXD) and an NAD(P)HX epimerase (NAXE). Mutations in NAXE or NAXD cause early onset progressive encephalopathy (PEBEL1 or PEBEL2), typically induced by fever or other triggers, and leading to premature death. To advance our comprehension of the disease mechanism and investigate potential therapeutic strategies, we generated zebrafish lines deficient in naxe or naxd using CRISPR/Cas9 technology. While both models accumulated NADHX, only naxd[-/-] larvae developed a severe phenotype, showing reduced locomotion and early death, which was partially rescued by nicotinic acid supplementation. Both mutant lines displayed signs of dysregulated immune function based on gene expression analyses and increased neutral red staining in the head region, indicating an increased number or activation of microglial cells. Our findings suggest that immune system perturbations play a role in PEBEL disease development, aligning with its inflammatory trigger-induced nature in humans. The naxd[-/-] model's responsiveness to nicotinic acid underscores its utility for preclinical drug screening. Overall, these models will be instrumental in furthering our understanding of PEBEL disease mechanisms and enhancing translational research efforts.},
}
@article {pmid41622198,
year = {2026},
author = {Speth, ZJ and Pokhrel, V and Featherston, KM and Rehard, DG and Reid, WR and Franz, AWE},
title = {Monoallelic knockout of r2d2 affects the antiviral RNAi response to Mayaro virus and the reproductive potential in Aedes aegypti.},
journal = {Parasites &amp; vectors},
volume = {19},
number = {1},
pages = {},
pmid = {41622198},
issn = {1756-3305},
support = {R01 AI134661/AI/NIAID NIH HHS/United States ; R56 AI167980/AI/NIAID NIH HHS/United States ; R01-AI134661 (awarded to A.W.E.F), R56-AI167980 (awarded to A.W.E.F), and R56-AI180215 (awarded to Dr. Stefan Rothenburg, UC Davis)//National Institutes of Health - National Institute for Allergy and Infectious Diseases (NIH-NIAID)/ ; },
mesh = {Animals ; *Aedes/virology/genetics/physiology/immunology ; *RNA Interference ; Female ; *Alphavirus/physiology/immunology ; Mosquito Vectors/virology/genetics ; Gene Knockout Techniques ; *Insect Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Reproduction ; Fertility ; Alleles ; Argonaute Proteins/genetics ; RNA, Small Interfering/genetics ; },
abstract = {BACKGROUND: Aedes aegypti is an important vector for several human-pathogenic arboviruses. RNAi is the principal antiviral immune pathway in mosquitoes. Key steps of antiviral RNAi are processing of long dsRNAs into siRNA duplexes by dicer-2; loading of the siRNA duplexes onto Argonaute-2 with the help of R2D2; RISC formation via incorporation of Argonaute-2, which contains an siRNA; RISC-mediated targeting and degradation of homologous viral RNAs. Here, we generated an r2d2 knockout mosquito line to reveal how RNAi impairment during RISC loading complex (RLC) formation would affect arbovirus infection of Ae. aegypti.<br><br>METHODS: CRISPR/Cas9 gene editing has been used to knock out r2d2 in Ae. aegypti. Crossing experiments were conducted to reveal the effects of loss of r2d2 function on fecundity and fertility. Mayaro virus (Togaviridae: MAYV) infection and RNAi pathway gene expression levels were monitored using time-course RT-qPCR assays. Small RNA profiling was conducted to determine small RNA abundance in &#x394;R2D2[(+/-)] mosquitoes.<br><br>RESULTS: We show that in Ae. aegypti, the r2d2 allele is linked to the sex determination locus on chromosome 1. It was not possible to generate homozygous &#x394;R2D2[(-/-)] mosquitoes, indicating that complete loss of r2d2 function is lethal to Ae. aegypti. Our observations suggest that r2d2 function is not limited to RNAi but also affects mosquito fecundity/fertility, likely through follicle development. Monoallelic disruption of r2d2 increased the replication of MAYV, and r2d2 expression was also increased in infected mosquitoes. MAYV infection of &#x394;R2D2[(+/-)] mosquitoes was associated with an increase in abundance of putative vpiRNAs. However, impairment of r2d2 did not affect the function of dicer-2, as there was no difference in the 21&#xa0;nt siRNA profiles between the &#x394;R2D2[(+/-)] mosquitoes and the non-transgenic control.<br><br>CONCLUSIONS: The RNAi pathway gene, r2d2, is an essential gene, and it is not possible to generate mosquitoes with biallelic (complete) loss of r2d2 function. Monoallelic impairment of r2d2 compromises the siRNA pathway downstream of dicer-2 function, at the point of RLC formation. In MAYV-infected mosquitoes, this defect in siRNA pathway function is compensated for by an increased piRNA pathway activity, which moderates increases in viral replication over a 10-day period.},
}
@article {pmid41622828,
year = {2026},
author = {Sen, MK and Roy, A and Varshney, RK and Chakraborty, A},
title = {Engineering next-generation crops through CRISPR-mediated horizontal gene transfer.},
journal = {The New phytologist},
volume = {249},
number = {6},
pages = {2683-2689},
pmid = {41622828},
issn = {1469-8137},
mesh = {*Crops, Agricultural/genetics ; *Gene Transfer, Horizontal/genetics ; Gene Editing ; *CRISPR-Cas Systems/genetics ; *Genetic Engineering/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Crops increasingly face overlapping stresses such as heat, drought, salinity, and pathogens that conventional breeding or genome editing rarely overcome in combination. To address this, we propose CRISPR-enabled horizontal gene transfer (CRISPR-HGT) as a programmable framework that recreates the evolutionary process by which plants historically acquired adaptive microbial genes. Microbial genes, refined under extreme environments, provide a naturally preadapted resource for multi-trait resilience. By integrating tools such as Cas12a, CasΦ, RNA-targeting, and dCas-based epigenome editors with AI-guided microbial gene discovery, CRISPR-HGT enables modular and inducible stress regulation. This approach shifts genome editing from allelic modification to evolution-guided design. We outline a conceptual pipeline spanning microbial gene mining to adaptive field deployment, highlighting the ecological, biosafety, and regulatory dimensions, from the European Union's cautious oversight to the UK's product-based framework. CRISPR-HGT thus introduces an evolution-informed paradigm for engineering crops that anticipate stress and sustain yield under climate uncertainty.},
}
@article {pmid41623178,
year = {2026},
author = {Hill, SF and Goldberg, EM},
title = {A hit for base editing: treatment of developmental epilepsy in a mouse model.},
journal = {The Journal of clinical investigation},
volume = {136},
number = {3},
pages = {},
pmid = {41623178},
issn = {1558-8238},
mesh = {Animals ; Mice ; Disease Models, Animal ; *Gene Editing ; *CRISPR-Cas Systems ; *Epilepsy/genetics/therapy ; *NAV1.6 Voltage-Gated Sodium Channel/genetics/metabolism ; Humans ; Mutation, Missense ; *Genetic Therapy ; },
abstract = {CRISPR/Cas9 base editing holds the potential to treat disease caused by single-nucleotide variants. In contrast with conventional CRISPR/Cas9 approaches, base editing enzymatically induces precise DNA alterations and can directly correct disease-causing variants. In this issue of JCI, Reever et al. used base editing to treat a mouse model of a severe neurodevelopmental disorder caused by a pathogenic missense variant in the voltage-gated sodium channel gene SCN8A. This work represents a starting point for the further refinement of base editing to treat genetic epilepsy.},
}
@article {pmid41623654,
year = {2026},
author = {Beh, JQ and Muzahid, NH and Mar, JH and Goh, CBS and Huët, MAL and Lim, SY and Rahman, S},
title = {Characterization of the CRISPR1-Cas array and its subtyping potential in Enterococcus faecalis from Malaysia.},
journal = {Access microbiology},
volume = {8},
number = {1},
pages = {},
pmid = {41623654},
issn = {2516-8290},
abstract = {Enterococcus faecalis is a gram-positive bacterium and a common cause of hospital-associated infections. Three major CRISPR loci have been discovered in this species, namely, CRISPR1-cas, CRISPR2 and CRISPR3-cas. We developed novel primers which target the CRISPR1-cas loci in E. faecalis and tested these primers on 26 E. faecalis isolates isolated from diverse settings from Segamat, Malaysia. Half of the isolates were found to carry the CRISPR1-cas9 locus, and the CRISPR1 array was successfully amplified in 12 out of 13 isolates that contained the cas9 gene. Characterization of the CRISPR array shows that CRISPR1-cas shares similar array length and typical repeat sequences with CRISPR2 but differs significantly in terms of spacer identities and terminal repeat (TR) sequences. Most CRISPR spacers encode for chromosomal DNA sequences. Genotype characterization based on ancestral spacer (AS) and TR sequences indicates that E. faecalis with the same CRISPR1-AS genotype do not always harbour the same CRISPR2-AS genotypes and vice versa. A combined CRISPR1-cas and CRISPR2 typing offers comparable discriminatory power to MLST, suggesting its potential to be used in short-term strain identification and epidemiological surveillance at a lower sequencing cost. Our study provides a genetic reference for future studies in Southeast Asia.},
}
@article {pmid41626318,
year = {2025},
author = {Sustek-Sánchez, F and Eelmets, E and Nigul, L and Kärblane, K and Laasmaa, M and Balode-Sausina, M and Berzina, SA and Ducis, D and Kaktina, E and Jaškūne, K and Rognli, OA and Rostoks, N and Sarmiento, C},
title = {Isolation and transformation of perennial ryegrass (Lolium perenne L.) protoplasts for the in vivo assessment of guide RNAs editing efficiency.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1744085},
pmid = {41626318},
issn = {1664-462X},
abstract = {Protoplasts are broadly used to perform different cellular and genetic assays. Transformation of protoplasts requires isolation methods that generate a large number of intact cells suitable for downstream applications. Lolium perenne L. is an important forage grass species for which gene editing techniques are in their early stages. Using protoplasts has previously been reported as a suitable approach to test the genome editing efficiency of guide RNAs in important grass species like wheat and rice. This approach can speed up and increase the chances of generating edited plants, especially when working with species for which stable transformation methods have not been established yet. Testing two different approaches regarding the processing of L. perenne L. tillers showed that using a blender for disintegrating the tissue was easier and faster than cutting the tillers with a razor blade. Conversely, the more classical strategy (cutting with a razor) provided a higher number of viable protoplasts. The use of an enzyme solution containing 2% cellulase during 8 h was shown to be the best experimental condition for protoplast isolation. The addition of a sucrose cushion improved the purification of alive cells, which were then positively transformed with guide RNA encoding vectors using polyethylene glycol. The presence of indels induced by these vectors was then confirmed through decomposition-based analysis of their sequenced genomic DNA. These results demonstrated the suitability of using protoplasts for the in vivo assessment of guide RNAs editing efficiency.},
}
@article {pmid41626796,
year = {2026},
author = {Jones, LE and Kellermeyer, R and Anand, R and Watson, J and Smith, L and Huang, X and Wang, Z and Yan, W and Zhang, H and Mastick, CC and Kidd, T and Mastick, GS},
title = {SLIT2 repellent is cleaved by TLL1 protease and promotes sensory axon fasciculation.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {16},
pages = {},
doi = {10.1242/dev.205124},
pmid = {41626796},
issn = {1477-9129},
support = {P30 GM145646/GM/NIGMS NIH HHS/United States ; RO1 NS114219/NH/NIH HHS/United States ; R56 NS114219/NS/NINDS NIH HHS/United States ; P30 GM145646; P20 GM103650/NH/NIH HHS/United States ; R01 NS114219/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; Ganglia, Spinal/metabolism/cytology/embryology ; *Axons/metabolism ; *Nerve Tissue Proteins/metabolism/genetics ; Mice ; *Intercellular Signaling Peptides and Proteins/metabolism/genetics ; Slit Homolog 2 Protein ; Mice, Knockout ; *Sensory Receptor Cells/metabolism ; CRISPR-Cas Systems ; Proteolysis ; *Tolloid-Like Metalloproteinases/metabolism/genetics ; },
abstract = {SLIT2 is a secreted protein that repels axons from the CNS midline. Full-length SLIT2 (SLIT2-FL) is proteolytically cleaved into two fragments, SLIT2-N and SLIT2-C. SLIT2-FL and SLIT2-N have opposing biological effects on cultured dorsal root ganglion (DRG) axons. This study identified SLIT2 cleavage mechanisms and functional significance for DRG axon guidance. The Tolloid-related protease TLL1 cleaved SLIT2 in cultured cells, with TLL1 requiring activation by furin/prohormone convertases. We used CRISPR editing in mice to produce a Slit2ΔTLS allele lacking the TLL1 cleavage site. Slit2ΔTLS embryos retained dorsal repulsion of DRG axons, in contrast to DRG midline invasion in Slit2 knockouts. However, DRG axons in Slit2 knockouts and Slit2ΔTLS mutants showed reduced fasciculation of rootlets and longitudinal DRG projections. In vitro, SLIT2-N promoted fasciculation of DRG axons. These results suggest that proteolytic cleavage generates additional SLIT2 biological functions for organizing DRG central axon projections.},
}
@article {pmid41626798,
year = {2026},
author = {Schuster, I and Shlipak, KK and Qin, PZ},
title = {Impacts of DNA Supercoiling on the Sequence-Dependent Nuclease Activity of CRISPR-Cas9 with Truncated Guides.},
journal = {Biochemistry},
volume = {65},
number = {4},
pages = {371-384},
pmid = {41626798},
issn = {1520-4995},
support = {R35 GM145341/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *DNA, Superhelical/chemistry/metabolism/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; Kinetics ; DNA Cleavage ; Nucleic Acid Conformation ; Base Sequence ; DNA/chemistry/metabolism ; Gene Editing ; },
abstract = {CRISPR-Cas9 is natively present in the adaptive immune systems of a multitude of bacteria and has been adapted as an effective genome engineering tool. The Cas9 effector enzyme, which is composed of a single Cas9 protein and a single-guide RNA (sgRNA), identifies and cleaves double-stranded DNA targets through a series of conformational changes that require DNA distortion and unwinding. While most studies of Cas9 specificity have focused on the DNA sequence, the role of intrinsic DNA physical properties ("DNA shape") in modulating Cas9 activity remains insufficiently defined. We previously showed that with a 16-nucleotide (-nt) truncated guide, the intrinsic DNA duplex dissociation energy at the PAM+(17-20) segment beyond the RNA-DNA hybrid tunes Cas9 cleavage rates of linear substrates. Here, we examined the impact of DNA supercoiling on Cas9 cleavage with the 16-nt truncated guide. Enzyme kinetic analysis revealed that PAM+(17-20) DNA sequences beyond the RNA/DNA hybrid preserve their effects on Cas9 cleavage in the supercoiled state. Furthermore, combining a novel asymmetric hairpin construct with a parallel-sequential kinetics model, rates for first-step nicking and second-step cleavage by Cas9 were obtained for both supercoiled and linear substrates. With both topologies, it was found that first-step nicking is clearly impacted by PAM+(17-20) DNA sequences, and the effects can be correlated with DNA unwinding, which dictates R-loop dynamics. This work expands our understanding of DNA target recognition by Cas9, and the methods developed, in particular those for analyzing the progression of Cas9-induced nicks, will aid in further in-depth mechanistic investigation.},
}
@article {pmid41627331,
year = {2026},
author = {Roy, N and Debnath, P and Srivastava, S and Gaur, HS},
title = {Recent developments in CRISPR/Cas9 genome editing research for edible fungiculture.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {36},
pmid = {41627331},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Genome, Fungal ; *Agaricales/genetics/growth &amp; development ; },
abstract = {Fungiculture refers to the deliberate cultivation or agricultural practice involving the growth and management of fungi. The practice encompasses the intentional culture of diverse species of macrofungi, including mushrooms and truffles, within controlled habitats or under specified conditions, in order to fulfill human requirements especially for food purpose. As the global market for edible mushrooms grows quickly, it is becoming increasingly necessary to grow novel and improved strains of edible fungi. Growing and breeding edible fungi using traditional methods is both time-consuming and difficult. So, there is a need for evolving advanced techniques at a molecular level which can help breeding of edible fungi with much better efficiency. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease 9) system is one of the most effective techniques for accurately cutting and modifying the genomes of edible fungi. In this review, we discuss how genome editing using CRISPR/Cas9 has been utilized in many edible fungal species such as Pleurotus ostreatus, Agaricus bisporus, Cordyceps militaris, Ganoderma lucidum, Flammulina filiformis, Lentinula edodes, and others for their target specific breeding. We also discuss the working mechanism of the above-mentioned system in these mushroom species, and also the advantages and limitations of using this system in mushrooms.},
}
@article {pmid41627751,
year = {2026},
author = {Nagamine, S and Oishi, R and Nakazawa, M},
title = {Engineering Wax Ester Composition in Euglena gracilis Using Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3014},
number = {},
pages = {37-49},
pmid = {41627751},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Euglena gracilis/genetics/metabolism ; CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Esters/metabolism/chemistry ; *Waxes/metabolism/chemistry ; },
abstract = {Genome editing technologies have significantly expanded the potential for metabolic engineering in non-model organisms. In Euglena gracilis, genome editing methods using Cas9 and Cas12a were reported in 2019 and 2024, respectively, and are increasingly being applied to modify metabolic functions. This chapter provides a detailed protocol for CRISPR/Cas9-based genome editing that enables stable modification of wax ester composition under anaerobic conditions. By targeting key enzymes in the reversed β-oxidation pathway, the method allows the generation of knockout mutants with altered wax ester chain lengths. Beyond this application, the protocol supports reproducible and stable genetic modification of E. gracilis metabolism. It can be extended to the engineering of other biosynthetic pathways and is compatible with future integration of knock-in strategies. The approach offers a practical basis for the broader use of E. gracilis as a green chassis organism in synthetic biology and biomanufacturing.},
}
@article {pmid41627753,
year = {2026},
author = {Arnould, K and El Kadri, M and Hervé, P and Asencio, C and Plazolles, N and Monic, S and Morel, CA and Rivière, L and Bringaud, F and Tetaud, E},
title = {Precision Without Selection: A Marker-Free CRISPR/Cas9-Based Protocol for Multiplexed Genome Editing in Trypanosomatids.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3014},
number = {},
pages = {65-78},
pmid = {41627753},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Protozoan ; Electroporation ; *Trypanosomatina/genetics ; Ribonucleoproteins/genetics ; },
abstract = {The CRISPR-Cas9 system has become a valuable tool for genome editing in trypanosomatid parasites such as Trypanosoma and Leishmania species. Although these organisms have been genetically engineered for a long time using homologous recombination, CRISPR/Cas9 offers improved efficiency for genome editing. However, conventional strategies employing stable Cas9 expression require the persistent use of a specific genetic background (i.e., strains expressing Cas9), depend on selectable resistance markers, compromise genomic stability, and are not readily applicable to diverse strain backgrounds. Herein, we report an optimized marker-free CRISPR/Cas9 method based on transient ribonucleoprotein (RNP) delivery that overcomes these drawbacks. Our method eliminates the need for plasmid integration or antibiotic selection while maintaining high editing efficiency. The protocol comprises the following steps: (1) design of the guide RNA (gRNA), (2) design of the repair template (cassette), (3) assembly of the ribonucleoprotein (RNP) complex, (4) delivery by electroporation, and (5) clonal screening through PCR and sequencing. The procedure permits rapid (≤3 weeks) production of homozygous mutant lines in wild-type strains, including low-density culture strains. The reproducibility and ease of the technique render it particularly suited for multiplexed editing of polyploid genomes, multi-gene families, and several different genes at once, as well as validation of the essential nature of genes. Although designed for trypanosomatids, the workflow can be adapted to other kinetoplastids, offering a flexible platform for functional genomics.},
}
@article {pmid41628256,
year = {2026},
author = {Luo, Y and Dong, Q and Yi, S and Zhang, W and Du, Y and Fang, Q and Zhang, W and Ouyang, K and Chen, Y and Yin, Y and Wei, Z and Qin, Y and Huang, W},
title = {Genome-wide CRISPR screening identifies Annexin A1 as a facilitator of porcine astrovirus entry.},
journal = {PLoS pathogens},
volume = {22},
number = {2},
pages = {e1013943},
pmid = {41628256},
issn = {1553-7374},
mesh = {Animals ; Swine ; *Virus Internalization ; CRISPR-Cas Systems ; *Annexin A1/metabolism/genetics ; *Swine Diseases/virology/genetics/metabolism ; *Astroviridae Infections/virology/metabolism/genetics ; *Mamastrovirus/physiology/genetics ; },
abstract = {Porcine astrovirus (PAstV) is an important and widespread pathogen in swine, linked to diarrheal outbreaks and extraintestinal disease. How PAstV enters host cells has remained unclear, and no cellular factor has been defined for PAstV entry. Here, a genome-wide CRISPR-Cas9 loss-of-function screen in porcine epithelial cells identifies Annexin A1 (ANXA1) as a host factor that facilitates PAstV entry. Genetic ablation or pharmacological/antibody blockade of ANXA1 reduces binding, lowers early viral RNA and capsid signals, and delays the rise of progeny, whereas re-expression restores susceptibility. Biochemical assays and surface plasmon resonance indicate a direct interaction between ANXA1 and the acidic C-terminal domain of the PAstV ORF2 capsid protein, and imaging shows ANXA1 co-localizes with incoming particles at the cell surface and supports attachment and uptake. Loss of ANXA1 does not alter infection by the non-astrovirus panel tested, indicating selectivity for PAstV under our conditions. Notably, infection is reduced but not abolished in ANXA1-deficient cells, consistent with additional entry factors acting alongside ANXA1. These findings position ANXA1 as an entry cofactor for PAstV and provide a mechanistic basis to refine models of astrovirus host-cell recognition.},
}
@article {pmid41628535,
year = {2026},
author = {Xie, K and Ren, H and Ban, D and Chen, L and Xin, X and Zhang, J and Tang, Q and Huang, L and Wei, J and Zhang, K and Liao, X},
title = {Silica-detoxified perovskite ECL: Cas13a-triggered signal-on sensing with CsPbBr3@SiO2@Au.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {170},
number = {},
pages = {109243},
doi = {10.1016/j.bioelechem.2026.109243},
pmid = {41628535},
issn = {1878-562X},
mesh = {*Silicon Dioxide/chemistry ; *Gold/chemistry ; *Biosensing Techniques/methods ; *Titanium/chemistry ; *MicroRNAs/analysis/blood ; *Oxides/chemistry ; *Calcium Compounds/chemistry ; Electrochemical Techniques/methods ; Limit of Detection ; Humans ; Luminescent Measurements/methods ; CRISPR-Cas Systems ; Nanocomposites/chemistry ; },
abstract = {Perovskite nanocrystals are attractive ECL emitters but suffer from poor water stability and potential toxicity. Here we report a signal-on electrochemiluminescent biosensor that integrates CsPbBr3@SiO2@Au nanocomposites with a CRISPR/Cas13a-Nb.BbvCI amplification cascade for ultrasensitive microRNA detection. The CsPbBr3 core provides bright emission, a conformal SiO2 shell enhances water compatibility and suppresses ion leakage, and surface Au nanoparticles offer abundant sites for thiolated ferrocene-hairpin (Fc-HP) immobilization. In the resting state, proximal Fc efficiently quenches the CsPbBr3 ECL. Target miRNA activates Cas13a to cleave a dumbbell probe and release an intermediate strand that hybridizes with Fc-HP; subsequent Nb.BbvCI nicking removes Fc from the electrode and is recycled, producing robust signal restoration. Morphology (TEM), composition (EDS/XPS), and stepwise electrochemistry (CV/EIS) verify a core-shell-Au architecture and a reliably assembled interface that follows the expected quench→restore behavior. Under optimized conditions (0.5 mg mL[-1] CsPbBr3@SiO2@Au, 2.0 μM Fc-HP, 40 min target incubation, 100 mM TPrA, 120 s pre-reaction), the assay affords a 1 aM-1.0 × 10[9] aM linear range with an estimated limit of detection (LOD) of 1.86 aM. The sensor shows high specificity against homologous sequences and achieves 95.22%-104.61% recoveries with RSD < 5% in spiked serum. Pilot measurements distinguish patient serum samples from healthy controls, underscoring clinical potential. This modular platform couples stable perovskite ECL emission with programmable CRISPR chemistry, offering a sensitive, selective, and water-compatible route for microRNA analysis and readily extensible nucleic-acid diagnostics.},
}
@article {pmid41628563,
year = {2026},
author = {Liang, L and Xu, B and Xiao, S and Mu, X and Zhao, S and Tian, J},
title = {A new split DNA-based activation of CRISPR/Cas12a for amplification-free and dual-stimulus responsive detection and precise imaging of miRNA-221.},
journal = {Talanta},
volume = {303},
number = {},
pages = {129443},
doi = {10.1016/j.talanta.2026.129443},
pmid = {41628563},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *DNA/chemistry/genetics/metabolism ; Biosensing Techniques/methods ; Fluorescent Dyes/chemistry ; DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The CRISPR/Cas12a system is a genome editing technology that has been widely applied in biosensing and molecular diagnostics. However, the detection and regulation of its core components remain challenging. Therefore, we constructed a new split DNA-based activation method for the regulation of CRISPR/Cas12a, and based on that, an APE1-assisted activation CRISPR/Cas12a system for miRNA detection and a precise imaging method was also developed without amplification and complex design. Two split DNA were used as activators and embedded in two hairpins. When APE1 and miRNA-221 were simultaneously input, the DNA logic gate was started, thus releasing the determinant activation chain to activate the trans-shearing activity of the CRISPR/Cas12a system, so that the fluorescent probe signal can be significantly recovered. Different cleavage-activated chain hairpins were designed, and the influence on the trans-shear activity of CRISPR/Cas12a and the activation effect were discussed. And the method was successfully applied to detect the expression levels of miRNA-221 in cell lysates. The detection limit for miRNA-221 is 9.71 pmol/L (S/N = 3). At the same time, the method was applied for precise imaging of miRNA-221 within different cells and can effectively distinguish tumor cells. This study combines the regulation of the CRISPR/Cas system by split activators with the advantages of dual-responsive DNA logic circuits. The dual-response activation design effectively reduces false positive signals, thereby enhancing the detection and imaging accuracy. This method provides a novel design concept for utilizing split-DNA activation of the CRISPR/Cas system for nucleic acid detection and cell imaging.},
}
@article {pmid41628878,
year = {2026},
author = {Gao, S and Wang, L and Hou, M and Zhang, M and Zhu, X and Luo, H and Yu, X and Lv, H and Chen, S and Huang, Y and Zhang, K and Wu, J},
title = {Gene insertion and transcriptional regulation of Escherichia coli based on CRISPR-associated transposases.},
journal = {International journal of biological macromolecules},
volume = {348},
number = {},
pages = {149850},
doi = {10.1016/j.ijbiomac.2025.149850},
pmid = {41628878},
issn = {1879-0003},
mesh = {*Escherichia coli/genetics ; *Gene Expression Regulation, Bacterial ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems ; *Transcription, Genetic ; Plasmids/genetics ; },
abstract = {Synthetic biology aims to construct robust microbial cell factories for sustainable biomanufacturing. A major obstacle lies in the difficulty of efficiently integrating large polycistronic expression cassettes into the genome and flexibly regulating gene expression. Here, a programmable tool MUSCULAR-CAST was developed based on type IF CRISPR-associated transposases (CAST) system Tn6677. Using MUSCULAR-CAST, we have achieved efficient genomic integration of various size polycistronic expression cassettes (1-10K). Among these, a human milk oligosaccharide 3-fucosyllactose (3-FL) chassis strain was successfully constructed, with similar yields and better growth compared with that of plasmid expression strain, and a plasmid-free cutinase recombinant expression strain was constructed, with enzyme activity higher than that of plasmid containing strain. Meanwhile, we developed a gene repression tool Tn-CRISPRi based on the targeting module of MUSCULAR-CAST, which achieved high single-gene repression across diverse PAM sequences and near-complete dual-gene suppression (98.6-99.8%). Applying Tn-CRISPRi to repress 17 genes competing with 3-FL biosynthesis or nonessential for growth revealed that knockdown of osmoregulated periplasmic glucans biosynthesis protein H (mdoH) and motility protein A (motA) increased 3-FL titers by 2.79- and 4.4-fold, respectively. This study establishes MUSCULAR-CAST and Tn-CRISPRi as efficient tools for genomic integration and transcriptional regulation, providing a scalable framework for advanced chassis strain engineering in synthetic biology.},
}
@article {pmid41629010,
year = {2026},
author = {Saini, A and Sharma, N and Sharma, N and Kumari, N and Sharma, M and Singh, B and Thakur, AK},
title = {Precision pest management: Genome editing tools, specifically CRISPR/Cas9 and future prospects.},
journal = {Pesticide biochemistry and physiology},
volume = {218},
number = {},
pages = {106941},
doi = {10.1016/j.pestbp.2026.106941},
pmid = {41629010},
issn = {1095-9939},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Insecta/genetics ; *Pest Control, Biological/methods ; *Pest Control/methods ; Insect Control/methods ; },
abstract = {The growing resistance to synthetic insecticides and Bt toxins, alongside persistent crop losses despite heavy pesticide application, highlights the urgent need for safer, sustainable and efficient pest management strategies. This review presents genome editing as a precise and versatile approach to reduce pest impact by altering fertility, feeding patterns or vulnerability, while protecting beneficial organisms. Among the genome editing tools, CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) is one of the most promising genome editing techniques in insects. It facilitates targeted functional studies, integration with RNAi and dual-expression systems and gene drive applications. Deployment is envisioned in two phases, initial laboratory modification followed by regulated field release, with a strong emphasis on biosafety through terminator genes, marked individuals for gene flow monitoring, optimized dosages, stringent screening and long-term ecological surveillance, along with transparency and adherence to international safety protocols. Significant challenges encompass delivery efficiency, identification of edits, off-target mutations, dose-related efficacy and sterility, unstable transmission and resistance development. Innovations such as base and prime editing minimize unintended mutations by circumventing double-stranded breaks (DSBs), while paratransgenic strategies targeting gut symbionts offer supplementary avenues; plant-mediated insect gene editing emerges as a promising frontier. Overall, carefully regulated trials aligned with policy frameworks and stakeholder involvement are vital to assess effectiveness in natural environments and achieve targeted, dependable and ecologically responsible pest control.},
}
@article {pmid41629044,
year = {2026},
author = {Sharma, S and Karna, SKL and Khanal, S and Pokharel, YR},
title = {Rapid detection of measles virus RNA from clinical specimens by using RT-LAMP coupled with CRISPR/Cas12b via fluorescence and lateral flow biosensor readouts: A proof-of-concept study.},
journal = {Analytica chimica acta},
volume = {1389},
number = {},
pages = {345081},
doi = {10.1016/j.aca.2026.345081},
pmid = {41629044},
issn = {1873-4324},
mesh = {*Measles virus/genetics/isolation &amp; purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/analysis/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Fluorescence ; *Measles/diagnosis/virology ; Proof of Concept Study ; Animals ; Chlorocebus aethiops ; Vero Cells ; },
abstract = {BACKGROUND: Rapid laboratory confirmation of suspected cases is essential for measles control, but current methods require a complex laboratory infrastructure. We developed loop-mediated isothermal amplification coupled with CRISPR-Cas-mediated diagnostic (LAmCaD), a novel two-pot diagnostic platform that integrates rapid nucleic acid extraction, RT-LAMP amplification, and in-house purified AapCas12b as a rapid test to detect measles RNA that could be used in settings lacking laboratory infrastructure.<br><br>METHODS: LAmCaD is based on dual detection modalities, fluorescence and lateral flow biosensor readouts. The LAmCaD assay was evaluated for analytical sensitivity and specificity using RNA from Vero/hSLAM-grown measles virus, and diagnostic evaluation was performed using patient samples, compared with standard RT-PCR. The cross-genotype detection capability was assessed across epidemiologically relevant measles genotypes D8, D4, and B3.<br><br>RESULTS: The in-house purified protein AapCas12b from Alicyclobacillus acidiphilus exhibited strong cis and trans cleavage activities, eliminating dependence on commercial enzyme preparations. The platform enables the use of clinical samples from patients through rapid nucleic acid extraction that eliminates the need for RNA purification steps, allowing direct use of extracted material in RT-LAMP reactions. RT-LAMP alone achieved analytical sensitivity of &#x223c;10[3] copies, while the complete protocol detected measles virus at &#x223c;10[5] copies by fluorescence and &#x223c;10[4] copies by lateral flow detection. Diagnostic evaluation demonstrated sensitivity of 64.00&#xa0;%, specificity of 92.59&#xa0;%, and negative predictive value of 99.95&#xa0;% with an overall accuracy of 92.56&#xa0;%. ROC curve analysis revealed an AUC of 0.717, indicating fair discriminatory performance. The assay demonstrated moderate agreement with RT-PCR (&#x3ba;&#xa0;=&#xa0;0.6) and successfully identified genotypes D8, D4, and B3. The entire testing process took 90&#xa0;min to complete.<br><br>CONCLUSIONS: This proof-of-concept LAmCaD platform establishes the foundation for a cost-effective, field-deployable diagnostic test without relying on commercial enzymes or complex sample processing. The platform could facilitate the rapid confirmation of measles cases in resource-limited and/or remote settings, thereby contributing to global measles elimination goals. Conducted within the WHO South-East Asia Region, this study is particularly relevant to the region's 2023 elimination target, addressing current surveillance gaps and specimen transport challenges that hinder efforts to eliminate the disease.},
}
@article {pmid41629462,
year = {2026},
author = {Nayfach, S and Bhatnagar, A and Novichkov, A and Kim, N and Hoffnagle, AM and Hussain, R and Estevam, GO and Hill, E and Ruffolo, JA and Silverstein, RA and Gallagher, J and Kleinstiver, BP and Meeske, AJ and Cameron, P and Madani, A},
title = {Customizing CRISPR-Cas PAM specificity with protein language models.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41629462},
issn = {1546-1696},
abstract = {CRISPR-Cas enzymes must recognize a protospacer-adjacent motif (PAM) to edit a genomic site, greatly limiting the range of targetable sequences in a genome. Although engineering strategies to alter PAM specificity exist, they typically require labor-intensive, iterative experimentation. We introduce an evolution-informed deep learning model, Protein2PAM, to efficiently guide the design of Cas protein variants tailored to recognize specific PAMs. Trained on a dataset of over 45,000 CRISPR-Cas PAMs, Protein2PAM rapidly and accurately predicts PAM specificity directly from Cas proteins across type I, II and V CRISPR-Cas systems. Using in silico mutagenesis, the model identifies residues critical for PAM recognition in Cas9 without using structural information. We use Protein2PAM to computationally evolve Nme1Cas9, generating variants with broadened PAM recognition and up to a 50-fold increase in PAM cleavage rates compared to the wild type in vitro. Our machine learning approach allows Cas enzymes to target sequences that were previously inaccessible because of PAM constraints, potentially increasing target flexibility in personalized genome editing.},
}
@article {pmid41629464,
year = {2026},
author = {},
title = {Reprogramming CRISPR-Cas enzymes for customized genome editing.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41629464},
issn = {1546-1696},
}
@article {pmid41629994,
year = {2026},
author = {Papaioannou, NY and Patsali, P and Klermund, J and Papasavva, PL and Andrieux, G and Koniali, L and Naiisseh, B and Christou, S and Sitarou, M and Kleanthous, M and Cathomen, T and Lederer, CW},
title = {Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {41629994},
issn = {1474-760X},
support = {EXCELLENCE/1216/0092//Research and Innovation Foundation/ ; CA21113 GenE-HumDi//COST Actions/ ; New Infrastructure for the Diagnosis and Therapy of Patients//Norway Grants 2014-2021/ ; },
mesh = {Humans ; *Gene Editing/methods ; *beta-Thalassemia/genetics/therapy ; *Hematopoietic Stem Cells/metabolism ; Promoter Regions, Genetic ; gamma-Globins/genetics ; CRISPR-Cas Systems ; Enhancer Elements, Genetic ; Repressor Proteins ; },
abstract = {BACKGROUND: Beta-thalassemia is among the most common monogenic disorders, posing a major global health challenge. Editing of genetic modifiers, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors (BE), are potentially safer and better suited for multiplexed application than DSB-dependent CRISPR/Cas technology. However, harmful on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements invisible to standard detection technologies.<br><br>RESULTS: Using primary patient-derived CD34[+] cells from three donors, we investigate simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115 bp) on the HBG promoter for DNA-level and functional studies at the RNA, protein, and morphological level. Analyses include direct comparison to DSB-based editing, the current clinically applied standard, and CAST-seq to assess recombination events, allowing wider inferences on relative safety. RNA-seq analyses for clones of primary CD34[+] cells across all treatments confirm peak HBG induction for duplex BE and comparable effects on apoptotic and immune response signatures. Overall, duplex BE produces robust &#x3b3;-globin and fetal hemoglobin induction, improves functional correction over simplex editing and results in low incidence of genomic alterations in both target loci.<br><br>CONCLUSIONS: Duplex BE targeting both BCL11A erythroid enhancer and HBG promoter enables functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach.},
}
@article {pmid41630603,
year = {2026},
author = {Rodriguez-Parks, A and Beezley, EG and Manna, S and Silaban, I and Almutawa, SI and Cao, S and Ahmed, H and Guyer, M and Baker, S and Richards, MP and Kang, J},
title = {Advancing knock-in approaches for robust genome editing in zebrafish.},
journal = {Biology open},
volume = {15},
number = {2},
pages = {},
pmid = {41630603},
issn = {2046-6390},
support = {7000320//National Institute of Food and Agriculture/ ; R35GM137878/NH/NIH HHS/United States ; R35 GM137878/GM/NIGMS NIH HHS/United States ; //NIH/ ; 2137434//National Science Foundation/ ; P30CA014520/NH/NIH HHS/United States ; R01 HL151522/HL/NHLBI NIH HHS/United States ; R21OD037634/NH/NIH HHS/United States ; GR000042507/NH/NIH HHS/United States ; 2019-67017-29179//National Institute of Food and Agriculture/ ; R21 OD037634/OD/NIH HHS/United States ; R01HL151522/NH/NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/genetics ; *Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Genome ; CRISPR-Cas Systems ; Genetic Vectors ; Exons ; },
abstract = {Precise genome editing remains a major challenge in functional genomics, particularly for generating knock-in (KI) alleles in model organisms. Here, we introduce the mini-golden system, a versatile Golden Gate-based subcloning platform that enables rapid assembly of donor constructs containing homology arms and a gene of interest. This system offers a library of middle entry vectors including diverse genes, enhancing the preparation of donor minicircles for KI applications. Using the mini-golden system, we efficiently generated a foxd3CreER KI zebrafish line, allowing conditional recombination in neural crest cells. To further improve genome editing precision, we developed a synthetic exon-based donor template strategy combined with fluorescence screening. Using this approach, we successfully engineered a targeted isoleucine-to-valine substitution (Ile-to-Val) in hbaa1.2, one of the two adult hemoglobin alpha genes in zebrafish. Importantly, despite the high sequence similarity between hbaa1.2 and its paralog hbaa1.1, our strategy specifically edited hbaa1.2, demonstrating the effectiveness of the synthetic exon approach. This method minimized undesired recombination and significantly improved the identification of lines carrying the edited genome. Together, we provide a robust toolkit for efficient and precise genome engineering in zebrafish, with broad applicability to other model systems.},
}
@article {pmid41630633,
year = {2026},
author = {Lee, SY and Park, HH},
title = {Structural Basis of Recognition of Anti-CRISPR Operon by Aca3.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {3},
pages = {e71512},
doi = {10.1096/fj.202502389RR},
pmid = {41630633},
issn = {1530-6860},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2025-16065724//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Operon ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Viral Proteins/genetics/metabolism/chemistry ; Bacteriophages/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Promoter Regions, Genetic ; },
abstract = {The CRISPR-Cas system equips bacteria with adaptive immunity by storing fragments of invading nucleic acids in CRISPR loci and deploying Cas proteins to recognize and degrade matching sequences. In turn, bacteriophages have evolved small anti-CRISPR (Acr) that neutralize diverse CRISPR-Cas types. Acr genes are often co-encoded with transcriptional regulators called anti-CRISPR-associated (Aca) proteins, which suppress acr expression. Although 13 Aca families have been identified through bioinformatic analysis, detailed information on their target DNA-binding mechanisms and the inhibition of acr expression remains limited. Here, we report the high-resolution structure of Aca3 and delineate its DNA-binding interface. We demonstrate that Aca3 selectively recognizes inverted repeats upstream of its cognate acr gene, AcrIIC1. Mutational analyses of key helix-turn-helix residues confirm their essential roles in promoter engagement. Together, these results reveal the molecular basis for Aca3-mediated control of anti-CRISPR expression and expand our understanding of regulatory strategies that phages employ to modulate host CRISPR-Cas immunity.},
}
@article {pmid41630767,
year = {2025},
author = {Molaghi, MAA and Atiyah, WR and Saeed, AAR},
title = {Novel CRISPR/Cas12-based assay for the rapid and accurate detection of donkey meat.},
journal = {Open veterinary journal},
volume = {15},
number = {11},
pages = {5682-5688},
pmid = {41630767},
issn = {2218-6050},
mesh = {Animals ; *Equidae/genetics ; *Meat/analysis ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Polymerase Chain Reaction/veterinary/methods ; },
abstract = {BACKGROUND: Precise identification of items originating from animals is required for monitoring the authenticity of livestock commodities. Nucleic acid-based detection methods, including polymerase chain reaction, are highly accurate tests for detecting meat fraud. However, these tests require costly devices and highly skilled personnel.<br><br>AIM: This study aims to develop a precise and rapid test based on deoxyribonucleic acid (DNA) for detecting meat from donkeys.<br><br>METHODS: The assay was developed by combining Clustered regularly interspaced short palindromic repeat /Cas12 with RPA and a lateral flow device. A conserved sequence of the mitochondrial D-loop gene was used as the target gene. The assessment of the assay focused on its sensitivity, specificity, and completion time.<br><br>RESULTS: The results show that the assay can detect donkey meat in the tested samples with 100% specificity with no cross-reactivity with other species, and no false-positive results were recorded. Furthermore, the assay has shown high sensitivity and to detect as little as 5 ng of input DNA. Moreover, the results, including DNA extraction, were obtained in less than 40 minutes and did not require any specialized equipment.<br><br>CONCLUSION: The assay is a good option for the rapid and precise detection of donkey meat. Further optimization and studies are needed to enable the direct detection of donkey meat without DNA extraction.},
}
@article {pmid41632281,
year = {2026},
author = {Li, J and Zhu, M and Hu, G and Chen, X and Xue, H and Zhang, Y and Wang, Y and Li, Z and Xu, D and Zhai, M and Zhou, G and Cui, C and Zhao, C and Qin, R and Wu, Y and Cui, F and Sun, H},
title = {Gene editing and association analysis of circadian clock gene TaPRR59 highlights its importance in yield-related traits in wheat.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {2},
pages = {58},
pmid = {41632281},
issn = {1432-2242},
support = {32272119//National Natural Science Foundation of China/ ; 32072051//National Natural Science Foundation of China/ ; ZR2022YQ19//Shandong Provincial Fund for Excellent Young Scholars/ ; 32101726//National Natural Science Foundation for Young Scholars of China/ ; ZR2022MC119//Natural Science Foundation of Shandong Province, China/ ; 20230119//Taishan scholar young expert/ ; ZR2019ZD16//Major Basic Research Project of Natural Science Foundation of Shandong Province, China/ ; 2022LZG002-2//Key R &amp; D Program of Shandong Province, China (Major Innovation Project)/ ; 2023ZDCX023//Yantai science and technology plan project/ ; IPGS2025-083//The Innovation Project for graduate students of Ludong University/ ; },
mesh = {*Triticum/genetics/growth &amp; development ; *Gene Editing ; *Plant Proteins/genetics/metabolism ; *Circadian Clocks/genetics ; Gene Expression Regulation, Plant ; Haplotypes ; Phenotype ; Plant Breeding ; *Genes, Plant ; CRISPR-Cas Systems ; },
abstract = {Mutations in TaPRR59 impact transcript levels of some key flowering genes and show earlier heading time and reduced plant height. Favorable haplotype TaPRR59-A1-Hapla was positively selected in wheat breeding programs. The circadian clock system is a crucial endogenous rhythmic regulatory mechanism with a significant role in plant growth and development. The pseudo-response regulator (PRR) family is a pivotal component of circadian networks. In the present study, we cloned the wheat PRR family member TaPRR59 and investigated its function using gene editing, transcriptome sequencing, haplotype analysis, and association analysis. The expression profile of TaPRR59 over a 24-h period exhibited a diurnal rhythmic expression pattern. Luciferase transient transcriptional assay demonstrated that TaPRR59 acts as a transcriptional repressor in the nucleus. The taprr59-ABD-KO gene-edited lines produced using the CRISPR/Cas9 genome-editing system had earlier heading time and reduced plant height. Overexpression of TaPRR59-D1 in rice significantly delayed the heading date, reduced plant height and thousand-grain weight, and increased the number of grains per panicle. Transcriptome analysis revealed the transcript levels of several key flowering genes and chlorophyll a-b binding protein-related genes were up- or down-regulated in the taprr59 mutant plants. Association analysis showed that natural variations at TaPRR59-A1, TaPRR59-B1, and TaPRR59-D1 were significantly associated with yield traits such as plant height, thousand-grain weight, and heading date. Geographical analysis showed distinctive distribution characteristics of TaPRR59 haplotypes in different agroecological production zones. Additionally, the significant difference in frequency of the favorable haplotype TaPRR59-A1-Hapla between landraces and modern cultivars indicates that it has been subject to directional selection during wheat breeding. This research provided novel insights into the influence of the circadian clock system on agronomic traits and provided useful molecular markers and genetic resources for wheat breeding.},
}
@article {pmid41632443,
year = {2026},
author = {Mašlaňová, I and Nováková, D and Švec, P and Kovařovic, V and Sedláček, I and Botka, T and Šedo, O and Neumann-Schaal, M and Vives, J and Doškař, J and Pantůček, R},
title = {Genomic and taxonomic characterization of strain CCM 2573: Uncovering unique genetic features and description of Macrococcus caseolyticus subsp. lactis subsp. nov.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41632443},
issn = {1874-9356},
abstract = {Strain CCM 2573 is a Gram-positive bacterium that has been intensively studied in the past due to its distinct chemotaxonomic properties, but its reliable taxonomic classification has not been satisfactorily clarified. Whole-genome sequencing and comparative genomic analyses performed in this study revealed that the strain belongs to the Macrococcus caseolyticus phylogenetic clade. Genome-to-genome comparisons confirmed the closest relationship to the type strains of M. caseolyticus subsp. hominis CCM 7927[T] and M. caseolyticus subsp. caseolyticus DSM 20597[T]. However, the strain harbored unique genomic elements distinguishing it from its nearest phylogenetic neighbors. Its accessory genome contains dozens of insertion sequences, a 92-kbp composite transposon with unique palindromic repeat loci associated with a CRISPR-Cas adaptive immune system, a pseudo-staphylococcal chromosome cassette, and several additional genomic islets. Unlike other macrococci, strain CCM 2573 exhibits a specific peptidoglycan composition (L-Lys-Gly2-Ser2-Gly) and shows a higher phylogenetic divergence of aminoacyltransferases (FemABX) involved in interpeptide bridge synthesis. In addition, it reveals distinct biochemical characteristics from both subspecies of M. caseolyticus, particularly in its ability to produce acid from galactose, cellobiose, melezitose, and turanose, as well as in its susceptibility to novobiocin. The MALDI-TOF mass spectra enable differentiation of the strain from other type strains of the genus Macrococcus. The results of polyphasic taxonomy obtained in this study showed that strain CCM 2573 belongs to the species M. caseolyticus, but it is distinct from both validly named M. caseolyticus subspecies. We propose to assign the analyzed strain as a new subspecies, Macrococcus caseolyticus subsp. lactis subsp. nov. The type strain is CCM 2573[T] (= DSM 20227[T]).},
}
@article {pmid41632863,
year = {2026},
author = {Shinoda, H and Makino, A and Yoshimura, M and Minagawa, N and Iida, T and Nakano, M and Noda, T and Toyoda, M and Watanabe, R},
title = {Multicolor Amplification-Free RNA Detection with Cas13a and Cas13b.},
journal = {Analytical chemistry},
volume = {98},
number = {6},
pages = {4705-4714},
doi = {10.1021/acs.analchem.5c06305},
pmid = {41632863},
issn = {1520-6882},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/analysis/genetics ; *COVID-19/diagnosis ; Influenza A virus/genetics/isolation &amp; purification ; Limit of Detection ; *CRISPR-Cas Systems ; Influenza, Human/diagnosis ; *CRISPR-Associated Proteins/genetics ; },
abstract = {The COVID-19 pandemic and recurring outbreaks of infectious diseases underscore the urgent demand for multiplex diagnostics capable of rapid and accurate pathogen identification. Although multiplex nucleic acid amplification tests (NAATs) are widely used for diagnosing diverse infectious diseases, their inherent amplification bias and long turnaround times highlight the demand for faster and reliable alternatives. Here, we present multicolor SATORI (mSATORI), an amplification-free single-molecule genetic test that leverages the complementary activities of CRISPR-Cas13a and Cas13b to achieve simultaneous detection of dual RNA targets. mSATORI identified Influenza A and SARS-CoV-2 RNAs within ∼10 min, with analytical limits of detection (LoD) of 86 aM and 52 aM, respectively. Validation using clinical specimens demonstrated robust diagnostic performance, achieving femtomolar limits of detection (550 aM for Influenza A and 640 aM for SARS-CoV-2), along with sensitivities exceeding 80% and specificities of 100%. Collectively, these results establish mSATORI as a platform for next-generation molecular diagnostics, with broad implications for clinical implementation, outbreak preparedness, and global infectious disease surveillance.},
}
@article {pmid41633939,
year = {2026},
author = {Lin, L and Zhang, JJ and Liu, BH and Du, S and Zhang, YQ and Yang, Y and Li, C and Dong, CC and He, YB and Wang, Q and Wang, HY and Shao, CW},
title = {Epigenetic editing of marine medaka (Oryzias melastigma) fgf2 using CRISPR/dCas9-Tet1CD.},
journal = {Zoological research},
volume = {47},
number = {1},
pages = {263-278},
doi = {10.24272/j.issn.2095-8137.2025.089},
pmid = {41633939},
issn = {2095-8137},
mesh = {Animals ; *Oryzias/genetics ; *Gene Editing/veterinary ; *Epigenesis, Genetic ; *Fibroblast Growth Factor 2/genetics/metabolism ; *CRISPR-Cas Systems ; Epigenome Editing ; },
abstract = {CRISPR/dCas9-mediated epigenetic editing offers a versatile approach for transcriptional regulation without introducing DNA strand breaks. Although this strategy has been explored in a limited number of species, its application in aquatic vertebrates remains largely uncharacterized. In this study, ten-eleven translocation methylcytosine dioxygenase 1 (tet1) was cloned and molecularly characterized in marine medaka (Oryzias melastigma). Decitabine treatment identified fibroblast growth factor 2 (fgf2) as a methylation-sensitive gene, with a regulatory CpG island located within its promoter region. Subsequently, a CRISPR/dCas9-Tet1CD activation system was constructed by fusing the catalytic domain of Tet1 (Tet1CD, Ala1352-Thr2034) to dCas9, enabling locus-specific DNA demethylation. Targeting fgf2, this CRISPR/dCas9-Tet1CD system induced efficient and selective demethylation of the CpG island, resulting in a maximal 2.41-fold increase in fgf2 transcript levels. Whole-genome bisulfite sequencing and transcriptomic analysis confirmed high on-target precision with minimal off-target effects. Epigenetic activation of fgf2 further modulated downstream gene networks associated with growth, promoting durable transcriptional enhancement and increased cellular proliferation. Collectively, these results establish a robust and highly specific epigenetic editing platform in marine medaka, providing a powerful tool for functional genomics studies and regulatory element analysis in aquatic models.},
}
@article {pmid41633991,
year = {2026},
author = {Kumari, B and Damodaran, AP and Guiblet, WM and Xiao, MS and Behera, AK and On, TA and McIntosh, CE and Teszler, M and Holloway, C and Le, S and Parab, N and Zhao, Y and Aregger, M and Gonatopoulos-Pournatzis, T},
title = {Single-cell exon deletion profiling reveals splicing events that shape gene expression and cell state dynamics.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {1218},
pmid = {41633991},
issn = {2041-1723},
support = {ZIA BC012033/ImNIH/Intramural NIH HHS/United States ; ZIA BC012101/ImNIH/Intramural NIH HHS/United States ; HHSN261200800001C/RC/CCR NIH HHS/United States ; HHSN261200800001E/CA/NCI NIH HHS/United States ; 1ZIABC012101//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 1ZIABC012033//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; 75N91019D00024/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Exons/genetics ; *Alternative Splicing/genetics ; *Single-Cell Analysis/methods ; CRISPR-Cas Systems/genetics ; Gene Expression Profiling/methods ; Cell Cycle/genetics ; Sequence Deletion ; Transcriptome ; Gene Expression Regulation ; },
abstract = {Alternative splicing is a pervasive gene regulatory mechanism critical for diversifying the human proteome. To systematically investigate its role in cell fate determination, we develop scCHyMErA-Seq, a scalable CRISPR-based exon deletion screening platform integrated with 10x Genomics single-cell transcriptomic readouts. This tool enables efficient exon deletion while simultaneously capturing Cas9/Cas12a guides and polyadenylated transcripts at single-cell resolution. Applying scCHyMErA-Seq to high-throughput profiling of alternative cassette exons, we identify numerous exons with pronounced regulatory effects on gene expression and cell cycle progression. Analysis of the alternative NRF1 exon-7 demonstrates that its inclusion modulates NRF1's regulatory function by influencing its recruitment to the promoters of target genes. Importantly, gene expression profiles generated using scCHyMErA-Seq accurately recapitulate findings from traditional, labor-intensive orthogonal methods, while offering enhanced scalability and efficiency. Overall, scCHyMErA-Seq represents a versatile platform for systematically unraveling the functional impact of alternative splicing by directly linking specific splicing variants to transcriptional phenotypes.},
}
@article {pmid41634489,
year = {2026},
author = {Araújo, MRB and Dos Santos, LS and Viana, MVC and Sousa, EG and Prates, FD and Perini, HF and da Silva, MV and da Silva Sousa, J and Brenig, B and Andrade, BS and Mattos-Guaraldi, AL and de Oliveira Sant'Anna, L and Ramos, JN and de Oliveira Vinhal, AL and de Castro Soares, S and Azevedo, V},
title = {Comparative genomics and molecular characterization of a multidrug-resistant Corynebacterium glucuronolyticum isolated for the first time from the human genitourinary tract in Latin America.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {57},
number = {1},
pages = {49},
pmid = {41634489},
issn = {1678-4405},
abstract = {UNLABELLED: Although Corynebacterium glucuronolyticum has been associated with human infections, its pathogenic potential remains poorly understood. Here, we describe the first case in Latin America of C. glucuronolyticum isolated from the human urogenital tract. The strain, designated IHP2022, was identified by MALDI-TOF MS (99% probability) and exhibited resistance to benzylpenicillin, clindamycin, and tetracycline, characterizing a multidrug-resistant (MDR) phenotype. Genomic analysis revealed a 2.88-Mb genome with 59.04% G + C content and no plasmids. Comparative genomic analysis, including 11 other publicly available genomes, demonstrated high genetic diversity and positioned IHP2022 close to strain p3-SID752 from the USA, suggesting a broad geographical distribution. The genome harbored multiple virulence and resistance genes, as well as a Type I-E CRISPR-Cas system. Functional annotation and pangenome analysis identified 4,027 gene families categorized into core, shell, and cloud components. By integrating phenotypic and genomic data, this study provides the first in-depth characterization of an MDR C. glucuronolyticum strain minimizing current knowledge gaps and contributing to a better understanding of its pathogenic potential.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s42770-025-01822-7.},
}
@article {pmid41634962,
year = {2026},
author = {Li, W and Duan, M and Sun, S and Li, J and Wang, M and Zhao, H},
title = {A CRISPR Switch Integrated with Strand Displacement Amplification for Binary Channel Detection of SARS-CoV-2 Gene Fragments and Infectious Diagnosis.},
journal = {Langmuir : the ACS journal of surfaces and colloids},
volume = {42},
number = {6},
pages = {5070-5078},
doi = {10.1021/acs.langmuir.5c06262},
pmid = {41634962},
issn = {1520-5827},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *COVID-19/diagnosis/virology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The development of sensitive, accurate, and multimodal approaches for the detection of viral gene fragments and the diagnosis of infections is essential for effective pandemic management across various contexts. This study introduces a CRISPR switch integrated with strand displacement amplification (SDA) for the binary channel detection of SARS-CoV-2 gene fragments and the diagnosis of SARS-CoV-2 infections. In the conventional channel, a specific single gene fragment can directly facilitate the formation of a three-way junction, thereby initiating the SDA process and resulting in the production of a substantial amount of single-stranded DNA. In the logical channel, two gene fragments can first induce the release of a substitute, which subsequently leads to the formation of the three-way junction and the ensuing SDA process. The single-stranded SDA product acts as the target sequence that activates the CRISPR switch, which performs reporter cleavage functions, thereby generating enhanced and detectable fluorescence signals. This method achieves sensitive and selective detection of SARS-CoV-2 gene fragments, with limits of detection (LODs) of 1.0 aM for the ORF1ab gene and 0.9 aM for the N gene in the conventional channel and 3.7 aM for simultaneous detection of both ORF1ab and N in the logical channel. Furthermore, accurate detection of these gene fragments in real samples obtained from patients exhibiting upper respiratory symptoms was successfully conducted, along with the corresponding diagnosis of SARS-CoV-2 infections. Consequently, this method represents a novel binary channel approach for viral gene detection and holds significant promise for clinical diagnosis and potential future epidemic control.},
}
@article {pmid41636073,
year = {2026},
author = {Zhu, X and Gu, G and Shen, Y and Abdurazik, M and Sun, G},
title = {CRISPR/Cas13a-induced self-priming cyclic amplification enables liquid biopsy of exosomal circular RNA in non-small cell lung cancer.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1413-1419},
doi = {10.1039/d5an01345c},
pmid = {41636073},
issn = {1364-5528},
mesh = {*RNA, Circular/genetics/blood ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; Humans ; *Lung Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems/genetics ; *Exosomes/genetics/chemistry ; Liquid Biopsy/methods ; *Nucleic Acid Amplification Techniques/methods ; DNA Primers/genetics ; },
abstract = {The precise and reliable identification of circular RNA (circRNA) is essential for both biological studies and clinical diagnostics of non-small cell lung cancer (NSCLC), especially the exosomal circRNA. In this study, we utilize a CRISPR/Cas13a system to specifically recognize the unique back-splice junction of target circRNA and develop a novel detection platform termed CRISPR/Cas13a-induced self-priming cyclic amplification. This method enables highly sensitive and specific circRNA detection. A pair of stem-loop DNA primers was carefully designed, each incorporating complementary single-stranded DNA sequences and five ribouridine (rU) residues at the 3' end serving as an overhang. When Cas13a binds to the target circRNA, its trans-cleavage activity is activated, leading to the cleavage of the rU residues. This cleavage permits the 3' ends of the stem-loop primers to extend along one another, generating multiple double stem-loop DNA structures that initiate successive cycles of self-priming chain elongation. By leveraging the sustained trans-cleavage activity of Cas13a and the high amplification efficiency of the self-priming cyclic reaction, the assay achieves sensitive detection of circRNA at concentrations as low as 564 aM within 90 min. In addition, the proposed method has been successfully applied for the analysis of exosomal hsa_circ_0003026 expression level in normal samples and NSCLC samples and demonstrated the potential of exosomal hsa_circ_0003026 in regulating the pathological progression. Owing to the high specificity of Cas13a, the proposed method can be directly applied to detect circRNA in complex biological samples without prior isolation of corresponding linear RNAs.},
}
@article {pmid41636718,
year = {2026},
author = {Xu, ZH and Hu, X and Weng, X and Lin, RM and Xu, W and Yu, LS and Gao, H},
title = {An "off-on" electrochemiluminescence biosensor based on CRISPR-Cas12a for ultrasensitive determination of aflatoxin B1.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1325-1331},
doi = {10.1039/d5an01369k},
pmid = {41636718},
issn = {1364-5528},
mesh = {*Aflatoxin B1/analysis ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems ; Aptamers, Nucleotide/chemistry/genetics ; Limit of Detection ; Quantum Dots/chemistry ; *CRISPR-Associated Proteins/genetics ; Food Contamination/analysis ; Electrodes ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Accurate and sensitive determination of aflatoxin B1 (AFB1) is of vital importance for food safety. Herein, an electrochemiluminescence (ECL) "off-on" switch sensor combined with CRISPR-Cas12a is fabricated for the quantitative analysis of AFB1. Initially, the DNA activator is effectively locked by two AFB1 aptamers. Once the target AFB1 is introduced, the activator is released in an open state and thus is recognized by the Cas12a-crRNA duplex. Black hole quencher (BHQ)-marked DNA strands are digested by the activated CRISPR-Cas12a system. Owing to the resonance energy transfer (RET) between aggregation-induced ECL active (AIECL-active) polymer dots (Pdots) and BHQ, the ECL signal of Pdots switches from signal "off" to "on" along with the detachment of BHQ from the electrode surface. The proposed ECL sensor thus achieves sensitive quantification of AFB1 with a detection limit of 0.06 pg mL[-1]. This work provides an effective strategy for mycotoxin determination.},
}
@article {pmid41636766,
year = {2026},
author = {Abbas, W and Hu, J and Zhu, Y and Xu, L and Mo, B and Liu, L},
title = {Decoding MicroRNA Networks in Plant Vegetative and Reproductive Branching: Mechanisms and Applications for Crop Improvement.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {5},
pages = {2795-2808},
doi = {10.1111/pce.70429},
pmid = {41636766},
issn = {1365-3040},
support = {32270878//National Natural Science Foundation of China/ ; JCYJ20220531101618038//Shenzhen Basic Research General Project/ ; JCYJ20230808105419038//Shenzhen Basic Research General Project/ ; 2024ZY0132//Central Guidance Funds for Local Science and Technology Development of Inner Mongolia/ ; 868-000003011001//Excellence 2035 Research Program of Shenzhen University/ ; 2023DFT005//Research Team Cultivation Program of Shenzhen University/ ; 2025M784036//China Postdoctoral Science Foundation of China/ ; 2025TQ09A772//Guangdong Special Support Program for Young Talents in Innovation Research of Science and Technology/ ; },
mesh = {*MicroRNAs/genetics/metabolism/physiology ; *Crops, Agricultural/genetics/growth &amp; development ; Gene Expression Regulation, Plant ; Gene Editing ; *RNA, Plant/genetics ; Gene Regulatory Networks ; Reproduction/genetics ; Plant Development/genetics ; CRISPR-Cas Systems ; },
abstract = {Plant branching, encompassing both vegetative and reproductive forms, is a complex and crucial process that shapes overall architecture and determines crop yield and biomass. MicroRNAs (miRNAs) have emerged as master regulators in fine-tuning the intricate genetic and hormonal networks that govern plant branching. This review systematically synthesises recent advances in understanding how miRNA-target gene modules regulate essential pathways to orchestrate the branching patterns. We highlight a central insight that specific miRNA families form hierarchical, stage-specific networks that facilitate the independent optimisation of vegetative and reproductive branching. Furthermore, we explore the potential applications of miRNA manipulation in optimising branching architecture to improve crop yield. By critically evaluating strategies such as artificial miRNAs, target mimics and CRISPR/Cas9 genome editing, we discuss how modulating miRNA networks can engineer ideal plant architecture. Finally, we provide a forward-looking perspective on overcoming challenges in miRNA-based crop improvement, emphasising the integration of single-cell omics and epigenetic insights to achieve precise genetic modifications. This review underscores the transformative potential of miRNAs in designing future crops for enhanced productivity.},
}
@article {pmid41637037,
year = {2026},
author = {Hao, J and Gao, X and Light, C and Sun, Y and Lu, S and Tian, Y and Gao, X and Su, Y and Gao, J and Huang, X and Zhang, Q and Wang, J and Hai, R and Hu, W and Wang, G},
title = {Genome-wide CRISPR/Cas9 knockout screen identifies host factors essential for bovine parainfluenza virus type 3 replication.},
journal = {Science China. Life sciences},
volume = {69},
number = {4},
pages = {1301-1316},
pmid = {41637037},
issn = {1869-1889},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems/genetics ; *Virus Replication/genetics ; *Parainfluenza Virus 3, Bovine/physiology/genetics ; *Respirovirus Infections/virology/veterinary/genetics ; Gene Knockout Techniques ; Cell Line ; *Host-Pathogen Interactions/genetics ; },
abstract = {Bovine parainfluenza virus type 3 (BPIV3) is a leading cause of respiratory illness in cattle and a primary component of the bovine respiratory disease complex (BRDC), resulting in significant economic losses. Understanding the mechanisms of BPIV3 infection, particularly the entry process, is essential for developing effective control measures. Identifying specific host factors that viruses exploit during their life cycle can reveal critical vulnerabilities for potential antiviral targets. We established a genome-wide CRISPR/Cas9 knockout screen in bovine cells to identify host factors involved in viral infections. Our screen identified several key host factors required for BPIV3 infection, including the sialic acid transporter SLC35A1 and the Sm-like protein LSM12. Further mechanistic analysis revealed that these factors played critical roles at distinct stages of the BPIV3 entry process. These findings not only advance our understanding of how BPIV3 infects host cells but also identify potential host targets for inhibiting infection and developing novel antiviral strategies.},
}
@article {pmid41637835,
year = {2026},
author = {Xie 谢飞, F and Liu 刘晓宙, X and Wang 王露露, L and Zhang 张聪, C and Liu 刘传宏, C and Huo 霍振庆, Z and Zhao 赵正东, Z and Zhao 赵清远, Q and He 贺秋月, Q and Guo 郭科男, K and Sun 孙宇, Y and Wang 王勇, Y},
title = {A porcine congenital deafness model with unconditional knockout of GJB2 generated by CRISPR/Cas9 genomic editing.},
journal = {Hearing research},
volume = {472},
number = {},
pages = {109552},
doi = {10.1016/j.heares.2026.109552},
pmid = {41637835},
issn = {1878-5891},
mesh = {Animals ; *CRISPR-Cas Systems ; Disease Models, Animal ; *Connexin 26/genetics ; *Gene Editing ; *Deafness/genetics/physiopathology/congenital/pathology ; *Connexins/genetics/deficiency ; Phenotype ; *Hearing/genetics ; Gene Knockout Techniques ; Sus scrofa ; Homozygote ; Female ; Organ of Corti/physiopathology/pathology ; Mutation ; Animals, Genetically Modified ; Hair Cells, Auditory/pathology/metabolism ; Genetic Predisposition to Disease ; Male ; },
abstract = {GJB2, the primary gene responsible for DFNB1, the most prevalent non-syndromic hearing loss (NSHL), has variants that account for over 50% of all prelingual hearing loss (HL). Mice are the main model for congenital hearing loss (CHL) research, but they have delayed auditory maturation postnatally, and unconditional Gjb2 knockout in mice causes embryonic lethality. Pigs have similar inner-ear anatomy to humans and, like humans, have matured auditory function and fully differentiated cochlea at birth. Currently, there is no GJB2 unconditional knockout animal model for GJB2-related CHLs research, and whether unconditional GJB2 deletion causes embryonic lethality in pigs or if GJB2-deficient pigs can recapitulate typical clinical pathological characteristics remains unclear. In this study, we employed CRISPR/Cas9 to establish the first unconditional GJB2 knockout pig model. The mutant GJB2 alleles in the founder pig were stably germline-transmitted to subsequent generations. Homozygous GJB2 knockout pigs exhibited no embryonic lethality and showed profound hearing loss, cochlear hair cell depletion and impaired Organ of Corti's development. This GJB2 unconditional knockout pig model has not been reported before and demonstrates GJB2 mutation pathological characteristics consistent with clinical patients, validating its potential in investigating the pathogenic mechanisms and therapeutic interventions of GJB2-deficient CHLs.},
}
@article {pmid41637875,
year = {2026},
author = {Wu, H and Merényi, Z and Virágh, M and Liu, XB and Hegedüs, B and Hou, Z and Ábrahám, E and Fürtön, A and Kristóffy, Z and Lipinszki, Z and Nagy, LG},
title = {Functional characterization of a Con7-related transcription factor in Coprinopsis cinerea indicates evolutionary conservation of morphogenetic roles.},
journal = {Microbiological research},
volume = {306},
number = {},
pages = {128462},
doi = {10.1016/j.micres.2026.128462},
pmid = {41637875},
issn = {1618-0623},
mesh = {*Transcription Factors/genetics/metabolism ; Fruiting Bodies, Fungal/growth &amp; development/genetics ; *Fungal Proteins/genetics/metabolism ; Gene Expression Regulation, Fungal ; *Agaricales/genetics/growth &amp; development ; Morphogenesis/genetics ; CRISPR-Cas Systems ; Phylogeny ; Gene Expression Profiling ; Gene Deletion ; Evolution, Molecular ; Zinc Fingers ; },
abstract = {Fruiting bodies of mushroom-forming fungi (Agaricomycetes) exhibit the highest degree of multicellular complexity in fungi, yet the molecular underpinnings of their developmental programs remain incompletely understood. Here, we characterize gcd1, a gene encoding a transcription factor in the Con7 subfamily of C2H2-type zinc finger proteins. This subfamily has previously been implicated in pathogenic morphogenesis in Ascomycota, but its role in Agaricomycetes has not previously been addressed. In Coprinopsis cinerea, CRISPR/Cas9-mediated deletion of gcd1 resulted in strains with severely impaired fruiting body morphogenesis, with malformed cap, stipe, and gill tissues. Gcd1 deletion strains lacked universal veil, resembling species with open (gymnocarpous) development. We find that GCD1/Con7 homologs are widely distributed in most Dikarya species and are mostly encoded by a single gene in each species' genome. Transcriptome analyses identified several misregulated genes in the Δgcd1 mutant, which pinpoint potential mechanisms underlying its developmental defects as well as provided insights into the morphogenesis of mushroom fruiting bodies. These findings establish GCD1 as a key regulator of multicellular development in C. cinerea and broaden the known functions of Con7-like transcription factors to include fruiting body morphogenesis in Agaricomycetes. Overall, our results and the morphogenetic role of Con7-like transcription factors of Ascomycota suggest functional conservation over half a billion years of evolution.},
}
@article {pmid41638184,
year = {2026},
author = {Li, Z and Zhang, W and Feng, Z and Liu, Z and Feng, Z and Shi, Y and Zhan, J and Zhang, J},
title = {A turn-on CRISPR/Cas12a strategy featuring a sterically-hindered activator for in situ fluorescence imaging of H2O2 in vivo.},
journal = {Biosensors &amp; bioelectronics},
volume = {300},
number = {},
pages = {118449},
doi = {10.1016/j.bios.2026.118449},
pmid = {41638184},
issn = {1873-4235},
mesh = {*Hydrogen Peroxide/isolation &amp; purification/analysis/chemistry ; *Biosensing Techniques/methods ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; Humans ; *Optical Imaging/methods ; Limit of Detection ; *Endodeoxyribonucleases/chemistry/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Hydrogen peroxide (H2O2) serves as a key biomarker of oxidative stress in pathological processes such as cancer and inflammation. However, its in vivo visualization remains challenging due to the lack of sensitive, rapid, and bioorthogonal imaging methods. Here, we present a H2O2-activatable CRISPR/Cas12a strategy, termed A-BO-CRISPR, for real-time fluorescence imaging in living systems. This biosensing strategy employs a 4-bromomethylphenylboronic acid pinacol ester-caged DNA activator whose binding to crRNA is initially blocked by steric hindrance, effectively suppressing Cas12a trans-cleavage activity. Upon encountering endogenous H2O2, the boronate ester is selectively hydrolyzed, restoring activator/crRNA hybridization and triggering amplified fluorescent signal generation via Cas12a-mediated collateral cleavage of a ssDNA reporter. The system achieves a detection limit of 0.64 μM and responds within minutes, enabling real-time monitoring of H2O2 fluxes in living cells and tumor-bearing mice. It exhibits high selectivity and robust stability in complex biological environments. By integrating a chemical gating mechanism with CRISPR-based signal amplification, this work paves the way for potential applications in probing redox biology, imaging-guided diagnostics and therapeutic monitoring.},
}
@article {pmid41638449,
year = {2026},
author = {Qu, Y and Li, Y and Shao, T and Kuang, J and Qi, Y and Yang, J and Liu, Y and Wang, J and Fu, X and Liu, J and Zhang, X and Peng, T and Yuan, Q and Zhu, L},
title = {Optimizing prime editing: Advances in efficiency enhancement.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108815},
doi = {10.1016/j.biotechadv.2026.108815},
pmid = {41638449},
issn = {1873-1899},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Protein Engineering/methods ; },
abstract = {Prime editing (PE) enables the precise installation of targeted insertions, deletions, and all possible base-to-base conversions without introducing double-strand breaks or donor DNA templates. However, its efficiency remains highly variable across genomic contexts. To address this, multi-faceted optimization strategies have been developed: protein engineering has yielded editor variants with enhanced reverse transcriptase activity and stability; structural refinements to pegRNA design improve its functional integrity and resistance to degradation; regulation of the PE-Flap-mismatch repair (MMR) process favors the retention of desired edits; and the development of protospacer adjacent motif (PAM)-relaxed Cas variants dramatically expands targetable sites. This review systematically consolidates these advances, illustrating how the integration of structural, mechanistic and targeting enhancements is overcoming fundamental bottlenecks. Together, these developments establish PE as a versatile and efficient system for precision genome engineering, paving the way for its reliable application in diverse biological settings.},
}
@article {pmid41638451,
year = {2026},
author = {Asemoloye, MD},
title = {The fungal cure: Harnessing mycelial approach as sustainable green solution for industrial waste treatment.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108834},
doi = {10.1016/j.biotechadv.2026.108834},
pmid = {41638451},
issn = {1873-1899},
mesh = {*Industrial Waste ; Biodegradation, Environmental ; *Fungi/metabolism/enzymology ; *Mycelium/metabolism ; },
abstract = {Industrialization has intensified releases of complex waste streams (e.g., synthetic dyes, petroleum hydrocarbons, heavy metals, and plastics) whose treatment can be costly, energy-intensive, and often incomplete using conventional physicochemical methods. 'Mycoremediation' defined as fungi mediated remediation, or their secreted materials/enzymes offers compelling advantages. These advantages stem across the extensive mycelial networks for matrix penetration, non-specific oxidative enzyme systems that transform lignin-like xenobiotics, and cell-wall chemistries that sorb metal ions. This review synthesizes mechanistic foundations on fungal enzymes (laccases; class II peroxidases such as manganese peroxidase and lignin peroxidase; biosorption and biomineralization), bioengineering strategies (CRISPR/Cas editing, artificial consortia), process intensification (immobilized-laccase reactors; whole-cell formats), and applications across textile dye effluents, petroleum-impacted soils/sediments, heavy-metal bearing wastewaters/soils, and polymer-rich wastes. Emerging evidence shows robust lab and mesocosm performance like rapid dye decolorization in fungal cartridge systems, significant alteration of petroleum (saturate, aromatic, resin and asphaltene-SARA) fractions under estuarine salinities, and high-capacity metal biosorption, while systematic verification for plastics remains a priority. Fungi sustainability assessments identify life-cycle hot spots in enzyme production and immobilization supports; techno-economic analyses suggest feasibility pathways when biocatalyst durability and reuse are optimized. This review also delves into regulatory frameworks for contained use and deliberate environmental release of engineered fungi, shaping the near-term deployments toward contained bioreactors. It concludes by projecting the combination of bioengineering (strain/secretome control), reactorization (immobilized catalysts, modular beds), and standardized metrics (toxicity, mass balance, life-cycle assessment-LCA/techno-economic analysis-TEA) for accelerating the transition of mycoremediation from promising prototypes to field-validated, scalable technologies for industrial waste treatment.},
}
@article {pmid41638522,
year = {2026},
author = {Niles, A and Kroening, K and Lauer, A and Chakravorty, A},
title = {Identity of protease-based biomarkers for viability and cytotoxicity revealed by CRISPR knockouts.},
journal = {Analytical biochemistry},
volume = {712},
number = {},
pages = {116073},
doi = {10.1016/j.ab.2026.116073},
pmid = {41638522},
issn = {1096-0309},
mesh = {Humans ; Cell Survival/drug effects ; *Gene Knockout Techniques ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Biomarkers/metabolism/analysis ; *Peptide Hydrolases/metabolism/genetics ; Gene Editing ; },
abstract = {A wide variety of assay chemistries are routinely employed to determine cell health within an in vitro test population. Each method relies on the consistent and robust measurement of specific biological surrogates for cell viability or cytotoxicity. Unfortunately, the precise cellular origin or identity of many of these biomarkers remain poorly characterized or unknown, and thus subject to a host of undetermined biological and chemical interferences. This work details efforts to pinpoint the enzymatic sources of a set of proteolytic activity profiles previously discovered in a phenotypic activity screen and measured in a multiplexed viability ("live cell") and cytotoxicity ("dead cell") assay. First, Clustered Regularly Interspaced Short Palindromic Repeats gene editing (CRISPR) was utilized to knockout (KO) genes encoding candidate enzymes in a human cell background to create clones for identity testing. Next, clones demonstrating discrete reduction of either the live or dead cell signals were further characterized by Western blot analysis for presence of immunogenic protein and by Sanger Sequencing of the targeted edit site. The KO data directed the sourcing of potent and selective inhibitors for orthogonal activity studies of the proteases in a parental population. Last, the utility of the multiplexed assay reagent was further explored in both non-human and human primary cell lines to characterize the universality of the application. Collectively, the positive identification of Cathepsin C (CatC) and Tripeptidyl peptidase II (TPP II) informs assay users about potential modulators of activity leading to possible interferences. Last, the work provides new information about assay performance in previously untested cell types.},
}
@article {pmid41639367,
year = {2026},
author = {Barreiro-Docío, E and Guerrero-Peña, L and Soni, P and Méndez-Martínez, L and Costas-Prado, C and Alvarado, MV and Vázquez, JA and Tort, L and Cerdá-Reverter, JM and Rotllant, J},
title = {Loss-of-function mutations in the melanocortin-2-receptor (mc2r) lead to skin hyperpigmentation in teleost fish.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41639367},
issn = {2045-2322},
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Receptor, Melanocortin, Type 2/genetics/metabolism ; *Hyperpigmentation/genetics/metabolism ; *Loss of Function Mutation ; *Skin Pigmentation/genetics ; *Zebrafish Proteins/genetics/metabolism ; Melanophores/metabolism ; Melanins/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {Melanocortins regulate pigmentation via melanocortin receptors (MCRs), which are highly conserved across vertebrates. Unlike other MCRs, the melanocortin 2 receptor (MC2R) is exclusively activated by ACTH; however, its role in pigmentation remains unclear. Using CRISPR/Cas9-generated mc2r knockout zebrafish, we demonstrated that the loss of mc2r in zebrafish results in impaired interrenal steroidogenesis and pronounced hyperpigmentation characterized by an increased number of melanophores and xanthophores while preserving normal patterning. Transcriptomic analyses revealed the upregulation of genes involved in melanosome formation, melanin synthesis, lipid metabolism, and carotenoid accumulation. These findings demonstrate that, in addition to controlling steroidogenesis, mc2r plays a key role in pigment cell development and metabolic regulation.},
}
@article {pmid41640077,
year = {2026},
author = {Xu, YC and Liu, WJ and Li, CC and Zhang, D and Ma, F and Zhang, CY},
title = {CRISPR/Cas13a-Engineered RNA-Based Fluorogenic Biosensor for Label-Free Quantification of RNA in Colorectal Tissues.},
journal = {Analytical chemistry},
volume = {98},
number = {6},
pages = {5076-5084},
doi = {10.1021/acs.analchem.5c07694},
pmid = {41640077},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Colorectal Neoplasms/genetics/diagnosis ; *CRISPR-Cas Systems ; *Fluorescent Dyes/chemistry ; *RNA/analysis/genetics ; Aptamers, Nucleotide/genetics/chemistry ; },
abstract = {Colorectal cancer (CRC) continues to represent a serious threat to global health, and its diagnosis faces significant challenges, especially in the early phases of malignant tumors. Herein, we demonstrate that BRD2 RNA can serve as a potent noninvasive CRC biomarker and construct a CRISPR/Cas13a-engineered RNA-based fluorogenic biosensor for label-free detection of BRD2 RNA in colorectal tissues. In this assay, the specific recognition of BRD2 RNA by the substrate probe activates Cas13a/crRNA, leading to the trans-cleavage of the substrate probe and the generation of the T7 promoter sequence. The resulting T7 promoter subsequently induces efficient transcription amplification to synthesize abundant Pepper RNA aptamers that can light up HBC620. Leveraging the synergistic advantages of Cas13a precision, efficient transcription amplification, and superior signal-to-noise ratio of RNA aptamer-fluorophore complex, this fluorogenic biosensor enables sensitive detection of BRD2 RNA down to 0.39 fM and accurate quantification of its expression at the single-cell level. In addition, this fluorogenic biosensor can successfully distinguish CRC patient tissues from adjacent normal tissues based on distinct BRD2 RNA expression profiles. Importantly, the programmability of crRNA makes this fluorogenic biosensor readily adapted for detecting a broad range of RNA targets (e.g., noncoding RNAs and viral RNAs) by simply modifying the spacer sequence of crRNA, providing a new paradigm for early clinical diagnostics.},
}
@article {pmid41640336,
year = {2026},
author = {Kim, YJ and Bang, S and Chung, AJ},
title = {Safe and Efficient CRISPR Genome Editing of Primary Human T Cells Using a Droplet-Based Cell Mechanoporation Platform.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {19},
pages = {e12553},
pmid = {41640336},
issn = {1613-6829},
support = {2021R1A2C2006224//Korean government (MSIT; Ministry of Science and ICT)/ ; RS-2023-00242443//Korean government (MSIT; Ministry of Science and ICT)/ ; RS-2023-00218543//Korean government (MSIT; Ministry of Science and ICT)/ ; A.J.C//Korean government (MSIT; Ministry of Science and ICT)/ ; },
mesh = {Humans ; *T-Lymphocytes/metabolism/cytology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cell Survival ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {T cell engineering is a transformative strategy for adoptive cell therapy, holding the key to treating a wide array of human diseases. However, clinical translation is limited by current intracellular delivery methods that compromise viability, induce stress responses, and restrict scalability. This study presents a microfluidic droplet mechanoporation system tailored for primary human T cells, enabling efficient, stable, and clinically scalable gene delivery. Delivery of 2000 kDa fluorescein isothiocyanate (FITC)-dextran achieves ∼98% efficiency and >90% post-treatment viability, even at high cell densities, supporting the rapid production of therapeutically relevant cell numbers. The platform efficiently delivers mRNA, achieving transfection efficiencies approaching 99%; further, chimeric antigen receptor (CAR)-encoding mRNA is successfully delivered to generate CAR-expressing T cells with tunable surface expression. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoproteins are effectively delivered for both single and multiplex knockouts (TRAC and PDCD-1), achieving up to a 2.35-fold higher efficiency than electroporation. Longitudinal analyses confirm preserved viability, proliferation, genome integrity, and T cell phenotypic stability. Collectively, these results establish microfluidic droplet mechanoporation as a safe, efficient, and scalable platform for the clinical manufacturing of engineered T cell therapies.},
}
@article {pmid41640466,
year = {2026},
author = {Bharti, N and Modi, U and Bhatia, D and Solanki, R},
title = {Engineering delivery platforms for CRISPR-Cas and their applications in healthcare, agriculture and beyond.},
journal = {Nanoscale advances},
volume = {8},
number = {4},
pages = {1137-1161},
pmid = {41640466},
issn = {2516-0230},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have transformed genome editing through unprecedented precision, and next-generation variants (base and prime editors) further enhance specificity by enabling targeted nucleotide changes without introducing double-strand DNA breaks. These technologies have unlocked broad applications in therapeutic gene correction, functional genomics, infectious disease management, diagnostics, agricultural engineering, environmental biotechnology, and synthetic biology. However, the targeted delivery of these systems remains a major challenge due to the large and chemically distinct nature of their components, including Cas protein or its base/prime editor fusions, guide RNA, and in some cases, DNA repair templates-which complicate packaging, stability, and cellular uptake. Additional hurdles arise from tissue and cell-type specificity, differential intracellular environments, variable editing efficiencies, and the persistent risk of off-target genome modifications. This review outlines the key challenges in the delivery of CRISPR technologies as well provides a comprehensive overview of both current and emerging delivery strategies, including viral vectors (adenovirus, adeno-associated virus, and lentivirus), non-viral physical approaches (microinjection, electroporation, ultrasound, and hydrodynamic tail-vein injection), and nanoparticle-based modalities (lipid and polymeric nanoparticles, gold nanoparticles, DNA nanostructures, and extracellular vesicles). We also discussed the diverse applications of CRISPR-Cas9 in gene therapy, immune cell engineering for cancer therapies, and agricultural innovation.},
}
@article {pmid41640776,
year = {2025},
author = {Di Pasquale, G and Ottaviani, L and Camardo Leggieri, M and Giorni, P and Marocco, A and Battilani, P and Lanubile, A},
title = {Plant oxylipins: adaptation to environmental stresses and impact on mycotoxin contamination.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1739321},
pmid = {41640776},
issn = {1664-462X},
abstract = {Due to increasingly frequent changes in climatic conditions and global warming, plants consistently deal with severe weather events including extreme temperature variations, floods and drought. These abiotic stressors resulting from climate change weaken host crop resistance, making them more exposed to fungal disease insurgences and mycotoxin contamination. Oxylipins are major players in the plant-environment interaction. Their synthesis begins with the oxygenation of polyunsaturated fatty acids by lipoxygenases (LOXs) to generate fatty acid hydroperoxides that in turn are converted into a huge assortment of bioactive compounds by specialized cytochrome P450 enzymes, known as CYP74. In the present review we focus on recent advances concerning oxylipin biosynthesis and the phylogenetic relationships among the main key enzymes of the oxylipin pathway considering five monocot and dicot plant species. Moreover, new information regarding the role of these signaling molecules on the plant physiology in response to abiotic stress and mycotoxin occurrence are provided along with the application of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) (CRISPR/Cas)-based tools. Here, we report the intervention of LOX, allene oxide synthase, OPDA reductase, JASMONATE (JA) resistant and JA ZIM domain genes along with the accumulation of JA and its conjugates, 12-OPDA, ketols and green leaf volatiles in response to abiotic stress. The modulation of LOX genes and the production of several fatty acids, oxylipins and sphingolipids is also required against mycotoxin contamination.},
}
@article {pmid41641699,
year = {2026},
author = {Fu, M and Wang, J and Li, J and Zhou, Y and Huang, X and Jia, Z and Luo, Y and Tan, X and Gao, Y and Yu, B and Duan, Y and Bu, Q and Li, X and Wang, Y and Takaya, N and Zhou, S},
title = {Dual-single-guide RNA strategy improves CRISPR-mediated homology-directed repair in Aspergillus.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41641699},
issn = {1362-4962},
support = {22077032//National Natural Science Foundation of China/ ; 21672065//National Natural Science Foundation of China/ ; K202415//State Key Laboratory of Natural and Biomimetic Drugs/ ; },
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics ; *Aspergillus nidulans/genetics ; *CRISPR-Cas Systems ; *Recombinational DNA Repair ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; Fungal Proteins/genetics/metabolism ; DNA, Fungal/genetics ; },
abstract = {CRISPR-Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3' ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks-including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis-and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.},
}
@article {pmid41642051,
year = {2026},
author = {Yang, Y and Yang, L and Ma, H and Zhang, S and Zhu, Y and Zhang, S and Lin, X and La, H and Gu, X and Ma, J and Zhao, S and Yang, Y and Lei, H and Yang, Y},
title = {Precision detection of rifampicin-resistant rpoB_L378R mutation in Mycobacterium tuberculosis with CRISPR-Cas12a.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {7},
pages = {1442-1453},
doi = {10.1039/d5ay01718a},
pmid = {41642051},
issn = {1759-9679},
mesh = {*Rifampin/pharmacology ; *Mycobacterium tuberculosis/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Bacterial/genetics ; *Bacterial Proteins/genetics ; *DNA-Directed RNA Polymerases/genetics ; Mutation ; Humans ; Antitubercular Agents/pharmacology ; Nucleic Acid Amplification Techniques/methods ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rifampicin is one of the most effective anti-tuberculosis drugs. However, certain strains of Mycobacterium tuberculosis (MTB) have developed resistance to rifampicin, making it crucial to identify alternative drugs for treating rifampicin-resistant MTB infections. Mutations in the rpoB gene play a pivotal role in MTB's resistance to rifampicin. Therefore, identifying these mutations is essential for effectively managing rifampicin-resistant MTB strains. Here, we developed a CRISPR-Cas12a platform integrated with recombinase polymerase amplification (RPA) and fluorescence detection, which was specifically designed to identify the rpoB_L378R mutation associated with rifampicin resistance in MTB. Our findings indicated that this detection technique exhibited high specificity and did not cross-react with reference samples constructed from the genomes of MTB H37Rv, Mycobacterium smegmatis, Mycobacterium aurum, and Escherichia coli. The RPA-CRISPR-Cas12a-based platform established in this research was simple, sensitive, and specific for detecting the rifampicin-resistant MTB strain with the rpoB_L378R mutation. These results suggest its potential applicability in clinical diagnosis for identifying the MTB rpoB_L378R mutation.},
}
@article {pmid41642528,
year = {2026},
author = {Shafique, MS and Liu, Y and Ji, Z},
title = {Genome Editing for Developing Disease-Resistant Plants.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3012},
number = {},
pages = {253-264},
pmid = {41642528},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Disease Resistance/genetics ; CRISPR-Cas Systems/genetics ; *Genome, Plant ; *Plant Diseases/genetics ; Plants, Genetically Modified/genetics ; Oryza/genetics ; Plant Breeding/methods ; Crops, Agricultural/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; },
abstract = {Genome editing tools, most notably the CRISPR-Cas9 system, have transformed modern crop breeding by allowing precise and efficient modification of plant genomes, particularly for enhancing resistance to disease. In this chapter, we present two major CRISPR-based approaches that have been successfully applied to develop disease-resistant crops: (1) gene knockout of host susceptibility (S) genes to block pathogen compatibility, and (2) targeted insertion of resistance alleles or synthetic genes using the homology-directed repair (HDR) pathway. Both strategies utilize the specificity and efficiency of CRISPR-Cas9 to introduce defined edits in crops such as rice. This chapter outlines comprehensive step-by-step protocols covering key elements of the process, including target site selection, construction of sgRNA and Cas9 vectors, plant transformation methods, and mutation validation techniques. Together, these methods provide a powerful platform for engineering plant immune responses and offer practical tools to accelerate the breeding of disease-resistant cultivars.},
}
@article {pmid41642668,
year = {2026},
author = {Takenaka, R and Albanese, EH and Simmerman, SM and Verghese, S and Maddox, MAE and de la Cruz, AF and Young, JM and Schmidt, CA and Rieder, LE and Malik, HS},
title = {The Drosophila maternal-effect gene abnormal oocyte (ao) does not repress histone gene expression.},
journal = {Genetics},
volume = {232},
number = {4},
pages = {},
pmid = {41642668},
issn = {1943-2631},
support = {T32 GM007270/GM/NIGMS NIH HHS/United States ; //NIH IRACDA/ ; F32GM140778//NIH NRSA/ ; R00HD092625//NIH NRSA/ ; R35GM142724//NIH NRSA/ ; R01GM074108//NIH NRSA/ ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; *Histones/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/genetics ; Female ; Heterochromatin/genetics/metabolism ; CRISPR-Cas Systems ; *Oocytes/metabolism ; Maternal Inheritance ; Male ; },
abstract = {The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created 2 new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that loss of ao causes maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. Our data indicate that ao genetically interacts with the heterochromatin, as previously suggested. However, contrary to a prior study, we detected neither Ao localization to histone genes nor ao repression of core histone transcript levels. Thus, the molecular basis for ao-associated maternal-effect lethality remains unknown.},
}
@article {pmid41642758,
year = {2026},
author = {Sun, L and Liu, Z and Dong, C and Chi, T and Chen, J and Cheng, D and Zhu, L and Hu, B},
title = {Method Development and Sensitivity Optimization Paradigm for One-Step RPA-CRISPR/Cas12a Rapid Detection of the Hard-to-Amplify Gene blaNDM.},
journal = {Environmental science &amp; technology},
volume = {60},
number = {6},
pages = {4552-4564},
doi = {10.1021/acs.est.5c10280},
pmid = {41642758},
issn = {1520-5851},
mesh = {*CRISPR-Cas Systems ; *beta-Lactamases/genetics ; Nucleic Acid Amplification Techniques ; },
abstract = {The presence and spread of antibiotic resistance genes (ARGs) across various habitats have increased the risks of antibiotic resistance, highlighting the urgent need for effective monitoring methods. One key challenge in method development lies in balancing sensitivity, speed, and portability. To address it, a one-step assay targeting the carbapenem resistance gene blaNDM was developed based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a. A sensitivity optimization paradigm─MOSAIC (multistrategy optimized sensitive assay via integrated CRISPR/Cas12a)─was proposed, incorporating component optimization, suboptimal-PAM-mediated CRISPR inhibition, and glycerol-assisted phase separation. The glycerol-assisted strategy exhibited the largest enhancement, followed by the suboptimal-PAM strategy and component optimization. When combined, these strategies demonstrated a synergistic effect, yielding greater improvement (10 000-fold) than a single strategy alone. MOSAIC reached a limit of detection (LOD) of 260 copies/μL, comparable to that of qPCR, and enabled faster quantification of blaNDM at 37 °C within 1 h on a standard plate reader. It achieved 100% diagnostic sensitivity and 95.45% specificity in clinical isolates, and 77.41-99.73% accuracy in environmental matrix-spiked samples, comparable to that of qPCR. It provides a technological foundation for on-site detection of blaNDM and offers an optimization paradigm and new insights for the development of one-step RPA-CRISPR/Cas12a assays targeting various genes.},
}
@article {pmid41642947,
year = {2026},
author = {Pelea, O and Tálas, A and Carrera, JF and Mathis, N and van de Venn, L and Yeh, CD and Kulcsár, PI and Marquart, KF and Weber, Y and Gerecke, SE and Harvey-Seutcheu, IF and Mailänder, D and Pfleiderer, MM and Chanez, C and Corn, JE and Schwank, G and Jinek, M},
title = {Programmable genome editing in human cells using RNA-guided bridge recombinases.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6790},
pages = {eadz1884},
doi = {10.1126/science.adz1884},
pmid = {41642947},
issn = {1095-9203},
mesh = {Humans ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; Plasmids ; Recombination, Genetic ; Genome, Human ; *DNA Nucleotidyltransferases/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; DNA/genetics ; },
abstract = {Site-specific insertion of gene-sized DNA fragments remains an unmet need in the field of genome editing. IS110-family serine recombinases have recently been shown to mediate programmable DNA recombination in bacteria by using a bispecific RNA guide (bridge RNA) that simultaneously recognizes target and donor sites. In this work, we have shown that the bridge recombinase ISCro4 is highly active in human cells and provided structural insights into its enhanced activity. Using plasmid- or all-RNA-based delivery, ISCro4 supports programmable multikilobase excisions and inversions and facilitates donor DNA insertion at genomic sites with efficiencies that exceed 6%. Last, we assessed ISCro4 specificity and off-target activity. These results establish a framework for the development of bridge recombinases as next-generation tools for editing modalities that are beyond the capabilities of current technologies.},
}
@article {pmid41643424,
year = {2026},
author = {Wang, S and Li, C and Zhu, L and Liu, K and Jing, L and Xie, L and Guo, H and Ge, S and Yu, J},
title = {Engineered DNA hydrogel paper chip biosensor by cascaded hybridization chain reaction-assisted CRISPR/Cas12a system for sensitive detection of miRNA 622.},
journal = {Biosensors &amp; bioelectronics},
volume = {300},
number = {},
pages = {118482},
doi = {10.1016/j.bios.2026.118482},
pmid = {41643424},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification/analysis ; *Biosensing Techniques/instrumentation ; CRISPR-Cas Systems/genetics ; Hydrogels/chemistry ; Limit of Detection ; Nucleic Acid Hybridization ; Humans ; Paper ; *DNA/chemistry/genetics ; Endodeoxyribonucleases/chemistry/genetics ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Electrochemical Techniques ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The integration of DNA hydrogel with electrochemiluminescence (ECL) technology represents a synergistic enhancement through molecular-level precision design and nanoscale coordination. This strategic integration confers biosensors novel functionalities including intelligent responsiveness and environmental adaptability. In this study, a cascaded hybridization chain reaction (HCR) and CRISPR/Cas12a-integrated DNA hydrogel paper chip was engineered for ultrasensitive microRNA 622 (miRNA 622) detection. Target miRNA 622 triggered HCR amplification via hairpin DNA assembly, while Cas12a recognized protospacer adjacent motif (PAM) sequences within the HCR-generated double-stranded products to activate its trans-cleavage ability. The DNA hydrogel was constructed through copolymerization of acrylamide-modified DNA single strands (SA and SB) with Ru (II) complex-functionalized linker DNA. Activated Cas12a cleaved single-stranded DNA within the DNA hydrogel network, thereby releasing Ru (II) complexes. AuPd nanoparticles (AuPd NPs) served as the co-reactant accelerator, amplifying the cathodic ECL signals of the liberated Ru (II) complexes. The developed platform demonstrated a dynamic detection range from 0.001 to 500 pM with a detection limit of 0.33 fM, establishing a groundbreaking approach for detecting miRNA 622 in clinical diagnostics.},
}
@article {pmid41643679,
year = {2026},
author = {Ferrández-Roldán, A and Piulachs, MD},
title = {Using DIPA-CRISPR for simple and efficient endogenous protein tagging in insects.},
journal = {Cell reports methods},
volume = {6},
number = {2},
pages = {101297},
pmid = {41643679},
issn = {2667-2375},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Insect Proteins/genetics/metabolism ; Genes, Reporter ; *Insecta/genetics ; *Blattellidae/genetics/metabolism ; },
abstract = {CRISPR-Cas9 is rapidly expanding across diverse organisms. Among these advances, in-frame knockins of reporter genes have become essential for studying gene expression and protein localization. However, in hemimetabolan insects such as the German cockroach Blattella germanica, a phylogenetically basal and relevant pest species, functional fusion proteins have remained technically difficult to obtain. We present a streamlined gene-editing strategy to knock in a reporter gene in-frame with the distal-less gene, generating a functional fusion protein in B. germanica. By combining direct parental CRISPR with donor constructs designed for homology-directed repair carrying the mCherry gene, we successfully achieved targeted integration at the distal-less locus. The resulting fusion protein was functional and heritable and enabled live visualization of Distal-less protein distribution, showing fluorescence in developing appendages and the nervous system. This simple and robust methodology opens the door to generating fusion proteins in non-model insects, providing a valuable molecular tool for ecological, developmental, and pest-management research.},
}
@article {pmid41643803,
year = {2026},
author = {Kim, C and Zhu, Z and Tagmount, A and Barbazuk, WB and Bacher, R and Vulpe, CD},
title = {Physiologically relevant 3D CRISPR screening enhances mechanistic insight into chemical toxicity compared to 2D screening.},
journal = {Toxicology},
volume = {522},
number = {},
pages = {154422},
doi = {10.1016/j.tox.2026.154422},
pmid = {41643803},
issn = {1879-3185},
mesh = {Humans ; Hep G2 Cells ; *Toxicogenetics/methods ; *CRISPR-Cas Systems ; *Doxorubicin/toxicity ; *Toxicity Tests/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Spheroids, Cellular/drug effects ; Bioreactors ; *Cell Culture Techniques, Three Dimensional ; },
abstract = {CRISPR-based approaches can complement other genomics-based toxicology studies by enabling causal interrogation of gene function modulating chemical-induced toxicity. Moreover, CRISPR screens enable scalable and systematic identification of functional pathways involved in cellular response to chemical exposure. Cell-based functional toxicogenomics approaches using CRISPR provide a potential powerful tool for the development of mechanism-driven new approach methodologies (NAMs) for toxicodynamic and toxicokinetic hazard screening to enable more effective risk assessment. To improve the physiological relevance of in vitro functional toxicogenomics, we developed a three-dimensional (3D) CRISPR screening platform using HepG2/C3A spheroids cultured in a continuously rotating bioreactor (ClinoStar). We evaluated the potential utility of a 3D CRISPR screen as compared to conventional 2D screen using a custom CRISPR sgRNA library representing common loss-of-function genetic variants in the human population and exposure to the well characterized DNA damaging toxicant, doxorubicin. The 3D platform identified more genes and pathways in which variants have previously been associated with doxorubicin toxicity in clinical studies than the 2D system. These results support the utility of 3D CRISPR screening to identify physiologically relevant genetic determinants underlying chemical toxicity.},
}
@article {pmid41644661,
year = {2026},
author = {Davydova, S and Liu, J and Kandul, NP and Antoshechkin, I and Mann, J and Braswell, WE and Akbari, OS and Meccariello, A},
title = {Generating cisgenic sexing strains in insect pests.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41644661},
issn = {2399-3642},
support = {101059523//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; AP23PPQS&amp;T00C108//United States Department of Agriculture | Agricultural Research Service (USDA Agricultural Research Service)/ ; },
mesh = {Animals ; Male ; Female ; *Ceratitis capitata/genetics ; CRISPR-Cas Systems ; *Pest Control, Biological/methods ; Gene Editing ; Introns ; },
abstract = {Insect pest population control via sterile insect technique markedly benefits from separation by sex prior to release. To simplify this process, traditional genetics has been deployed to develop genetic sexing strains (GSSs) for several disease vectors and agricultural pests of vast economic significance, although very few are applied in the field due to associated fitness costs and instability. In this study, we generated a method to engineer cisgenic GSS (CGSS) in insects. We use CRISPR/Cas9-mediated homology-directed repair to seamlessly translocate a sex-specific alternatively spliced intron into a dominant phenotypic gene generating a genetically stable strain that enables sex-sorting by eye. To achieve this feat, we use Ceratitis capitata as our model and relied on the sex-specifically spliced intron of its endogenous transformer gene, which we seamlessly inserted a copy into the pupal colouration white pupae gene. This minimal modification resulted in the generation of a homozygous strain we term IMPERIAL that was genetically and phenotypically stable where all female pupae are brown while male pupae are white with overall good fitness. By minimally editing the genome, our novel CGSS approach can be applied to other pests that may aid more efficient and economically suitable pest control.},
}
@article {pmid41644834,
year = {2026},
author = {Zhao, C and Guo, H and Chen, J and Tan, Y and Wu, Z and Zhang, Y and Li, Y and Wang, J and Wang, R and Zhang, H and Wang, D},
title = {An AuPt nanozyme-assisted CRISPR/Cas12a system for visual nucleic acid detection of pathogens.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {7},
pages = {1997-2009},
pmid = {41644834},
issn = {1618-2650},
mesh = {*CRISPR-Cas Systems ; *Gold/chemistry ; Solanum tuberosum/microbiology ; *Platinum/chemistry ; *Alternaria/genetics/isolation &amp; purification ; *Metal Nanoparticles/chemistry ; *Plant Diseases/microbiology ; Biosensing Techniques/methods ; Limit of Detection ; *DNA, Fungal/analysis/genetics ; },
abstract = {Potato early blight, caused by Alternaria solani, presents a significant threat to the potato industry. Existing detection methods for A. solani often fail to simultaneously achieve simplicity and accuracy. A gold-platinum (AuPt) nanozyme-assisted CRISPR/Cas12a system, termed the nanoparticle enzyme-assisted CRISPR detection (NACD assay) was developed. By integrating the precise target recognition of CRISPR with the enzyme-like activity of AuPt nanozymes, this system achieves simple, sensitive, and visual detection of A. solani. The NACD assay provided visual results through a distinct color change produced by the substrate catalyzed by the AuPt nanozyme. It can detect 100 copies/μL of the target dsDNA (A. solani 5.8S rRNA gene) and 10[-][3] ng/μL A. solani genomic DNA. This detection method demonstrates high specificity, with no cross-reactivity observed with three other pathogens. Moreover, the incorporation of a filter paper-based readout enables straightforward visual detection by the naked eye, making it particularly suitable for on-site testing. Overall, these features make it an effective on-site diagnostic tool, allowing the potato industry to manage early diseases more efficiently.},
}
@article {pmid41644992,
year = {2026},
author = {Oishi, S and Doi, S and Sekida, T and Yamashita, K and Yamada, Y and Muramoto, T},
title = {Genome editing across Dictyostelia species enables comparative functional genetics of social amoebas.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41644992},
issn = {2045-2322},
support = {23KJ1977//Japan Society for the Promotion of Science/ ; 23K05785//Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Dictyostelium/genetics ; *Genome, Protozoan ; Phylogeny ; },
abstract = {Gene manipulation is essential for understanding biological mechanisms, yet genetic modification in the social amoebas (Dictyostelia) has been largely limited to Dictyostelium discoideum. Here, we aimed to establish a CRISPR/Cas9-based genome-editing system applicable across the phylogenetic breadth of Dictyostelia, spanning Groups 1-4. Using an extrachromosomal CRISPR/Cas9 vector from D. discoideum, we disrupted stlA and pkaC in Polysphondylium violaceum and pkaC in two early-branching species, Heterostelium pallidum and Cavenderia fasciculata. In D. discoideum, co-introduction of donor oligos with the CRISPR vector enabled selection-free knockout generation of pkaC with 28.6% efficiency. In H. pallidum, where genome editing is typically inefficient, co-electroporation of donor oligos with the CRISPR/Cas9 vector followed by 4 days of drug selection increased the frequency of pkaC disruption from 0.9% to 8.3%. These results demonstrated that the D. discoideum CRISPR/Cas9 system can be extended across Dictyostelia, providing a versatile platform for comparative genetic and evolutionary developmental studies.},
}
@article {pmid41645678,
year = {2026},
author = {Ren, Y and Xu, J and Luo, Q and Li, P and Jiang, M and Cheng, Y and Zhang, H and Wei, D and Bai, W and Liu, S and Wang, G and Xie, Y and Li, J and Lu, W},
title = {A Recombinant Targeted Bionanoparticle In Vitro Expressing a Gene-Editing Protein for Epidermal Growth Factor Receptor Mutant Lung Cancer.},
journal = {ACS nano},
volume = {20},
number = {6},
pages = {4920-4941},
pmid = {41645678},
issn = {1936-086X},
mesh = {*Lung Neoplasms/genetics/therapy/pathology ; *ErbB Receptors/genetics ; Humans ; *Gene Editing/methods ; Animals ; *Nanoparticles/chemistry ; Mice ; Mutation ; HEK293 Cells ; *CRISPR-Associated Protein 9/genetics/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; Recombinant Proteins/genetics ; Mice, Nude ; },
abstract = {Epidermal growth factor receptor (EGFR) mutation is a significant driving factor in the occurrence and progression of lung cancer. How to effectively block the abnormal expression of EGFR remains a key issue that urgently needs to be addressed, as it is of vital importance for the effective treatment of this cancer. Here, we report a recombinant targeted bionanoparticle that in vitro expresses Cas9 protein (tBioNP vitro-Cas9) for gene editing of EGFR mutant lung cancer. The nanoparticle was developed by transfection of four plasmids (Gag-Cas9, Gag, sgRNA, VSV-G Azi) into 293T cells to form a type of bionanoparticle and modifying it with a targeted polymer material (DBCO-PEG-FA), and it showed a cancer-targeted property, faster cancer cellular uptake, higher gene editing efficiency with lower off-target effects, and therapy efficacy in mice, indicating a translational prospect. In conclusion, the study provides a recombinant bionanoparticle in vitro expressing a Cas9 gene editing system and offers a potential strategy for gene therapy of EGFR mutant lung cancer.},
}
@article {pmid41645777,
year = {2026},
author = {Wu, H and Lin, S and Zuo, X and Hua, W and Sun, T and Shao, G and Li, F and Zhao, D and Feng, Z and Zhu, D and Wang, L},
title = {RAA-CRISPR-HCR cascade amplification for ultrasensitive visual detection of African swine fever virus DNA.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {14},
pages = {4329-4332},
doi = {10.1039/d5cc06815k},
pmid = {41645777},
issn = {1364-548X},
mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Colorimetry/methods ; Limit of Detection ; Swine ; *CRISPR-Cas Systems ; African Swine Fever/diagnosis/virology ; },
abstract = {An RAA-CRISPR-HCR (RCH) cascade amplification system was developed, which generates an ultrasensitive colorimetric response to low concentrations of African swine fever virus (ASFV) DNA with a detection limit of 1 copy µL[-1]. It demonstrates excellent accuracy and specificity in real samples, facilitating efficient on-site visual early detection of ASFV.},
}
@article {pmid41645934,
year = {2026},
author = {Wang, Z and Wang, Y and Ji, Q},
title = {Protocol for Discovery and Characterization of Miniature Cas12 Systems.},
journal = {ACS chemical biology},
volume = {21},
number = {2},
pages = {401-409},
doi = {10.1021/acschembio.6c00016},
pmid = {41645934},
issn = {1554-8937},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Dependovirus/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Efficient delivery remains a major challenge for therapeutic genome editing because many widely used CRISPR nucleases are large and leave limited space for regulatory elements or additional payloads in a single adeno-associated virus (AAV) vector. Miniature Cas12 nucleases are particularly appealing, as their reduced size alleviates packaging constraints while preserving RNA-guided DNA cleavage. Here, we outline a workflow that links large-scale sequence mining with phylogenetic and structural filtering, followed by PAM profiling, in vitro cleavage, bacterial genome interference, and genome-editing assays in human cells to confirm activity. This protocol is intended to distill broad sequence collections into a small set of compact Cas12 nucleases with demonstrated functions that can serve as starting points for further engineering in delivery-limited settings.},
}
@article {pmid41648339,
year = {2026},
author = {Phan, PT and Ozturk, M and Dougherty, EM and Ravishankar, J and Xue, C and Sashital, DG},
title = {Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41648339},
issn = {2692-8205},
support = {R35 GM140876/GM/NIGMS NIH HHS/United States ; },
abstract = {Type I-E CRISPR-Cas systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the PAM and PAM-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated, but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four E. coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 2 and 4. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cas8 conformation, and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.},
}
@article {pmid41648761,
year = {2025},
author = {Lu, Z and Bukhari, STH and Azeem, M and Tariq, N and Shabbir, MAB},
title = {A narrative review of precision and ethical considerations in cardiovascular health: CRISPR-Cas9, telemedicine, and lifestyle interventions.},
journal = {Frontiers in public health},
volume = {13},
number = {},
pages = {1737251},
pmid = {41648761},
issn = {2296-2565},
mesh = {Humans ; *Telemedicine/ethics ; *Cardiovascular Diseases/prevention &amp; control/therapy ; *CRISPR-Cas Systems ; *Life Style ; *Precision Medicine ; Gene Editing ; },
abstract = {BACKGROUND: Cardiovascular diseases (CVDs) remain the leading cause of global morbidity and mortality, influenced by lifestyle, socioeconomic status, and genetic factors. Emerging innovations, including wearable health technologies, telemedicine, and CRISPR-Cas9 gene editing, provide new possibilities for rapid prevention and personalized management.<br><br>METHODS: This narrative review collected evidence from Scopus, PubMed, and Google Scholar, using keywords such as cardiovascular (CV) prevention, lifestyle determinants, digital health, telemedicine, CRISPR-Cas9, and public health ethics. Eligible peer-reviewed studies, clinical guidelines, and policy documents were included to assess behavioral, technological, and genomic strategies for CVD care.<br><br>RESULTS: Modifications in lifestyle, such as quitting smoking, regular physical activity, following a heart-healthy diet plan, and getting adequate sleep, can significantly reduce the risk of CVD. Additionally, telemedicine and wearable devices facilitate early detection, better self-management, and treatment adherence, especially in underserved communities. CRISPR-Cas9 holds a significant potential for correcting genetic variants related to lipid disorders and inherited cardiomyopathies, but its clinical translation remains in early stages. However, existing evidence is limited by heterogeneity in study design, brief follow-up, particularly for digital health and CRISPR applications. Additional challenges, such as health inequities, digital access, data privacy, and ethical oversight, further influence their real-world implementation.<br><br>CONCLUSION: Effective integration of behavioral, digital and genomic innovations requires policy frameworks that ensure equity, ethical governance, and long-term sustainability. Combining precision medicine with efforts to address social determinants of health will be crucial in reducing the global burden of CVD and shaping the future of CV care.},
}
@article {pmid41648912,
year = {2026},
author = {Wang, R and Li, J and Zheng, Y and Wei, N and Li, W},
title = {CRISPR/Cas12a platform activated by a protospacer adjacent motif-engineered DNA circuit for specific target sensing.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {7},
pages = {1397-1405},
doi = {10.1039/d5ay02079d},
pmid = {41648912},
issn = {1759-9679},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA/genetics ; Circulating Tumor DNA/genetics/analysis/blood ; *Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas systems have found extensive applications in nucleic acid diagnostics. However, the generalizability of this approach, particularly for the sensing of non-nucleic acid targets, remains a challenge. This study presents the development of a CRISPR/Cas12a platform activated by a protospacer adjacent motif (PAM)-engineered DNA circuit. Initially, the influence of the presence or absence of the PAM on the DNA circuit and the subsequent CRISPR/Cas12a system was investigated, demonstrating that a PAM-engineered DNA circuit functions as an effective activator of Cas12a, whereas a DNA circuit lacking the PAM does not induce activation. Subsequently, through the strategic design of recognition elements, sensitive and selective detection of specific targets is achieved, with limits of detection (LODs) of 0.023 fM for circulating tumor DNA (ctDNA), 0.00004 U mL[-1] for uracil-DNA glycosylase (UDG), and 0.12 fM for acetamiprid (ACE). This approach exemplifies a two-stage signal amplification mechanism, achieving improved sensitivity relative to either the CRISPR/Cas12a system or the DNA circuits alone. Moreover, quantitative assays for these targets were successfully conducted in real samples, suggesting the practical applicability of the proposed method. This research establishes a versatile sensing platform for various targets, which holds significant promise for advancements in molecular diagnostics, food safety assessment, and environmental monitoring.},
}
@article {pmid41649152,
year = {2026},
author = {Gallo, D and Bes, M and Mounier, T and Calatayud, C and Meunier, AC and Périn, C},
title = {A Streamlined CRISPRa Architecture with Dual Pol II/Pol III Promoter and Optimized scRNA Enables Robust and Tunable Gene Activation.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {36-48},
doi = {10.1177/25731599251408333},
pmid = {41649152},
issn = {2573-1602},
mesh = {*Promoter Regions, Genetic ; Oryza/genetics ; *RNA Polymerase II/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *RNA Polymerase III/genetics ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; Gene Expression Regulation, Plant ; },
abstract = {CRISPR activation (CRISPRa) offers a powerful approach to upregulate endogenous genes; yet, existing systems in plants can be complex or difficult to integrate with CRISPR interference (CRISPRi). Here, we present a streamlined and flexible CRISPRa platform that enables robust gene activation. Using a dual-luciferase reporter, we benchmarked a range of guide RNA scaffolds, effector proteins, and promoters. We developed a novel single-guide RNA (sgRNA) architecture, harboring two MS2 aptamers inserted into the tetraloop and driven by a composite Pol II/Pol III promoter, as the most efficient configuration. This scaffold outperformed gR2.0- and SunTag-based constructs, reaching up to 100-fold activation of a minimal 35S promoter and up to 215-fold induction of three endogenous rice genes in protoplast assays. In contrast, scaffold RNAs (scRNAs) with aptamers at the 3' end or in excessive copy numbers were ineffective. Exploratory AlphaFold modeling supports a possible role for aptamer positioning and MCP-VP64 dimerization, although this remains a working hypothesis. This modular design enables tunable gene regulation in rice protoplasts and provides a practical platform for high-throughput screening and synthetic gene circuit prototyping in plants. Given that scRNA geometry and promoter architecture are universal features of CRISPR-based transcriptional modulation, the system is expected to be broadly portable across species. While the architecture is intended to be compatible with CRISPRi, future studies will be needed to establish its practical use in combined CRISPRa/i settings.},
}
@article {pmid41649281,
year = {2026},
author = {Zhu, Z-J and Cui, M-L and Liu, Y and Yao, X-Q and Lu, M-J and Wang, M-C and Liu, J-H and Li, J-F and Li, E-Z},
title = {CRISPR/Cas14a combined with RPA for visual detection of Marek's disease virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {3},
pages = {e0262525},
pmid = {41649281},
issn = {2165-0497},
support = {22A310017//Key Scientific Research Projects of Universities in Henan/ ; },
mesh = {Animals ; *Marek Disease/diagnosis/virology ; *CRISPR-Cas Systems ; *Herpesvirus 2, Gallid/isolation &amp; purification/genetics ; *Poultry Diseases/virology/diagnosis ; *Nucleic Acid Amplification Techniques/methods ; Chickens/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; Recombinases/metabolism/genetics ; *Mardivirus/isolation &amp; purification/genetics ; },
abstract = {Marek's disease, a highly contagious avian immunosuppressive disorder caused by the α-herpesvirus MDV-1, poses a significant threat to poultry health. The development of rapid visual detection methods capable of distinguishing epidemic MDV-1 strains from vaccine strains is crucial for early disease warning, vaccine efficacy evaluation, and precise disease control. We developed a novel isothermal detection system that integrates recombinase polymerase amplification (RPA) with CRISPR/Cas14a technology for the visual identification of epidemic MDV-1 strains. This method operates at a constant temperature of 37°C and allows for either real-time analysis or endpoint visual readout without the need for complex instrumentation. Our results showed no cross-reactivity with Newcastle disease virus, infectious bursal disease virus, MDV-1 vaccine strains, or herpesvirus of turkeys. Plasmid DNA standards were used to determine the sensitivity of the assay, and the detection limit was 24.6 copies/μL. Clinical evaluation using 24 field samples confirmed that the method successfully identified all Marek's disease virus-positive cases, demonstrating its diagnostic reliability. In conclusion, we have developed a rapid, highly specific nucleic acid detection platform for MDV-1 that enables visual readout without complex instrumentation by combining the sensitivity of RPA with the specificity of CRISPR/Cas14a technology, offering promising potential for field-based diagnostics and disease surveillance.IMPORTANCEMarek's disease virus (MDV-1) is a highly contagious and economically important avian pathogen. Existing diagnostic methods are unable to reliably distinguish between epidemic and vaccine strains in field settings, which hampers effective surveillance and evaluation of vaccination programs. To address this challenge, we developed a portable isothermal detection assay that combines recombinase polymerase amplification with CRISPR/Cas14a technology. This approach enables highly sensitive (24.6 copies/μL) and specific visual detection of epidemic MDV-1 strains without cross-reactivity with vaccine strains or related viruses. The assay demonstrated 100% agreement with reference methods when evaluated using clinical samples. As a cost-effective method that avoids the need for complex detection instruments, it offers a practical solution for rapid on-site diagnosis, facilitating enhanced outbreak control and improved poultry health management globally.},
}
@article {pmid41649621,
year = {2026},
author = {Alavian, F and Ghasemi, S},
title = {CRISPR-Based Therapy for Ischemic Stroke: A Narrative Review.},
journal = {Cellular and molecular neurobiology},
volume = {46},
number = {1},
pages = {45},
pmid = {41649621},
issn = {1573-6830},
mesh = {Humans ; Animals ; *Ischemic Stroke/therapy/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {Ischemic stroke (IS) is one of the most common neurological diseases worldwide and is caused by the blockage of cerebral blood vessels, leading to reduced blood flow and neuronal damage. Given the limitations of existing treatments, CRISPR gene-editing technology has emerged as a promising strategy to precisely target the molecular pathways underlying IS pathophysiology. By enabling intervention in genes regulating inflammation, apoptosis, and repair, CRISPR enables more precise and effective therapies. Various CRISPR delivery systems, including viral vectors, nanocarriers, and extracellular vesicles, play crucial roles in the effective access of this tool to neural cells. Studies have shown that the use of CRISPR-Cas9 to modulate key pathogenic pathways, including those governing inflammation, oxidative stress, and cell death, can prevent neuronal damage and improve neurological function. Additionally, targeting ncRNAs and RNA methylation with CRISPR-based systems plays a role in regulating oxidative stress and stress granule formation. The use of CRISPR to modulate cell communication and organelle transfer and correct mitochondrial mutations has also been considered a neuroprotective mechanism. Despite persistent challenges in targeted and safe delivery, substantial preclinical advances, primarily in rodent models, underscore the potential for CRISPR-based therapies to transform future stroke treatment. These findings suggest that CRISPR-based strategies could evolve into precision neurotherapeutics that address root molecular pathologies, potentially complementing or surpassing current stroke interventions.},
}
@article {pmid41649714,
year = {2024},
author = {Dong, G and Fan, Z},
title = {CRISPR/Cas-mediated germplasm improvement and new strategies for crop protection.},
journal = {Crop health},
volume = {2},
number = {1},
pages = {2},
pmid = {41649714},
issn = {2948-1945},
support = {SYND-2021-03//Administration Bureau of Sanya Yazhou Bay Science and Technology City/ ; CARS-02//China Agricultural Research System of MOF and MARA/ ; },
abstract = {Global agriculture and food security are encountering unprecedented challenges from both the ever-growing population and rapidly changing climate conditions. CRISPR/Cas-mediated genome editing technology has revolutionized plant functional genetic research and precision crop breeding with robustness, high target specificity and programmability. Furthermore, numerous emerging biotechnologies based on the CRISPR/Cas platform provide the opportunity to create new crop germplasms with durable resistance against disease or insect pests, herbicide tolerance, and other stress-tolerant improvements, reshaping crop protection to increase agricultural resilience and sustainability. In this review, we briefly describe the CRISPR/Cas toolbox, including base editing, prime editing, compact genome manipulation, transcriptional regulation and epigenetic editing, and then overview the most important applications of CRISPR/Cas-mediated crop genetic improvement, highlighting crop protection-based stress resistance engineering. In addition, we enumerate global regulations on genome-edited crops. Finally, we discuss some bottlenecks facing this cutting-edge technology and infinite possibilities for the future.},
}
@article {pmid41649900,
year = {2026},
author = {Zakiyyah, SN and Irkham, and Wulandari, DA and Zein, MIHL and Gaffar, S and Ozsoz, M and Ogata, G and Einaga, Y and Hartati, YW},
title = {CRISPR-Cas13a-Powered Electrochemical Biosensor for Label-Free Detection of Chikungunya Virus RNA Using a Ceria-Modified Screen-Printed Carbon Electrode.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {923-938},
doi = {10.1021/acssensors.5c01924},
pmid = {41649900},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods/instrumentation ; *Chikungunya virus/genetics/isolation &amp; purification ; *RNA, Viral/analysis/genetics ; *Cerium/chemistry ; Electrodes ; *Electrochemical Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Carbon/chemistry ; Limit of Detection ; Humans ; },
abstract = {This study aims to develop a label-free electrochemical biosensor for the detection of chikungunya virus (CHIKV) RNA, based on CRISPR-Cas13a integrated with a cerium oxide (ceria)-modified screen-printed carbon electrode (SPCE). The ceria film was deposited through cathodic electrodeposition, forming a uniform, needle-like film, as observed by SEM, and a crystalline fluorite structure was confirmed by XRD with characteristic (111), (200), and (220) reflections. The results showed that Raman spectroscopy demonstrated a dominant F2g band at ∼463 cm[-1], indicative of cubic ceria, while XPS analysis displayed the presence of 13.65% Ce[3+], contributing to favorable surface reactivity for biomolecule immobilization. This interface enhanced the attachment of a biotinylated RNA probe through streptavidin binding. Furthermore, a guide RNA (gRNA) was rationally designed to target the conserved region of the CHIKV E1 gene, with high specificity confirmed through in silico arrangement against related viruses. Upon target recognition, the activated Cas13a enzyme triggered collateral cleavage of the immobilized probe, leading to a measurable reduction in the guanine oxidation signal, detected by differential pulse voltammetry (DPV). This detection strategy was entirely label-free and amplification-free, simplifying both sensor fabrication and operation. The biosensor achieved a detection limit of 1.325 ppt, had a linear response in the range of 1-10,000 ppt, and showed excellent selectivity against DENV and SARS-CoV-2. It also retained signal stability over 45 days and yielded a recovery rate of 94.98% in spiked human serum. In conclusion, this study represents a modular and programmable sensing platform for direct RNA detection that integrates RNA-guided molecular recognition and signal transduction without the need for labeled substrates or amplification, simplifying CRISPR-based diagnostics supporting good health and well-being through field-deployable applications.},
}
@article {pmid41649928,
year = {2026},
author = {Yoon, DE and Yang, J and Jeong, TY and Park, J and Lee, H and Seong, JK and Kim, K},
title = {Protocol for non-invasive delivery of CRISPR RNPs via virus-like particles for mouse model generation.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104352},
pmid = {41649928},
issn = {2666-1667},
mesh = {Animals ; Mice ; *Gene Editing/methods ; *Ribonucleoproteins/genetics ; *CRISPR-Cas Systems/genetics ; Female ; Models, Animal ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-virus-like particle (VLP)-induced targeted mutagenesis (CRISPR-VIM) enables genome editing in mouse embryos through non-invasive delivery of CRISPR ribonucleoproteins (RNPs) via VLPs, eliminating the need for physical manipulation and specialized expertise. We detail protocols for VLP production, titration, and treatment for diverse genome edits. This protocol is compatible with zygotes and in vitro fertilization (IVF)-derived embryos via simple co-culture, facilitating high-efficiency and heritable mutations with minimized off-target effects, independent of specialized equipment and conducive to reduced animal use. For complete details on the use and execution of this protocol, please refer to Jeong et al.[1].},
}
@article {pmid41650957,
year = {2026},
author = {Shi, B and Li, J and Wang, X and Liu, D and Xiang, J and Wang, H and Xu, C and Zou, X and Wang, Z and Huang, T and Min, Q and Wang, K and Yang, Y and Li, J and Wang, B and Zhao, C and Pei, D},
title = {Generating high-quality porcine iPSCs with the new medium cocktail LACID.},
journal = {Stem cell reports},
volume = {21},
number = {3},
pages = {102790},
pmid = {41650957},
issn = {2213-6711},
mesh = {Animals ; *Induced Pluripotent Stem Cells/cytology/metabolism/drug effects ; Swine ; Cellular Reprogramming ; *Culture Media/pharmacology/chemistry ; Blastocyst/cytology/metabolism ; *Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Nuclear Transfer Techniques ; Cells, Cultured ; Cell Differentiation ; },
abstract = {Pigs are important for disease model generation, xenotransplantation, and interspecies organogenesis. Porcine induced pluripotent stem cells (piPSCs) should enable these efforts, but have not been generated to meet the attributes, such as feeder-free culture, robust development potential, and blastocyst generation through nuclear transfer. We report an improved strategy to generate such piPSCs. We show that chemically defined medium 3 promotes the formation of epithelium-like colonies in porcine reprogramming, which allows further reprogramming under the new medium cocktail LACID. The resulting piPSCs have key features, including flat morphology with feeder-free culture, generating robust teratoma and blastoids, forming chimeric blastocysts, and readily edited with CRISPR-Cas9. Lastly, nuclear transfer with piPSCs can develop into blastocysts. Despite maintaining a primed pluripotent state, our results suggest that the newly established LACID piPSCs may be ideal for applications in regenerative medicine. This method may be further improved to generate naive or totipotent stem cells.},
}
@article {pmid41651087,
year = {2026},
author = {Cao, W and Lu, J and Jiang, Y and Yuan, M and Wang, M and Mu, X and Gao, Z and Yang, S and Li, T and Wei, X and Guo, L},
title = {Recent advances in metabolic engineering of purple non‑sulfur photosynthetic bacteria for enhanced biohydrogen production.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108832},
doi = {10.1016/j.biotechadv.2026.108832},
pmid = {41651087},
issn = {1873-1899},
mesh = {*Hydrogen/metabolism ; *Metabolic Engineering/methods ; *Photosynthesis ; },
abstract = {Photo-fermentative hydrogen production from lignocellulosic biomass offers a sustainable and carbon-neutral route for bioenergy conversion, providing an effective strategy to mitigate fossil resource depletion and greenhouse gas emissions. Among diverse microbial candidates, purple non‑sulfur bacteria (PNSB) have emerged as promising photosynthetic platforms owing to their broad substrate utilization, intrinsic tolerance to lignocellulose-derived inhibitors, and high hydrogen yields under light-anaerobic conditions. Their metabolic versatility allows dynamic redistribution of carbon and electron fluxes, facilitating efficient energy conversion. Recent progress in metabolic engineering has substantially advanced the hydrogen-producing capacity of PNSB through targeted manipulation of photosynthetic metabolism, redox balance and stress response pathways. Engineering strategies have focused on optimizing pigment biosynthesis to enhance light harvesting, reinforcing redox homeostasis and adenosine triphosphate (ATP) generation, and improving tolerance to environmental stresses such as ammonia, pH, and temperature fluctuations. These efforts have led to engineered strains exhibiting extraordinary improvement in hydrogen yield, stability and robustness. This review provides an overview of the fundamental mechanisms underlying photo-fermentative hydrogen metabolism in PNSB, summarizes recent advances in the metabolic and systems-level engineering strategies, and outlines the prospects of developing strains capable of approaching the theoretical limit of hydrogen yield through integrated engineering strategies, advanced tools such as CRISPR-Cas, and adaptive laboratory evolution methods.},
}
@article {pmid41651998,
year = {2026},
author = {Natsagdorj, ME and Hara, H and Uosaki, H and Nakahara, F and Inoue, M and Hanazono, Y},
title = {ATM Inhibition Enhances Knock-in Efficiency by Suppressing AAV-Induced Activation of Apoptotic Pathways.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {177},
pmid = {41651998},
issn = {2399-3642},
support = {JP23bm1123020//Japan Agency for Medical Research and Development (AMED)/ ; JP22bm0804018//Japan Agency for Medical Research and Development (AMED)/ ; JP23bm1123020//Japan Agency for Medical Research and Development (AMED)/ ; JP18am0301002//Japan Agency for Medical Research and Development (AMED)/ ; JP22ae0201007//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {*Ataxia Telangiectasia Mutated Proteins/antagonists &amp; inhibitors/genetics/metabolism ; *Dependovirus/genetics ; Animals ; Mice ; *Apoptosis/genetics ; *Gene Knock-In Techniques/methods ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Morpholines/pharmacology ; Gene Editing/methods ; Mouse Embryonic Stem Cells/metabolism ; Thioxanthenes ; },
abstract = {CRISPR/Cas9-mediated genome editing has expanded the possibilities for precise gene modifications; however, the efficiency of targeted insertion remains suboptimal. In this study, we describe a triple-reporter system in mouse embryonic stem cells that simultaneously tracks double-strand break (DSB) induction, homology-directed repair (knock-in), and end-joining-mediated targeted insertion (EJ-TI). Using both plasmid and adeno-associated virus (AAV) donor vectors, our results demonstrate that ataxia telangiectasia and Rad3-related kinase (ATR) activity is essential for knock-in regardless of the donor type, whereas ataxia telangiectasia mutated (ATM) inhibition exhibits a donor-dependent role. In cells receiving circular plasmid donors, ATM inhibition with AZD1390 markedly reduced the knock-in and EJ-TI efficiencies, consistent with its canonical role in DSB repair. In contrast, with linear AAV donors, ATM inhibition enhanced the knock-in efficiency by suppressing the overactivation of the ATM-p53-caspase 3 apoptotic pathway and partially suppressing classical non-homologous end-joining. These findings highlight the critical influence of donor DNA configuration on DNA damage response signaling and provide a strategy for optimizing genome editing efficiency by selectively modulating the ATM pathways, an approach that may have significant implications for gene therapy, cell engineering, and other applications.},
}
@article {pmid41652090,
year = {2026},
author = {Pan, X and Yildiz, U and Armstrong, SK and Bissonnette, K},
title = {Status and advancement of root-knot nematode management strategies and the emerging CRISPR/Cas biotechnology application.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {38},
pmid = {41652090},
issn = {1438-7948},
mesh = {*CRISPR-Cas Systems ; Animals ; *Plant Diseases/parasitology/genetics/prevention &amp; control ; Gene Editing ; *Tylenchoidea/pathogenicity/genetics ; Crops, Agricultural/genetics/parasitology ; Disease Resistance/genetics ; Plant Roots/parasitology/genetics ; Gossypium/parasitology/genetics ; },
abstract = {Root-knot nematodes (RKNs), Meloidogyne spp., exhibit a broad host range, threatening more than 3000 species of plants, including agriculturally important crops such as cotton (Gossypium hirsutum), tomato (Lycopersicon esculentum) and rice (Oryza sativa). Among the over 90 RKN species, the four most prevalent are M. incognita, M. arenaria, M. javanica, and M. hapla, with M. incognita being the most damaging. This paper reviewed the current RKN management strategies, including chemical nematicides, biological control, crop rotation, and resistant varieties, with a focus on the application of the revolutionary CRISPR/Cas genome editing tool in developing RKN resistance in plants. CRISPR/Cas has been widely utilized for improving crop traits due to its specificity, streamline, and inheritability. Recent progress has demonstrated the simplicity and robustness of CRISPR/Cas technology in improving plant traits. Among these, the development of nematode resistance by CRISPR/Cas knocking out of plant compatibility factors in model and commercial plants, has achieved significant progress. This review summarizes the RKN parasitism mechanisms and plant compatibility factors that would be promising CRISPR/Cas targets. The fundamentals and key aspects of CRISPR/Cas genome editing technology are addressed and discussed, and an example experimental pipeline for developing nematode resistance in cotton is described.},
}
@article {pmid41652115,
year = {2026},
author = {Omelchenko, DO and Barkovskaya, AM and Omelchenko, LV and Klepikova, AV and Penin, AA and Logacheva, MD},
title = {Optimization of functional genetics tools for a model tetraploid Capsella bursa-pastoris, with focus on homoeolog-aware gene editing.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {6},
pmid = {41652115},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Capsella/genetics/growth &amp; development ; Tetraploidy ; *Plants, Genetically Modified/genetics/growth &amp; development ; Genome, Plant/genetics ; Agrobacterium tumefaciens/genetics ; CRISPR-Cas Systems ; },
abstract = {Capsella bursa-pastoris is a recent allotetraploid and a promising model for studying early consequences of polyploidy. One of the intriguing questions in polyploid research is how new functions arise from initially identical or nearly identical homoeologous genes. Functional genetics tools, including genetic editing, can help to understand this process, but they have not been developed for C. bursa-pastoris yet. We present here the results of our study aimed at filling this gap. In particular, we compared the efficiency of floral dip transformation in six accessions of C. bursa-pastoris representing distant populations. The Asian clade accession PGL0025 had the highest efficiency of transformation (~ 1.1%). Comparison of Agrobacterium tumefaciens strains EHA105 and GV3101 (pMP90) showed that the latter is more effective. Also, we created a genome-wide gRNA database for all pairs of homoeologs of the PGL0001 accession of C. bursa-pastoris and integrated it into publicly available genome browser: https://t2e.online/igv_capsella_bursa-pastoris/ . We assessed the possibility of differential editing for two pairs of homoeologous genes with high sequence similarity (> 90%) both in vitro and in silico. Despite the test results that indicated off-target activity, we have succeeded in obtaining lines of plants with homozygous frameshift mutations in each of the homoeologs separately in vivo. We expect that these findings and resources will promote the use of C. bursa-pastoris as a model in functional genetics experiments, in particular, the studies of the fate of duplicated gene after polyploidization event.},
}
@article {pmid41652143,
year = {2026},
author = {Fu, T and Zhao, D and Wang, Q and Peng, N and Zhang, L and Chen, S and Ren, J and Tang, F and Pu, K and Wu, Q and Bi, C and Zhang, X and Li, Q},
title = {A rapid and ultrasensitive CRISPR-Cas12a assay for clinical detection of pathogens and mutations.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {8},
pages = {2455-2469},
pmid = {41652143},
issn = {1618-2650},
mesh = {*CRISPR-Cas Systems ; Humans ; *Mutation ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; Isocitrate Dehydrogenase/genetics ; Glioma/genetics/diagnosis ; DNA/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-Cas12a-driven nucleic acid diagnostics offer considerable potential for highly specific and rapid detection. However, their practical applications are limited by the necessity for pre-amplification of target DNA to enhance sensitivity. To overcome this limitation, we developed Auto-catalyst, a novel one-pot, amplification-free DNA detection platform employing a two-stage autocatalytic Cas12a cascade. This approach integrates a positive feedback amplification mediated by a circular crRNA-DNA nanostructure with an asymmetric CRISPR reaction driven by split crRNA. Without external amplification, this system detects DNA fragments at concentrations as low as 80 aM within 30 min at room temperature and maintains high specificity, accurately distinguishing single-base mutations down to 1 fM. Clinical validation demonstrated successful detection of pathogenic DNA in cerebrospinal fluid samples from patients with intracranial infections, highlighting its potential for rapid bedside diagnostics essential for timely clinical decision-making. Additionally, Auto-catalyst accurately identified the clinically significant isocitrate dehydrogenase 1 (IDH1) gene R132H mutation from glioma tissue samples. This integrated two-stage autocatalytic Cas12a strategy represents a powerful, convenient, and promising diagnostic tool suitable for point-of-care applications.},
}
@article {pmid41652223,
year = {2026},
author = {Zhang, J and Zhang, J and Tang, S and Liu, C and Cai, Y and Zeng, H and Meng, X and Liu, B and Zhang, Y and Wang, Y},
title = {Single-round evolution of RNA aptamers with GRAPE-LM.},
journal = {Nature biotechnology},
volume = {},
number = {},
pages = {},
pmid = {41652223},
issn = {1546-1696},
support = {62302311//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82273967//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M742397//China Postdoctoral Science Foundation/ ; 2024T170585//China Postdoctoral Science Foundation/ ; },
abstract = {The directed evolution of biomolecules is an iterative process. Although advancements in language models have expedited protein evolution, effectively evolving RNA remains a challenge. RNA aptamers, selected for their binding properties, provide an ideal system to address this challenge, yet traditional aptamer discovery still relies on labor-intensive, multi-round screening. Here we introduce GRAPE-LM (generator of RNA aptamers powered by activity-guided evolution and language model), a generative artificial intelligence framework designed for the one-round evolution of RNA aptamers. GRAPE-LM integrates a transformer-based conditional autoencoder with nucleic acid language models and is guided by CRISPR-Cas-based aptamer screening data derived from intracellular environments. We validate GRAPE-LM on three disparate targets: the human T cell receptor CD3ε, the receptor-binding domain of the SARS-CoV-2 spike protein and the human oncogenic transcription factor c-Myc (an intracellular disordered protein). GRAPE-LM, informed with only a single round of CRISPR-Cas-based screening, successfully obtains RNA aptamers that outperform those driven from multiple rounds of human selection and optimization.},
}
@article {pmid41652229,
year = {2026},
author = {Xu, X and Fang, J and Chen, J and Yang, J and Yang, X and Hou, S and Sun, W and Champer, J},
title = {Assessing target genes for homing suppression gene drive.},
journal = {The EMBO journal},
volume = {45},
number = {6},
pages = {2074-2094},
pmid = {41652229},
issn = {1460-2075},
support = {32302455//MOST | National Natural Science Foundation of China (NSFC)/ ; 32270672//MOST | National Natural Science Foundation of China (NSFC)/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; Female ; *Gene Drive Technology/methods ; Male ; *Fertility/genetics ; CRISPR-Cas Systems ; Drosophila Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Gene drives are engineered alleles that bias their own inheritance in offspring, enabling the spread of specific traits throughout a population. Targeting female fertility genes in a gene drive can be an efficient strategy for population suppression. In this study, we investigated nine female fertility genes in Drosophila melanogaster using CRISPR-based homing gene drives. Employing a multiplexed gRNA approach to prevent the formation of functional resistance alleles, we aimed to maintain high drive-conversion efficiency with low fitness costs in female drive-carriers. Drive efficiency was assessed in individual crosses and had varied performance across different target genes. Notably, drives targeting the octopamine β2 receptor (oct) and stall (stl) genes exhibited the highest drive-conversion rates and were further tested in cages. A drive targeting stl successfully suppressed a cage population with a high release frequency, though suppression failed in another replicate cage with a lower initial release frequency. Fitness costs in female drive carriers were observed in test cages, impacting the overall efficiency of population suppression. Further tests on the fertility of these lines using individual crosses indicated that some fitness costs were due to maternal deposition of Cas9 combined with new gRNA expression, which would only occur in progeny of drive males when testing split drives with separate Cas9 (when mimicking cages with complete drives) but not for complete drive systems. This could enable success in complete drives with higher maternal Cas9 deposition, even if cage experiments in split drives fail. Overall, our findings identify oct and stl as promising fertility targets and demonstrate both the potential and the constraints of fertility-based suppression drives, providing empirical evidence to guide the design and assessment of more efficient population control strategies.},
}
@article {pmid41652748,
year = {2026},
author = {Lei, Y and Ma, W and Wang, L and Chen, Y and Chen, X},
title = {Genomic insights into prophage and CRISPR-Cas system present in Lactobacillus delbrueckii subsp. bulgaricus strains.},
journal = {Food research international (Ottawa, Ont.)},
volume = {227},
number = {},
pages = {118213},
doi = {10.1016/j.foodres.2025.118213},
pmid = {41652748},
issn = {1873-7145},
mesh = {*Prophages/genetics ; *Lactobacillus delbrueckii/virology/genetics ; *CRISPR-Cas Systems/genetics ; Genomics ; Yogurt/microbiology ; Base Composition ; Genome, Bacterial ; Genome, Viral ; },
abstract = {As a key starter culture for yogurt fermentation, Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) was subjected to bioinformatics analysis to investigate the distribution of prophages and the structure of the CRISPR-Cas system in 119 L. bulgaricus strains, and explored the targeting relationship between them and annotated the functional genes of targeted prophages. A total of 1704 prophage fragments were identified, of which 8.74 % (149/1704) were classified as complete prophages, none of them carried virulence factor genes and antibiotic resistance genes. Among all complete prophage genomes, the GC content was significantly affected by geographical sources (Asia, Europe, and North America) of host bacteria (P < 0.01). The 123 CRISPR-Cas systems detected in L. bulgaricus, type I-E (69) and type II-A (40) systems were predominant. Spacers-prophages targeting analysis revealed a negative correlation (P < 0.05), with 23.43 % (1055/4503) spacers showing homology to prophages. Functional gene annotation revealed that the diversity of prophage functional genes showed significant differences in under different grouping conditions (P < 0.05). Nevertheless, in-depth analysis of targeted prophage genomes revealed a conserved functional gene--ABC transporter. This study provides data support for exploring the genomic diversity and adaptive evolution mechanism of prophages in L. bulgaricus, and providing insights for screening phage-resistant strains and developing novel anti-phage strategies in dairy industry.},
}
@article {pmid41653514,
year = {2026},
author = {Li, M and He, L and Wang, Z and Wang, L and Pan, Q and Sun, P and Li, D and Zhang, C},
title = {Amplified ferroptosis and immunomodulation triggered by NIR-II photothermal-controllable CRISPR/Cas9 nanoplatform to treat osteosarcoma and prevent postsurgical implant-associated infections.},
journal = {Biomaterials},
volume = {330},
number = {},
pages = {124043},
doi = {10.1016/j.biomaterials.2026.124043},
pmid = {41653514},
issn = {1878-5905},
mesh = {*Ferroptosis/drug effects ; Humans ; Animals ; *CRISPR-Cas Systems ; *Osteosarcoma/therapy/immunology ; *Immunomodulation ; Mice ; Cell Line, Tumor ; Infrared Rays ; HSP70 Heat-Shock Proteins/genetics ; NF-E2-Related Factor 2/genetics/metabolism ; },
abstract = {Ferroptosis has been proven as a promising therapeutic approach with immunomodulatory effect; however, intracellular antioxidant system maintains redox balance and diminishes its efficacy. Nuclear factor erythroid 2-related factor 2 (Nrf2) is identified as a central transcription factor for regulating oxidative homeostasis. Herein, we have developed a thermal-controllable genome-editing nanoplatform BF/pHCN. Specifically, a CRISPR/Cas9 plasmid with an upstream HSP70 promoter sequence (HSP70-Cas9-sgNrf2, named pHCN) was constructed. Subsequent Fe(II) and pHCN were co-loaded into organic small-molecule BTP with near infrared II (NIR-II) absorption and coated with DSPE-mPEG2000. The generated BF/pHCN (BTP@Fe/pHCN) could be internalized within osteosarcoma cells. Subsequent NIR-II laser-triggered hyperthermia at 42 °C activated the HSP70 promoter and facilitated the precise inhibition targeting Nrf2 genomic sequences while promoting Fe(II) release, ultimately disrupting oxidative stress states. Moreover, the amplified ferroptosis fully triggered immunogenic cell death, thus reprogramming macrophages, promoting maturation of dendritic cells, and activating cellular antitumoral immunity. Additionally, BF/pHCN exhibited direct bactericidal activity against planktonic bacteria, and effectively eliminated intracellular bacteria through iron metabolic disorders strategy targeting macrophages, thereby initiating adaptive antimicrobial immunity. Overall, our NIR-II thermal-controllable genome-editing nanoplatform BF/pHCN exhibits remarkable antitumoral properties alongside robust antiinfection and immunomodulation, providing feasible strategies toward effective management of osteosarcoma, and preventing postsurgical implant-associated infections.},
}
@article {pmid41653624,
year = {2026},
author = {Sun, Y and Demachi-Okamura, A and Shinohara, S and Wang, Y and Guo, Z and Yamaguchi, R and Matsushita, H and Nabekura, T and Muraoka, D},
title = {Dual knockout of Fas and TCRα in Jurkat reporter cells enables highly sensitive identification of antigen-specific TCRs.},
journal = {Biochemical and biophysical research communications},
volume = {805},
number = {},
pages = {153400},
doi = {10.1016/j.bbrc.2026.153400},
pmid = {41653624},
issn = {1090-2104},
mesh = {Humans ; Jurkat Cells ; *fas Receptor/genetics/immunology ; CRISPR-Cas Systems ; *Gene Knockout Techniques ; CD8-Positive T-Lymphocytes/immunology ; Lung Neoplasms/immunology/genetics ; *Receptors, Antigen, T-Cell, alpha-beta/genetics/immunology ; *Receptors, Antigen, T-Cell/genetics ; },
abstract = {T-cell receptors (TCRs) that target tumor antigens are crucial for antitumor immunity; however, tumor-specific TCRs often exhibit low affinity for their cognate antigens, making the identification of functional TCRs challenging due to the limited sensitivity of current detection methods. In this study, we established a high-sensitivity TCR screening platform by generating Jurkat cell reporter clones with dual knockout (DKO) of endogenous Fas and TCRα via CRISPR-Cas9 system. In a viral antigen model system, these DKO Jurkat cells exhibited approximately 100-fold greater sensitivity to antigen stimulation compared with parental Jurkat cells. Notably, our DKO Jurkat-based platform enabled the identification of tumor-specific CD8[+] T cells from a lung cancer patient that could not be detected using parental Jurkat cells. Moreover, the identified tumor-specific T-cell clone exhibited a unique phenotype characterized by robust cytotoxic T lymphocyte (CTL) activity and natural killer-like properties. Together, these findings demonstrate that dual deletion of Fas and TCRα in Jurkat cells enables highly sensitive functional TCR screening. Integration of this platform with single-cell analysis facilitates the discovery of previously uncharacterized tumor-reactive TCRs and provides a powerful tool for advancing TCR-based cancer immunotherapy.},
}
@article {pmid41653634,
year = {2026},
author = {Jung, KM and Klein, R and Mony, SI and Chen, PR and Lee, K and Lee, HJ},
title = {Highly efficient gene editing via targeted Cas9 insertion into chicken housekeeping gene.},
journal = {Poultry science},
volume = {105},
number = {4},
pages = {106585},
pmid = {41653634},
issn = {1525-3171},
mesh = {Animals ; *Chickens/genetics ; *Gene Editing/veterinary/methods ; *Genes, Essential ; *CRISPR-Cas Systems ; Animals, Genetically Modified/genetics ; Transgenes ; *Avian Proteins/genetics/metabolism ; Cell Line ; Mutagenesis, Insertional ; },
abstract = {Achieving stable and efficient transgene expression is a key challenge in advancing avian genome engineering. Although viral vector-based and piggyBac-mediated transgenesis have been widely used in chickens, both approaches are prone to epigenetic silencing, leading to inconsistent, tissue-specific, and often diminished expression over time. This variability limits used of transgenes requiring robust and long-term expression across multiple tissues. In mammals, site-specific integration into genomic safe harbor loci, such as Rosa26, has enabled stable and predictable transgene expression without disrupting endogenous gene function; however, such strategy has not been established in birds. In this research, we hypothesized that integrating Cas9 into endogenous housekeeping genes (the ACTB and GAPDH) could achieve efficient gene editing in chickens through stable and ubiquitous transgene expression. Using two different approaches, 3'-targeted gene insertion and gene tagging, we inserted Cas9 and GFP cassettes into defined genomic loci in chicken DF-1 cells. Both approaches exhibited stable expression of transgenes in the cells, and functional assays confirmed that Cas9 showed highly efficient nuclease activity following guide RNA delivery. Additionally, we derived single-cell clones stably expressing Cas9, enabling uniform and reproducible genome editing in downstream applications. Targeted insertion of transgenes into active housekeeping genes as candidate safe harbor loci mitigates the limitations of random integration and promoter silencing, offering a robust platform for consistent transgene expression in poultry biotechnology and genome engineering.},
}
@article {pmid41653672,
year = {2026},
author = {Bai, T and Qu, X and Pan, J and Tang, Y and Wang, L and Zhou, P and Hu, Z and Guo, Z and Zhu, Y and Zhang, Y},
title = {CRISPR-initiated exponential amplification on fluorescently-barcoded microspheres for deep learning-assisted multiplexed HPV detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {300},
number = {},
pages = {118488},
doi = {10.1016/j.bios.2026.118488},
pmid = {41653672},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/methods ; Deep Learning ; *CRISPR-Cas Systems/genetics ; Microspheres ; *Papillomavirus Infections/virology/diagnosis ; Nucleic Acid Amplification Techniques/methods ; *DNA, Viral/genetics/isolation &amp; purification ; *Papillomaviridae/isolation &amp; purification/genetics ; Limit of Detection ; Quantum Dots/chemistry ; Fluorescence ; Human papillomavirus 16/isolation &amp; purification/genetics ; },
abstract = {Rapid, low-cost, and multiplexed nucleic acid testing is essential for human health but remains challenging. Although CRISPR-Cas systems offer high specificity, their integration into Multiplexed platforms suitable for near-patient testing has been limited. Here, we introduce a biosensing platform that combines CRISPR-initiated enzymatic amplification with quantum-dot encoded microbeads and deep-learning based image analysis for Multiplexed detection of human papillomavirus (HPV) DNA. The high specificity of CRISPR/Cas9 first triggers an exponential amplification. The products are then specifically captured on the microbead surface for localized fluorescent readout. A custom deep-learning algorithm automatically quantifies the bead fluorescence, enabling robust and automated analysis. The integrated approach achieves simultaneous detection of HPV16, HPV18, and HPV33 with detection limits as low as 0.2 pM. By using recognition-triggered amplification and a simple deep-learning assisted fluorescence readout, the workflow is significantly simplified. The platform establishes a universal and practical strategy for molecular diagnostics, demonstrating strong potential for near-patient testing.},
}
@article {pmid41653914,
year = {2026},
author = {Chandrasekaran, SS and Tau, C and Fu, BXH and Nemeth, M and Bartie, L and Pawluk, A and Konermann, S and Hsu, PD},
title = {Rewriting endogenous human transcripts with dual CRISPR-guided 3' trans-splicing.},
journal = {Cell systems},
volume = {17},
number = {2},
pages = {101487},
doi = {10.1016/j.cels.2025.101487},
pmid = {41653914},
issn = {2405-4720},
mesh = {Humans ; *Trans-Splicing/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; RNA, Messenger/genetics ; RNA Editing/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; HEK293 Cells ; RNA Precursors/genetics ; Exons/genetics ; },
abstract = {Unlike genome editing, RNA editing offers the ability to transiently alter cells with minimal risk from off-target effects. While exon-skipping technologies can influence splice site selection, many desired perturbations to the transcriptome require replacement or addition of exogenous exons to target mRNAs, such as replacing disease-causing exons, repairing truncated proteins, or engineering protein fusions. Here, we report the development of RNA-guided trans-splicing with Cas editor (RESPLICE). RESPLICE uses two orthogonal RNA-targeting CRISPR effectors to co-localize a trans-splicing pre-mRNA and to inhibit the cis-splicing reaction, respectively. We demonstrate efficient, specific, and programmable trans-splicing of RNA cargo (up to 2.1 kb) into 11 endogenous transcripts across 3 cell types, achieving up to 45% trans-splicing efficiency in bulk or 90% when sorting for high effector expression. Our results present RESPLICE as a mode of RNA editing that could provide fine-tuned and transient control of cellular programs.},
}
@article {pmid41654238,
year = {2026},
author = {Jo, SJ and Park, SC and Kim, SG},
title = {CRISPR/Cas9-engineered Salmonella phage displaying antimicrobial peptide LL37 for enhanced antibacterial activity.},
journal = {International journal of antimicrobial agents},
volume = {67},
number = {4},
pages = {107734},
doi = {10.1016/j.ijantimicag.2026.107734},
pmid = {41654238},
issn = {1872-7913},
mesh = {Animals ; *Salmonella typhimurium/virology/drug effects ; *Cathelicidins/genetics/pharmacology ; *CRISPR-Cas Systems ; *Salmonella Phages/genetics ; *Anti-Bacterial Agents/pharmacology ; Moths/microbiology ; *Antimicrobial Cationic Peptides/genetics/pharmacology ; Epithelial Cells/microbiology ; Humans ; Larva/microbiology ; Phage Therapy ; *Antimicrobial Peptides/genetics/pharmacology ; Salmonella Infections/therapy ; Gene Editing ; },
abstract = {OBJECTIVES: The increasing prevalence of antibiotic-resistant Salmonella Typhimurium has highlighted the urgent need for alternative therapeutic strategies. This study engineered a lytic S. Typhimurium bacteriophage to present the antimicrobial peptide LL-37 on the virion surface, followed by evaluation of its enhanced antibacterial efficacy.<br><br>METHODS: A recombinant lytic bacteriophage displaying LL-37 on its capsid was constructed using CRISPR/Cas9-mediated genome editing. The engineered phage was characterized for structural stability, adsorption kinetics, and lytic activity. Antibacterial efficacy was evaluated through bacterial growth inhibition assays, assessment of phage resistance rates, and host-pathogen interaction studies using intestinal epithelial cells. Intracellular bacterial survival was assessed in vitro, and prophylactic efficacy was further examined in a Galleria mellonella infection model.<br><br>RESULTS: The engineered phage exhibited thermal and pH stability comparable to that of the wild-type phage, while demonstrating enhanced adsorption efficiency. In a cell lysis assay, the engineered phage sustained bacterial suppression for 24-72 h, whereas the wild-type phage permitted bacterial regrowth due to the emergence of phage-resistant mutants. The engineered phage significantly reduced bacterial attachment and intracellular survival in an intestinal epithelial cell model. Furthermore, it improved larval survival in a Galleria mellonella infection model in a dose-dependent manner, without inducing significant cytotoxicity.<br><br>CONCLUSIONS: LL-37-engineered bacteriophages demonstrated improved antibacterial activity and intracellular infection control against S. Typhimurium. These findings support antimicrobial peptide-armed phages as a promising strategy for enhancing phage therapy and mitigating antibiotic-resistant bacterial infections.},
}
@article {pmid41654255,
year = {2026},
author = {Saini, H and Yadav, J and Pandey, S and Kumar, A and Nanda, D and Sachan, S and Kaushik, JJ},
title = {Field-deployable CRISPR-cas variants for rapid on-site detection of plant pathogens.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {365},
number = {},
pages = {113028},
doi = {10.1016/j.plantsci.2026.113028},
pmid = {41654255},
issn = {1873-2259},
mesh = {*CRISPR-Cas Systems/genetics ; *Plant Diseases/virology/microbiology ; Biosensing Techniques/methods ; *Plants/microbiology/virology ; RNA Viruses/genetics/isolation &amp; purification ; },
abstract = {Rapid, field-deployable diagnostics are essential for effective plant disease management. Although CRISPR-Cas systems offer high sensitivity and programmability, their use in on-site plant pathogen detection has been hindered by the lack of standardized, practical workflows. Here we present implementable CRISPR-Cas diagnostic protocols using Cas12a, Cas13a, and miniature Cas variants for rapid detection of major plant pathogens. Three field-ready assays are described: (i) an RPA-Cas12a lateral-flow test for DNA pathogens, (ii) a Cas13a RT-RPA assay for RNA viruses, and (iii) an amplification-free Cas12a electrochemical biosensor suited for portable laboratories. Each protocol includes sample preparation steps, reagent formulations, incubation conditions, and troubleshooting guidance. Across platforms, detection limits of 1-100 copies µL[-1] were achieved within 20-45 min, demonstrating analytical sensitivity comparable to conventional PCR-based diagnostics while offering substantially reduced assay time and improved field deployability. We also address practical constraints including sample inhibitors, reagent stability, and biosafety and propose solutions for field implementation. These standardized workflows translate recent advances in CRISPR diagnostics into reproducible, field-deployable tools for plant health surveillance and rapid disease detection.},
}
@article {pmid41654278,
year = {2026},
author = {Wang, X and You, J and Li, X and Xu, Y and Li, Z and Wang, L},
title = {Limitations of traditional mycotoxin control and biotechnological advances toward sustainable solutions.},
journal = {Biotechnology advances},
volume = {88},
number = {},
pages = {108836},
doi = {10.1016/j.biotechadv.2026.108836},
pmid = {41654278},
issn = {1873-1899},
mesh = {*Biotechnology/methods ; *Mycotoxins/metabolism ; Fungi/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Food Contamination/prevention &amp; control ; },
abstract = {Mycotoxins are harmful fungal metabolites that contaminate food and feed, posing serious health and economic risks worldwide. Traditional control methods often fall short due to inefficiency and safety concerns, prompting the development of innovative biotechnological approaches. This review explores recent advances in mycotoxin management, focusing on engineered microbes for targeted degradation, nanotechnology-based detection and removal systems, phage therapy targeting toxin-producing fungi, CRISPR-Cas gene editing of mycotoxin biosynthesis pathways, and plant-microbe interactions that suppress fungal growth. Additionally, enzyme immobilization strategies are highlighted for improving enzyme stability and reusability in detoxification processes. These integrated biotechnological tools offer promising, sustainable solutions to mitigate mycotoxin contamination, enhancing food safety and agricultural productivity. The review also discusses current challenges and future directions for translating these advances into practical applications. Highlighting that biotechnological tools are technically feasible and increasingly close to industrial deployment in food and feed chains.},
}
@article {pmid41654520,
year = {2026},
author = {Zhang, J and Hołubowicz, R and Smidak, R and Hu, Y and Du, SW and Felgner, JH and Palczewska, G and Rodrigues Menezes, C and Risaliti, E and Dong, Z and Ma, X and Shayegan, MH and Chen, PZ and Xing, L and Hołubowicz, M and Li, B and Liu, DR and Felgner, PL and Tochtrop, GP and Palczewski, K},
title = {A combinatorial synthetic strategy for developing genome-editing protein-delivery agents targeting mouse retina.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41654520},
issn = {2041-1723},
support = {T32GM008620; F30EY033642//U.S. Department of Health &amp; Human Services | NIH | National Eye Institute (NEI)/ ; R01EY009339; R01EY030873, P30EY034070//U.S. Department of Health &amp; Human Services | NIH | National Eye Institute (NEI)/ ; CHE-1904530//National Science Foundation (NSF)/ ; P30CA062203//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; P30 CA062203/CA/NCI NIH HHS/United States ; //Knights Templar Eye Foundation/ ; //University of California, Irvine School of Medicine/ ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {Animals ; *Gene Editing/methods ; Liposomes/chemistry ; Mice ; CRISPR-Cas Systems/genetics ; *Retina/metabolism ; Ribonucleoproteins/genetics/metabolism/administration &amp; dosage ; Disease Models, Animal ; Rosaniline Dyes/chemistry ; Retinal Pigment Epithelium/metabolism ; },
abstract = {CRISPR/Cas9-based gene-editing technologies offer promise for treating inherited retinal diseases (IRDs), however safe and efficient ocular delivery of precision editors remains challenging. To address this challenge, we report a class of Coomassie brilliant blue (CBB)-derived lipidoids that bind and deliver proteins. Subretinal injection of Cre complexed with these lipidoids into mT/mG mice leads to robust recombination in the retinal pigment epithelium and photoreceptors. We employ the CBB-lipidoid platform to deliver adenine base editor (ABE) ribonucleoproteins (RNP). Incorporating CBB lipidoids into liposomes improves delivery efficiency. CBB11 stands out for facilitating precise in vivo ABE-mediated gene editing. Delivery of liposome-CBB11-RNP complexes results in a 120-fold increase in base editing compared to RNP alone and restores the scotopic ERG b-wave response in the rd12 mouse model. These results demonstrate the potential of CBB-augmented, liposome-RNP systems for therapeutic gene editing in the eye, paving the way for single-dose precision medicines to treat IRDs.},
}
@article {pmid41655242,
year = {2026},
author = {Liu, J and He, N and Cheng, Z and Lin, S and Fu, S and Wang, M and Huang, F and Abou-Elwafa, SF and Al Aboud, NM and Huang, C and Yang, D},
title = {Genetic identification of Pid3-1 and its regulatory role in promoting blast resistance in rice.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {4},
pages = {},
pmid = {41655242},
issn = {2160-1836},
support = {2023R1021006//Special Fund for Agro-scientific Research in the Public Interest of Fujian Province/ ; 2024R1022001//Special Fund for Agro-scientific Research in the Public Interest of Fujian Province/ ; CSTB2022TIAD-KPX0018//Chongqing Technology Innovation and Application Development Special Key Project/ ; 32402387//National Natural Science Foundation of China/ ; GJYS202302//National Natural Science Foundation Project Extension Research Project/ ; XTCXGC2021001//5511 Collaborative Engineering Project/ ; },
mesh = {*Oryza/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; *Plant Proteins/genetics/chemistry/metabolism ; Magnaporthe/pathogenicity ; Chromosome Mapping ; Phenotype ; Genes, Plant ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; Alleles ; Ascomycota ; },
abstract = {Rice blast is a destructive rice disease caused by the fungus Magnaporthe oryzae. Here, we identified a resistance gene from the rice cultivar Wanhui 66 which is resistant to the rice blast Guy11 isolate. Genetic mapping positioned a blast resistance locus to chromosome 6. Employing map-based cloning approach ultimately mapped the novel blast resistance locus to a genomic region of 117 kb that contains the Pid3 gene. Candidate gene prediction and cDNA sequencing indicate that the target resistance gene in the Wanhui 66 is allelic to Pid3, thus it was designated Pid3-1. Further analysis showed that the Pid3-1 has 3 nucleotide substitutions, resulting in 3 amino acid substitutions in the Pid3-1 protein, which significantly affect the structure of the Pid3-1 protein as indicated by the 3D structure simulation. The CRISPR/Cas9 system was employed to generate a Pid3-1 knockout mutants that confirmed that the Wanhui 66 resistant phenotype is controlled by Pid3-1. A molecular marker, Indel-6-34, cosegregates with Pid3-1 was identified that could have a great impact on rice breeding against blast disease resistance.},
}
@article {pmid41655494,
year = {2026},
author = {Kantor, I and Wright, JL and Amor, DJ and Lockhart, PJ},
title = {Generation of two tetracycline-inducible NGN2 iN iPSC lines carrying a heterozygous floating-Harbor syndrome SRCAP truncating mutation.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103922},
doi = {10.1016/j.scr.2026.103922},
pmid = {41655494},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology/drug effects ; Humans ; *Nerve Tissue Proteins/genetics/metabolism ; *Mutation/genetics ; *Tetracycline/pharmacology ; Heterozygote ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; },
abstract = {Floating-Harbor syndrome (FHS) is a rare neurodevelopmental disorder caused by truncating variants in the last two exons of the gene encoding the chromatin remodeler SRCAP. We used CRISPR-Cas9 genome editing to introduce a monoallelic c.7330C > T (p.Arg2444*) truncating mutation into a published WTC11 iPSC line containing a tetracycline-inducible NGN2 transgene. We characterised two independent lines that maintained a normal karyotype, pluripotency and the ability to differentiate in vitro into all three embryonic germ layers. These lines can be rapidly differentiated into cortical neurons through the addition of doxycycline, making them a useful model for understanding the pathogenic mechanisms underlying FHS.},
}
@article {pmid41655694,
year = {2026},
author = {Saito, A and Tankou, S and Ishii, K and Sakao-Suzuki, M and Oh, EC and Murdoch, H and Namkung, H and Adelakun, S and Furukori, K and Fujimuro, M and Salomoni, P and Maul, GG and Hayward, GS and Tang, Q and Yolken, RH and Houslay, MD and Katsanis, N and Kosugi, I and Yang, K and Kamiya, A and Ishizuka, K and Sawa, A},
title = {Cytomegalovirus-encoded immediate early 1 protein perturbs neural progenitor proliferation via interfering with host PML-DISC1 interaction.},
journal = {The Journal of biological chemistry},
volume = {302},
number = {3},
pages = {111269},
pmid = {41655694},
issn = {1083-351X},
mesh = {*Neural Stem Cells/metabolism/pathology/virology/cytology ; Humans ; Cell Proliferation ; *Nerve Tissue Proteins/metabolism/genetics ; *Promyelocytic Leukemia Protein/metabolism/genetics ; *Cytomegalovirus/metabolism/genetics ; *Immediate-Early Proteins/metabolism/genetics ; Animals ; *Cytomegalovirus Infections/metabolism/congenital/pathology/genetics ; Mice ; CRISPR-Cas Systems ; },
abstract = {Congenital cytomegalovirus (CMV) infection is the most common perinatal infection, affecting up to 0.5% of infants. This elicits long-term disabilities that include neuropsychiatric manifestations, such as intellectual disability, microcephaly. Despite its high prevalence, the underlying mechanism of how congenitally acquired CMV infection causes brain pathology remain unknown. Here, we discovered the molecular interplay of key host (DISC1 and promyelocytic leukemia [PML]) and viral (immediate early 1 [IE1]) proteins within the neural progenitor cells, which underlay an attenuated neural progenitor proliferation in congenital CMV infection. Abolishing the viral IE1 protein by delivering IE1-targeting CRISPR/Cas9 to fetal brain rescued this progenitor cell deficit, a key pathology in congenital CMV infection. A selective targeting to a viral-specific protein by the CRISPR/Cas9 system is minimal in off-target effects. We further observed that CMV-encoded IE1 protein interferes with host PML-DISC1 interaction, resulting in disturbance of the Notch pathway in vitro and in embryonic brains. Therefore, we believe that a pivotal role of IE1 in an attenuated neural progenitor proliferation in the developing cortex through its interfering with interaction between host DISC1 and PML proteins.},
}
@article {pmid41656178,
year = {2026},
author = {McLamarrah, T and Aral, E and Hoffman, M and Tedstone, J and King, T and Vitko, J and Sebastião, MJ and Escandell, JM and Dias, MM and McCall, I and Machado, D and Cairns, V and DeMaria, C and Scarcelli, JJ},
title = {Evaluation of gene editing in CHO cells using the Cas-CLOVER system.},
journal = {Biotechnology progress},
volume = {42},
number = {2},
pages = {e70108},
pmid = {41656178},
issn = {1520-6033},
mesh = {CHO Cells ; Animals ; Cricetulus ; *Gene Editing/methods ; *Glutamate-Ammonia Ligase/genetics ; *CRISPR-Cas Systems/genetics ; Recombinant Proteins/genetics/biosynthesis ; Cricetinae ; Antibodies, Monoclonal/genetics/biosynthesis ; Gene Knockout Techniques ; },
abstract = {Recent advances in gene editing technologies have transformed the genetic engineering of Chinese hamster ovary (CHO) hosts, enabling the development of cell lines with improved stability and productivity. In this study, we employed the programmable nuclease (PN) Cas-CLOVER to precisely target the Glutamine synthetase (GS) locus in CHO cells. A total of 30 unique serum-free, suspension-adapted CHO-K1 candidate host cell lines were subjected to Cas-CLOVER-mediated gene editing, generating over one hundred potential GS knockout (GSKO) clones. A subset of the GSKO clones was subsequently validated using three orthogonal methods to confirm complete knockout of the GS gene in 98 clones. Randomly selected GSKO clones were utilized to produce standard monoclonal antibodies. The resulting pools demonstrated enhanced productivity, with up to a 14.5-fold increase in titer compared to their wild-type parental hosts. These findings highlight the potential of gene editing approaches to significantly improve recombinant protein production in CHO expression systems, offering valuable insights for biopharmaceutical manufacturing applications.},
}
@article {pmid41656257,
year = {2026},
author = {Ruis, BL and Ward, H and Myers, CL and Bielinsky, AK and Hendrickson, EA},
title = {SETDB1/ATF7IP regulate the precise genome engineering of HUSH-regulated genes.},
journal = {Epigenetics &amp; chromatin},
volume = {19},
number = {1},
pages = {10},
pmid = {41656257},
issn = {1756-8935},
support = {R21 AG077174/AG/NIA NIH HHS/United States ; R35GM141805//National Institutes of General Medical Sciences/ ; R01 CA154461/CA/NCI NIH HHS/United States ; CA154461/CA/NCI NIH HHS/United States ; R35 GM141805/GM/NIGMS NIH HHS/United States ; AG077174//National Institute of Aging/ ; GM088351/GM/NIGMS NIH HHS/United States ; R01 GM088351/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Histone-Lysine N-Methyltransferase/metabolism/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems ; Recombinational DNA Repair ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {BACKGROUND: The use of programmable nucleases has transformed genome editing and functional genomics. Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) was developed such that targeted genomic lesions [usually DNA double-stranded breaks (DSBs)] could be introduced in vivo with ease and precision. In the presence of homology donors, these lesions facilitate high-efficiency precise genome editing (PGE) via homology-directed repair (HDR) pathways. Because DSBs can lead to genomic instability, however, a large amount of effort has been invested in methodologies (e.g., base editors) that only require nicking the chromosomal DNA on one strand. Indeed, we have demonstrated in human cells that oligodeoxynucleotide (ODN)-mediated PGE using nickase variants of Cas9 can proceed by at least two HDR subpathways termed synthesis-dependent strand annealing (SDSA) and single-stranded DNA incorporation (ssDI). Which pathway is utilized is determined by which chromosomal strand (sense or anti-sense/Watson or Crick) is nicked and by the strandedness (sense or anti-sense/Watson or Crick) of the donor ODN.<br><br>RESULTS: While the mechanism of mammalian SDSA is moderately well understood, that of ssDI is not. To gain genetic insight into ssDI, we carried out a genome-wide CRISPR knockout screen to identify those genes which, when absent, enable increased ssDI. This screen identified the protein lysine methyl transferase (PKMT) Su(var)3-9, enhancer-of-zeste and trithorax (SET) domain bifurcated histone lysine methyltransferase 1 (SETDB1):activating transcription factor 7-interacting protein (ATF7IP) heterodimer and the downstream human silencing hub (HUSH) complex as strong negative regulators of ssDI. Consistent with their well-known biological effects, the negative regulation of ssDI by SETDB1/ATF7IP and HUSH was specific for transgenic reporters and for a HUSH-regulated single-copy gene, but was not observed at other (non-HUSH regulated) single-copy endogenous loci.<br><br>CONCLUSIONS: In toto, these experiments underscore the profound impact that chromatin modifiers - and by extension, chromatin structure - have on PGE outcomes. Specifically, we have identified SETDB1/ATF7IP and the HUSH complex as major negative regulators of the HDR subpathway, ssDI, when the target is a transgene. These experiments are a proof-of-principle that chromatin can act as a potent barrier to genetic recombination and they strongly support the feasibility of extending similar chromatin modulating strategies to enhance PGE efficiency at endogenous single-copy loci.},
}
@article {pmid41656299,
year = {2026},
author = {Fang, T and Bogensperger, L and Feer, L and Allam, A and Bezshapkin, V and Balázs, Z and von Mering, C and Sunagawa, S and Krauthammer, M and Schwank, G},
title = {Uncovering Cas9 PAM diversity through metagenomic mining and machine learning.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41656299},
issn = {2041-1723},
support = {//SNSF/ ; //Swiss National Science Foundation (SNSF)/ ; /ERC_/European Research Council/International ; },
mesh = {*Machine Learning ; *CRISPR-Cas Systems/genetics ; *Metagenomics/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; Genome, Archaeal ; Gene Editing/methods ; Genome, Bacterial ; Bacteriophages/genetics ; Data Mining ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Recognition of protospacer adjacent motifs (PAMs) is crucial for target site recognition by CRISPR-Cas systems. In genome editing applications, the requirement for specific PAM sequences at the target locus imposes substantial constraints, driving efforts to search for novel Cas9 orthologs with extended or alternative PAM compatibilities. Here, we present CRISPR-PAMdb, a comprehensive and publicly accessible database compiling Cas9 protein sequences from 3.8 million bacterial and archaeal genomes and PAM profiles from 7.4 million phage and plasmid sequences. Through spacer-protospacer alignment, we infer consensus PAM preferences for 8003 unique Cas9 clusters. To extend PAM discovery beyond traditional alignment-based approaches, we develop CICERO, a machine learning model predicting PAM preferences directly from Cas9 protein sequences. Built on the ESM2 protein language model and trained on the CRISPR-PAMdb database, CICERO achieves an average cosine similarity of 0.69 on test data and 0.75 on experimentally validated Cas9 orthologs. For Cas9 clusters where alignment-based predictions are infeasible, CICERO generates PAM profiles for an additional 50,308 Cas9 proteins, including 17,453 high-confidence predictions with CICERO confidence scores above 0.8. Together, CRISPR-PAMdb and CICERO enable large-scale exploration of PAM diversity across Cas9 proteins, accelerating design of next-generation CRISPR-Cas9 tools for precise genome engineering.},
}
@article {pmid41656303,
year = {2026},
author = {Lee, CJ and Nam, Y and Rim, YA and Ju, JH},
title = {Harnessing induced pluripotent stem cells and organoids for disease modeling and precision medicine.},
journal = {Stem cell research &amp; therapy},
volume = {17},
number = {1},
pages = {},
pmid = {41656303},
issn = {1757-6512},
support = {HI22C1314//Ministry of Health and Welfare/ ; },
mesh = {Humans ; *Organoids/metabolism/cytology ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Precision Medicine/methods ; Gene Editing ; CRISPR-Cas Systems ; Animals ; },
abstract = {The convergence of CRISPR genome editing, patient-derived organoids, and induced pluripotent stem cells (iPSCs) has reshaped in vitro disease modeling by enabling mechanistic investigations of human pathophysiology within genetically matched, tissue-relevant systems. Together, these technologies provide a synergistic platform for precise manipulation of disease-associated variants and support the generation of isogenic organoid models that reproduce key phenotypic and functional hallmarks across cancer, neurodegenerative, inflammatory, and monogenic disorders. In this review, we highlight how diverse CRISPR modalities-including knock-out, knock-in, CRISPRa/i, and genome-scale screening-have been applied to dissect gene function, model disease progression, and guide therapeutic development using iPSC- and organoid-based systems. We further discuss the application of these platforms in genotype- and phenotype-driven precision medicine, enabling patient stratification, drug-response prediction, and individualized treatment design. We illustrate these convergent applications with representative case studies spanning mechanistic research and early clinical translation. By combining the scalability of genome engineering with the physiological fidelity of organoids, CRISPR-integrated platforms are redefining the frontiers of experimental medicine. These approaches accelerate the discovery of disease mechanisms and actionable therapeutic targets while establishing individualized clinical strategies for complex human diseases. Collectively, they position CRISPR-enabled organoid systems as a foundational infrastructure that bridges genome editing to individualized therapy and supports next-generation precision medicine.},
}
@article {pmid41656481,
year = {2026},
author = {Laxmi, V and Verma, S and Kumar, M and Venkatesh, V and Mohit, and Maury, J and Mohd, S and Tripathi, S},
title = {Lactic Acid Bacteria-derived Bacteriocins: A Promising Antimicrobial Strategy against Multidrug-resistant for Neonatal Sepsis Pathogens.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41656481},
issn = {1867-1314},
abstract = {Neonatal sepsis continues to pose rising critical challenges to global health, particularly in low and middle-income countries, where it accounts for a substantial proportion of neonatal morbidity and mortality. The increasing prevalence of multidrug-resistant (MDR) pathogens in neonatal sepsis has significantly weakened the efficacy of conventional antibiotics, necessitating urgent exploration of alternative antimicrobial therapies for better clinical outcomes. Emerging research interest is growing to develop the microbial-derived peptides as novel antimicrobial agents, particularly "bacteriocins." In comparison to traditional antibiotics, many bacteriocins exhibit narrow-spectrum action, enabling them to inhibit specific pathogens without disrupting the host microbiota. Recent studies have highlighted the remarkable potential of lactic acid bacteria (LAB) derived bacteriocins in combating MDR pathogens responsible for neonatal sepsis. In this review, we compile current literature on the in vitro antimicrobial activity of LAB-derived bacteriocins, molecular diversity, mechanisms of action and clinical potential. Special attention is given to bacteriocins produced by LAB associated with the milk-derived microbiota, whose natural protective functions could be particularly beneficial for immunocompromised newborns. Additionally, we discuss the physicochemical properties of microbial peptides, including thermal stability, enzymatic resistance, and pH tolerance, which support their suitability for pharmaceutical applications. Overall, LAB-derived bacteriocins represent a novel, biocompatible, and complementary antimicrobial strategy integrated with conventional antibiotics to fight against MDR in neonatal sepsis. Further research and multicentric clinical trials are necessary to fully explore its compliance and efficacy as a future antimicrobial agent in neonatal medicine to underscore their potential as adjunct or preventive biotherapeutics in NICU settings.},
}
@article {pmid41658434,
year = {2026},
author = {Han, Y and Chen, R and Shentu, X},
title = {Advances and challenges of CRISPR/Cas gene editing for corneal diseases.},
journal = {Advances in ophthalmology practice and research},
volume = {6},
number = {1},
pages = {68-79},
pmid = {41658434},
issn = {2667-3762},
abstract = {BACKGROUND: Corneal diseases are a major cause of global visual impairment, and current treatments remain inadequate for severe or refractory cases. The CRISPR/Cas system offers robust and precise gene-editing capabilities, yet its therapeutic potential for corneal disorders remains largely unexplored.<br><br>MAIN TEXT: This narrative review introduces the CRISPR/Cas system and summarizes its recent advances in treating various corneal diseases, including inherited corneal dystrophies, infectious keratitis, corneal injury, and pathological neovascularization. We outline emerging preclinical and clinical studies, and analyze key issues that should be addressed for translation, including administration strategies, vector platform optimization and the mitigation of off-target toxicity.<br><br>CONCLUSIONS: This review provides a comprehensive and integrated overview of the current translational directions and challenges of CRISPR/Cas technology in corneal diseases from a novel perspective. It offers valuable guidance for future research and may accelerate the development of gene-editing therapies toward clinical application.},
}
@article {pmid41658853,
year = {2026},
author = {Rahimian, M and Aghazadeh-Soltan-Ahmadi, M and Panahi, B},
title = {In silico exploration of the genomic repertoire of Iranian aquatic bacteria: Prophage carriage, bioactive compound potential, CRISPR-Cas immunity, and integrated defensive-metabolic islands.},
journal = {Biochemistry and biophysics reports},
volume = {45},
number = {},
pages = {102452},
pmid = {41658853},
issn = {2405-5808},
abstract = {The unique and underexplored aquatic ecosystems of Iran represent a significant reservoir of microbial diversity. This study presents the first comprehensive genomic survey of 38 native Iranian bacterial strains from hypersaline lakes and wetlands, integrating in silico analyses of their secondary metabolome, bacteriocin potential, resident prophages, and genomic architecture. Our genome mining revealed a prolific capacity for secondary metabolite production, identifying dozens of biosynthetic gene clusters (BGCs). Ectoine biosynthesis was ubiquitous, underscoring its role as a key osmoprotectant, while diverse BGCs for terpenes, polyketides, and hybrid metabolites were also prevalent. Concurrently, we identified a wide array of ribosomally synthesized and post-translationally modified peptides (RiPPs), including known bacteriocins. Furthermore, we characterized eight high-quality prophages integrated within these genomes, encoding auxiliary genes such as carbohydrate-active enzymes (CAZymes) and putative anti-CRISPR (ACR) proteins. The bacterial hosts themselves were equipped with robust defense systems, with CRISPR-Cas loci, predominantly Type I, detected in most strains. Crucially, we identified multi-functional genomic islands that physically link BGCs with defense systems (e.g., CRISPR-Cas, restriction-modification) and prophage regions. We propose the "Fortress Hypothesis" to explain this architecture, wherein the co-localization of metabolite production and defense machinery protects metabolic investment against phage predation and genetic loss. This integrative genomic arrangement highlights a sophisticated co-evolutionary strategy for survival in extreme environments. Our findings position these indigenous bacteria as a promising genetic repository for discovering novel bioactive compounds, enzymes, and biotechnological tools, with implications for antibiotic discovery, CRISPR modulation, and understanding adaptive microbial genomics in extreme niches.},
}
@article {pmid41659011,
year = {2025},
author = {Wang, B and Lu, J and Zhang, X and Hu, R and Ma, H},
title = {Advances in nanomaterial-mediated CRISPR/Cas delivery: from lipid nanoparticles to vesicle-derived systems.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1669104},
pmid = {41659011},
issn = {2296-4185},
abstract = {Gene and genome editing therapies are increasingly connected with nanomaterials, which protect and transport fragile nucleic acids and CRISPR/Cas systems through biological barriers safely and accurately. This review discusses how different nanocarriers, including lipid-based, polymeric, inorganic, and vesicle-derived systems, can improve delivery efficiency, cell targeting, endosomal escape, and intracellular movement for gene and genome editing. It summarizes findings from early clinical and preclinical studies, comparing several carrier types such as ionizable lipid nanoparticles, polymeric nanoparticles, micelles, gold and silica nanostructures, and engineered extracellular vesicles. The review also explains how specific design factors, such as surface ligands, charge modification, PEGylation, and stimulus-responsive behaviors, influence biodistribution, and improve on-target efficiency while lowering immune responses and off-target effects. Ethical and regulatory concerns for in vivo editing are highlighted, along with current methods used to study nano-bio interactions. Among these carriers, ionizable lipid nanoparticles show the most advanced performance for delivering nucleic acids and CRISPR systems. However, new polymer-based and exosome-inspired carriers are progressing rapidly for repeated and targeted applications. Hybrid and responsive systems may also enable better spatial and temporal control of editing. Future research should focus on stronger in vivo potency testing, improved biocompatibility evaluation, and standardized manufacturing to ensure clinical safety and reliability.},
}
@article {pmid41660305,
year = {2026},
author = {Zhang, W and Jiang, A and Jia, BK and Jin, Y and Chen, Y and Li, Z and Liao, Y and Zhang, H and Lin, Z and Fang, X and Wang, L},
title = {Progress in RNA-Targeted Therapeutics for Human Diseases.},
journal = {MedComm},
volume = {7},
number = {2},
pages = {e70607},
pmid = {41660305},
issn = {2688-2663},
abstract = {RNA-targeted therapy is reshaping molecular medicine by shifting the traditional "protein-centric" view toward an "RNA-regulatory network" paradigm. Beyond carrying genetic information, RNA plays essential roles in posttranscriptional regulation, signaling pathways, and epigenetic modulation. Advances in high-throughput sequencing, structural biology, and delivery technologies have accelerated the development of diverse RNA therapeutics, including antisense oligonucleotides (ASOs), small interfering RNA (siRNA), microRNA (miRNA) modulators, messenger RNA (mRNA) therapeutics, aptamers, short hairpin RNA, and CRISPR/Cas-guided single-guide RNAs. However, a concise comparison of these major RNA modalities and the translational barriers that limit their broader clinical application is still lacking. This review outlines the mechanisms and representative applications of these RNA-based strategies in gene silencing, editing, protein replacement, immune activation, and targeted drug delivery. Special emphasis is placed on ASOs and siRNAs for neurological, metabolic, and infectious diseases, as well as mRNA therapeutics that are transforming vaccine development. Common challenges-such as in vivo stability, delivery efficiency, and immune activation-are also discussed. Finally, we highlight how chemical modification, nanotechnology, and artificial intelligence-assisted design are enhancing the specificity, stability, and safety of RNA therapeutics, providing a framework for advancing next-generation precision RNA medicine.},
}
@article {pmid41661055,
year = {2026},
author = {Bu, L and Yuan, A and Zhao, M and Pang, B and Li, J and Shang, Y and Xie, W and Peng, H},
title = {An Asymmetric Stem-Loop Translator Enhances Specificity of One-Pot Isothermal CRISPR/Cas12a Assay.},
journal = {Analytical chemistry},
volume = {98},
number = {7},
pages = {5513-5522},
doi = {10.1021/acs.analchem.5c07049},
pmid = {41661055},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *MicroRNAs/genetics/analysis ; Limit of Detection ; Humans ; *CRISPR-Associated Proteins/metabolism ; Aptamers, Nucleotide/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {Sensitive and specific detection of molecular biomarkers is fundamental to clinical diagnostics and biomedical research, yet existing CRISPR-based assays often suffer from nonspecific activation and cross-reactivity. Here, we introduce an asymmetric stem-loop translator that markedly enhances the specificity of a one-pot isothermal strand displacement amplification (SDA)-CRISPR/Cas12a assay. The asymmetric probe enables precise molecular recognition and controlled signal transduction, converting diverse targets into amplifiable DNA intermediates. Within the integrated one-tube system, SDA-generated DNA products directly trigger Cas12a trans-cleavage, yielding rapid fluorescence responses without thermal cycling or manual intervention. Systematic optimization of reaction parameters effectively mitigated enzyme inhibition and aerosol contamination. The resulting assay achieves highly specific and sensitive detection of miRNA with a detection limit of 500 fM, accurately distinguishing single- and double-base mutations. Owing to its modular design, the asymmetric stem-loop translator is readily adaptable to other analytes. By coupling to an aptamer-based recognition element, the system enables sensitive aflatoxin B1 detection. This work establishes a generalizable framework for enhancing CRISPR/Cas12a specificity through asymmetric molecular translation, offering a versatile platform for rapid nucleic acid and small-molecule diagnostics in clinical and point-of-care settings.},
}
@article {pmid41661284,
year = {2026},
author = {Li, Z and Chen, L and Luo, J and Lu, Y and Zhang, H and Zhao, P},
title = {One-pot recombinase polymerase amplification and CRISPR/Cas12a assay for Cryptococcus neoformans.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {61},
pmid = {41661284},
issn = {1432-0614},
mesh = {*Cryptococcus neoformans/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Recombinases/genetics/metabolism ; *Cryptococcosis/diagnosis/microbiology ; Humans ; Real-Time Polymerase Chain Reaction/methods ; Point-of-Care Testing ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cryptococcus neoformans, an opportunistic fungal pathogen, can induce central nervous system infections, posing a life-threatening risk and imposing substantial global health challenges and economic burdens. Given the significant reduction in mortality achieved through early monitoring of C. neoformans, there is an urgent demand for a rapid detection method for this pathogen. Herein, we developed a rapid, sensitive, and specific assay for the detection of C. neoformans based on a one-pot recombinase polymerase amplification (RPA) and CRISPR/Cas12a system, which can be read using a real-time fluorescent PCR instrument or lateral flow strips. This assay exhibits high sensitivity, with a detection limit of 1 copy/µL for C. neoformans, and no cross-reactivity was observed across different fungal strains. Notably, the assay can be performed in harsh environments without reliance on complex equipment, making it suitable for point-of-care testing (POCT). Collectively, this method not only provides a robust alternative for C. neoformans detection but also offers valuable insights for the identification of other fungal pathogens. KEY POINTS: • We developed a one-pot RPA and CRISPR/Cas12a assay to detect Cryptococcus neoformans. • This assay exhibits high sensitivity and specificity. • Detection results can be obtained by three ways which is suitable for POCT.},
}
@article {pmid41661420,
year = {2026},
author = {Zhang, H and Feng, G and Feng, Y},
title = {Quinoa as a naturally stress-resistant crop: current status and future promises.},
journal = {Stress biology},
volume = {6},
number = {1},
pages = {12},
pmid = {41661420},
issn = {2731-0450},
support = {2022B02010-1//Science and Technology Department of Xinjiang Uygur Autonomous Region/ ; 2022YFF1003403-4//Key Technologies Research and Development Program/ ; 2021YFA1300401//Key Technologies Research and Development Program/ ; 32441015//National Natural Science Foundation of China/ ; },
abstract = {Quinoa (Chenopodium quinoa Willd.), a semi-domesticated halophyte originating in the Andean region, has emerged as a promising crop for exploiting marginal lands, valued for its exceptional nutritional profile and remarkable resilience to high salinity and drought. This review analyzes the current status and future potential of quinoa as a model halophytic crop. We begin by examining the physiological mechanisms that enable quinoa to thrive in marginal environments, which have been the subject of extensive study. Thanks to the advancement in high-throughput sequencing technology, genomic resources - including the recent development of high-quality reference genomes and a Chenopodium pangenome - are rapidly expanding. Sequence-based genetic mapping techniques hold the promise to dissect the molecular basis of complex traits in combination with the utility of functional genomics tools such as virus-induced gene silencing (VIGS) and stable genetic transformation. Ultimately, the application of modern breeding technologies, such as phenomics, genomic selection (GS), and CRISPR/Cas, will expedite the development of locally adapted, climate-resilient quinoa cultivars worldwide.},
}
@article {pmid41662274,
year = {2026},
author = {Tek, MI and Budak Tek, K and Sarikaya, P and Ahmed, AR and Fidan, H},
title = {Choosing the best route: Comparative optimization of wheat transformation methods for improving yield by targeting TaARE1-D with CRISPR/Cas9.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0342491},
pmid = {41662274},
issn = {1932-6203},
mesh = {*Triticum/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Proteins/genetics ; Plant Breeding/methods ; },
abstract = {Wheat (Triticum aestivum L.) is one of the most important crops worldwide, supplying a major share of calories and protein for the global population. Incorporating gene editing into breeding programs is critical to improve yield and stress tolerance, yet wheat remains difficult to transform and regenerate efficiently. These bottlenecks limit the full application of CRISPR/Cas9 for improvement yield in wheat. To address this, transformation parameters were optimized for three methods: immature embryo transformation, callus transformation, and injection-based in planta transformation. Systematic optimization of Agrobacterium strain, bacterial density, acetosyringone concentration, and incubation conditions resulted in substantially improved transformation success. Efficiencies of 66.84% for immature embryos, 55.44% for callus, and 33.33% for in planta transformation were achieved, representing more than tenfold increase compared with previously reported rate of ~3%. A key innovation was the shortening of the callus induction stage for immature embryos, reducing the time required for plant regeneration by approximately one month while maintaining high transformation efficiency. The protocols were validated through CRISPR/Cas9-mediated knockout of TaARE1-D, a negative regulator of nitrogen uptake and yield. Generated mutants exhibited increased grain number, spike length, grain length, and thousand-grain weight, as well as the characteristic stay-green phenotype associated with loss of TaARE1-D function. The optimized protocols provide robust platforms to accelerate gene-editing in wheat to increase yield and stress-tolerance.},
}
@article {pmid41663224,
year = {2026},
author = {Li, Y and Chen, X and Yang, Z and Wang, Z and Wang, R},
title = {CRISPR/Cas12a empowered electrochemical biosensor for ultrasensitive detection of Vibrio parahaemolyticus in seafood samples.},
journal = {Analytica chimica acta},
volume = {1391},
number = {},
pages = {345158},
doi = {10.1016/j.aca.2026.345158},
pmid = {41663224},
issn = {1873-4324},
mesh = {*Vibrio parahaemolyticus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Seafood/microbiology/analysis ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; Limit of Detection ; Food Contamination/analysis ; Animals ; DNA, Bacterial/genetics/analysis ; },
abstract = {Rapid and ultrasensitive detection of Vibrio parahaemolyticus (V. parahaemolyticus, Vp) is of great significance for the early prevention of foodborne disease. Traditional methods for detecting Vp are time-consuming, exhibiting low sensitivity and specificity. In this study, CRISPR/Cas12a system is integrated with electrochemical sensing and polymerase chain reaction (PCR) to establish a PCR-based E-CRISPR biosensor for Vp detection. The target DNA extracted from Vp is amplified by PCR, then activate CRISPR/Cas12a system to cleave methylene blue (MB)-labeled hairpin DNA probes on electrode, resulting in great changes in current. The employment of hairpin DNA probes reduces the steric hindrance for Cas12a trans-cleavage, acquiring a better cleavage efficiency and sensing performance. Under optimal conditions, the limit of detection reaches 1.17 copies/μL (genomic DNA), 1.23 CFU/mL (standard bacteria), and 12.3 CFU/g (artificially contaminated shrimp samples) respectively. Moreover, the PCR-based E-CRISPR biosensor demonstrates superior reproducibility and specificity. Most importantly, the E-CRISPR biosensor were in 100 % agreement with real time quantitative PCR for the detection of 18 seafood samples, which confirms the biosensor's broad applicability for monitoring Vp in complex food matrix. Our developed E-CRISPR biosensor demonstrates to be a simple, rapid and ultrasensitive method for Vp detection in the food supply chain, and can be extended to other foodborne pathogens.},
}
@article {pmid41663226,
year = {2026},
author = {Yin, X and Zhang, Z and Luo, H and Qin, X and Chen, Y and Chen, W and Zheng, H},
title = {Amplification-free one-pot RNA detection by pairing CRISPR-Cas13a with cascade amplification circuit-driven DNAzyme (RAPID).},
journal = {Analytica chimica acta},
volume = {1391},
number = {},
pages = {345138},
doi = {10.1016/j.aca.2026.345138},
pmid = {41663226},
issn = {1873-4324},
mesh = {*DNA, Catalytic/metabolism/chemistry/genetics ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Neisseria gonorrhoeae/genetics/isolation &amp; purification ; *RNA, Bacterial/analysis/genetics ; Humans ; },
abstract = {RNA has become a versatile target for diagnosing a wide range of pathogens. The demand for rapid and accurate diagnostics in point-of-care (POC) or resource-limited settings is growing. However, most RNA-based assays depend on reverse transcription and complex instruments (e.g., RT-qPCR), restricting their use in these settings. Isothermal amplification methods provide a simpler alternative with reduced instrumentation requirements, but their high amplification efficiency raises concerns about nucleic acid carry-over contamination. To address these challenges, we developed RAPID (CRISPR-Cas13a with a cascade amplification circuit-driven DNAzyme), an isothermal, one-pot RNA detection biosensing platform that eliminates the need for sample pre-amplification. RAPID integrates the precise target recognition by CRISPR-Cas13a with robust signal amplification by a toehold-mediated strand-displacement DNA circuit, eliminating the need for reverse transcription and thermal cycling. This platform enables quantitative RNA detection within 30 min at 37 °C. By reprogramming RAPID crRNAs, we successfully detected both bacterial (e.g., Treponema pallidum and Neisseria gonorrhoeae) and viral (e.g., herpes simplex virus) targets. The RAPID platform is designed for versatile detection, being compatible with both fluorescence-based (RAPID-Flu) and lateral flow assay (RAPID-LFA) readouts. The RAPID-Flu and RAPID-LFA both demonstrated a sensitivity of 5 fM per reaction, exhibiting comparable detection limits. Both methods showed excellent specificity and high concordance with clinical diagnoses of Neisseria gonorrhoeae. In summary, the RAPID platform provides rapid, programmable, and visually interpretable solutions with strong potential for POC diagnostics. Its flexibility and portability make it particularly suitable for early diagnosis and on-site monitoring of diverse infectious pathogens.},
}
@article {pmid41663312,
year = {2026},
author = {Eraña, H and Vidal, E and Fernández-Borges, N and Charco, JM and Díaz-Domínguez, CM and Sampedro-Torres-Quevedo, C and Galarza-Ahumada, J and Fernández-Muñoz, E and San-Juan-Ansoleaga, M and Pérez-Castro, M&#xc1; and Gonçalves-Anjo, N and Piñeiro, P and Giler, S and González-Martín, N and Lorenzo, NL and Manero-Azua, A and Perez de Nanclares, G and Geijo, M and Sánchez-Martín, MA and Requena, JR and Castilla, J},
title = {The L108I polymorphism in mouse prion protein drives spontaneous disease and enhances transmission of atypical and classical prion strains.},
journal = {Brain pathology (Zurich, Switzerland)},
volume = {},
number = {},
pages = {e70083},
doi = {10.1111/bpa.70083},
pmid = {41663312},
issn = {1750-3639},
support = {PID2024-160022OB-I00//Agencia Estatal de Investigaci&#xf3;n/ ; PID2021-122201OB-C21//Agencia Estatal de Investigaci&#xf3;n/ ; PID2020-117465GB-I00//Agencia Estatal de Investigaci&#xf3;n/ ; PID2021-1222010B-C22//Agencia Estatal de Investigaci&#xf3;n/ ; CEX2021-001136-S//Ministerio de Ciencia e Innovaci&#xf3;n/ ; EFA031/01 NEURO-COOP//Interreg/ ; PT23/00123//Instituto de Salud Carlos III/ ; BN661-FTPGB-2023//Fundaci&#xf3;n Tatiana P&#xe9;rez de Guzm&#xe1;n el Bueno/ ; //Creutzfeldt-Jakob Disease Foundation-2022/ ; },
abstract = {Prion diseases are fatal neurodegenerative disorders that can be idiopathic, associated with genetic mutations, or acquired by infection with misfolded prion protein. We developed two complementary transgenic mouse models to investigate how the L108I substitution in mouse prion protein (PrP) influences spontaneous prion formation and transmission characteristics. The transgenic mouse model overexpressing the variant at approximately three times wild-type (WT) PrP levels (TgMo(L108I)3x) consistently developed a spontaneous neurodegenerative disorder between 219 and 536 days of age with 100% penetrance. This spontaneous disease exhibited biochemical and neuropathological characteristics of atypical prion disorders, featuring a distinctive 7-10 kDa protease-resistant PrP fragment and pathology comparable to small ruminants' atypical scrapie and certain forms of Gerstmann-Sträussler-Scheinker syndrome (GSS). In contrast, the knock-in model expressing the same variant at physiological levels (TgMo(L108I)1x) showed no spontaneous disease beyond 600 days, demonstrating that both the specific amino acid substitution and elevated expression levels are necessary for spontaneous prion formation. The spontaneously generated prions transmitted efficiently to models expressing the I108 variant and to Tga20 mice overexpressing WT PrP but encountered a robust transmission barrier toward WT mice, indicating strain-specific replication requirements. The TgMo(L108I)3x model demonstrated exceptional versatility as a universal acceptor for heterogeneous prion isolates, demonstrating superior efficiency in propagating atypical variants like GSS A117V (57 ± 0.6 days) and rapid propagation of classical scrapie-derived mouse prion strains, including Rocky Mountains Laboratory mouse prion strain (RML) (85 ± 3.8 days) and 22L (95 ± 1 days). Comparative analysis revealed that the L108I substitution differentially impacts strain propagation, with greater acceleration of RML (~33% shorter incubation) than 22L (~0.5% shorter) compared to WT mice. These complementary systems offer powerful experimental platforms for investigating the molecular determinants of spontaneous prion formation, strain selection and transmission barriers, providing insights into idiopathic prion pathogenesis and developing therapeutic interventions.},
}
@article {pmid41663678,
year = {2026},
author = {Zhu, Y and Zhang, J and Ruan, Y and Lei, T and Li, S and Cao, L and Zou, X and He, Y and Li, Q and Chen, S and Peng, A},
title = {Application of compact CRISPR/Cas nucleases for citrus genome editing.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {7},
pmid = {41663678},
issn = {1573-9368},
support = {CSTB2024TIAD-CYKJCXX0021//Sichuan-Chongqing Science and Technology Innovation Cooperation Program Project/ ; CSTB2023TIAD-KPX0044//the Technology Innovation and Application Development Key Project of Chongqing/ ; CARS-26//Earmarked Fund for China Agriculture Research System/ ; CSTB2023NSCQ-MSX1085//Chongqing Natural Science Foundation Project/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Citrus/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; },
abstract = {Gene editing technology continues to advance, and the range of available editing tools is steadily expanding. Recently, several compact and ultracompact systems have been developed, gaining considerable attention because their components can be efficiently packaged into viral vectors. To identify compact tools suitable for efficient genome editing in citrus, Casπ, CoCas9, along with their respective single guide RNAs, were synthesized, and CRISPR/Casπ and CRISPR/CoCas9 constructs were designed to assess their editing efficiency in 'Wanjincheng' orange (Citrus sinensis Osbeck). The Casπ was able to mediate genome editing in the citrus genome, although with low efficiency. In comparison, CoCas9 showed a transformation efficiency three times higher than that of the widely used SpCas9. Moreover, while the gene editing efficiency of CoCas9 was comparable to that of SpCas9, the significantly elevated transformation efficiency resulted in a significantly higher overall editing efficiency for CoCas9 relative to SpCas9. Mutation profiles generated by CoCas9 and SpCas9 were highly similar, and both nucleases displayed comparable target specificity at three potential off-target sites. These results indicate that Casπ is not suitable for application in citrus genome editing, whereas CoCas9 represents a promising alternative to SpCas9 for efficient and precise genome modification in citrus.},
}
@article {pmid41663776,
year = {2026},
author = {Pearce, JC and Campbell, JS and Prior, JL and Titball, RW and Wakefield, JG},
title = {PiggyBac-mediated transgenesis and CRISPR-Cas9 knockout in the greater wax moth, Galleria mellonella.},
journal = {Lab animal},
volume = {55},
number = {3},
pages = {95-102},
pmid = {41663776},
issn = {1548-4475},
support = {NC/W002388/1//National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs)/ ; NC/T001518/1//National Centre for the Replacement Refinement and Reduction of Animals in Research (NC3Rs)/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Moths/genetics ; *DNA Transposable Elements/genetics ; *Gene Knockout Techniques/methods ; Larva/genetics ; Animals, Genetically Modified/genetics ; *Gene Transfer Techniques ; },
abstract = {The larvae of the greater wax moth, Galleria mellonella, are gaining prominence as a versatile nonmammalian in vivo model to study host-pathogen interactions. Their ability to be maintained at 37 °C, coupled with a broad susceptibility to human pathogens and a distinct melanization response that serves as a visual indicator for larval health, positions G. mellonella as a powerful resource for infection research. Despite these advantages, the lack of genetic tools, such as those available for zebrafish and Drosophila melanogaster, has hindered development of the full potential of G. mellonella as a model organism. Here we describe a robust methodology for generating transgenic G. mellonella using the PiggyBac transposon system and for precise gene knockouts via CRISPR-Cas9 technology. These advances significantly enhance the utility of G. mellonella in molecular research, paving the way for its widespread use as an inexpensive and ethically compatible animal model in infection biology and beyond.},
}
@article {pmid41664621,
year = {2026},
author = {Ullah, Q and Haider, W and Zeshan, M and Waqar, M and Arshad, MT and Parveen, H and Mukhtar, S and Zahir, A},
title = {Advancing climate adaptation in saffron through CRISPR-based modulation of stress tolerance and photoperiodic flowering control.},
journal = {GM crops &amp; food},
volume = {17},
number = {1},
pages = {2626180},
pmid = {41664621},
issn = {2164-5701},
mesh = {*Flowers/genetics/physiology/growth &amp; development ; *Crocus/genetics/physiology/growth &amp; development ; Gene Editing ; *CRISPR-Cas Systems ; Photoperiod ; Stress, Physiological ; Plants, Genetically Modified/genetics ; Adaptation, Physiological ; Droughts ; Climate Change ; },
abstract = {Saffron (Crocus sativus L.) is a high-value crop known for its intricate harvesting process and limited production due to factors like triploid sterility and specific climatic needs. This review discusses biotechnological methods, particularly CRISPR/Cas9 genome editing, aimed at improving heat and drought tolerance and achieving year-round flowering. Such genetic edits as evidenced with experimental CRISPR/Cas9 systems that reach up to 70% callus initiation in saffron. Cultivation efficiency and quality are increased in Hydroponic systems and synthetic bioreactors, which have been proven in trials in non-traditional areas such as North Bengal, India. Nonetheless, internationalization threatens the market value and cultural integrity of saffron, and such measures as fair-trade labels, GI laws, and cooperatives of stakeholders must be implemented fairly and equally.},
}
@article {pmid41664627,
year = {2026},
author = {Hancock, MB and Ruby, DR and Bieler, RA and Cole, DC and Marsden, KC},
title = {Multi-omic analyses identify molecular targets of Chd7 that contribute to CHARGE syndrome model phenotypes.},
journal = {Disease models &amp; mechanisms},
volume = {19},
number = {3},
pages = {},
pmid = {41664627},
issn = {1754-8411},
support = {R21NS120079-01/NS/NINDS NIH HHS/United States ; R01NS116354-01/NS/NINDS NIH HHS/United States ; R21NS120079-01/NS/NINDS NIH HHS/United States ; R01NS116354-01/NS/NINDS NIH HHS/United States ; //North Carolina State University/ ; },
mesh = {*CHARGE Syndrome/genetics/pathology ; Animals ; Zebrafish/genetics/metabolism ; Phenotype ; *Zebrafish Proteins/metabolism/genetics ; Disease Models, Animal ; *DNA Helicases/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; *Proteomics ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Transcriptome/genetics ; CRISPR-Cas Systems/genetics ; Humans ; Mutation/genetics ; Gene Expression Profiling ; Multiomics ; },
abstract = {CHARGE syndrome is a developmental disorder that affects 1 in 10,000 births, and patients exhibit both physical and behavioral characteristics. De novo variants in chromodomain helicase DNA binding protein 7 (CHD7) cause 67% of CHARGE syndrome cases. CHD7 is a DNA-binding chromatin remodeler with thousands of predicted binding sites in the genome, making it challenging to define molecular pathways linking loss of CHD7 to CHARGE syndrome phenotypes. To address this problem, here, we used a previously characterized zebrafish model of CHARGE syndrome to generate transcriptomic and proteomic datasets from larval zebrafish head tissue at two developmental time points. By integrating these datasets with differential expression, pathway and upstream regulator analyses, we identified multiple consistently dysregulated pathways and defined a set of candidate genes that link loss of chd7 with disease-related phenotypes. Finally, we identified that CRISPR/Cas9-mediated knockdown of capgb, nefla or rdh5 phenocopies behavioral defects seen in chd7 mutants, functionally validating the roles of these genes. Our data provide a resource for further investigation of molecular mediators of CHD7 and a template to reveal functionally relevant therapeutic targets to alleviate specific aspects of CHARGE syndrome.},
}
@article {pmid41665608,
year = {2026},
author = {Hanson, E and Gold, R and Lee, DH and Yigit, MV},
title = {Cas12a-Programmed Modular CRISPR Cascade Reaction on Paper Supports for Dual-Mode Detection of Pathogenic Genomes.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2774-2783},
doi = {10.1021/acssensors.5c04848},
pmid = {41665608},
issn = {2379-3694},
mesh = {*Paper ; *CRISPR-Cas Systems/genetics ; *Listeria monocytogenes/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Campylobacter jejuni/genetics/isolation &amp; purification ; *Endodeoxyribonucleases/genetics/metabolism ; *Genome, Bacterial ; *Bacterial Proteins/genetics ; Escherichia coli/genetics/isolation &amp; purification ; Metal Nanoparticles/chemistry ; Silver/chemistry ; DNA, Bacterial/genetics ; },
abstract = {We developed an optical biosensing platform using cost-efficient and scalable paper support for the detection and identification of three major bacterial pathogens using fluorescent DNA-templated silver nanoclusters (FNPs) and an innovative CRISPR-Cas12a cascade reaction. The sensors were fabricated as ∼5 mm letter-shaped paper cutouts, with each letter representing a specific pathogen: "C" for Campylobacter jejuni, "E" for Shiga toxin-producing Escherichia coli, and "L" for Listeria monocytogenes. Detection was initially achieved via an ON-to-OFF mechanism, wherein target recognition by Cas12a led to FNP degradation and fluorescence loss using target strands identified from the conserved genomic regions from each pathogen. This platform successfully detected individual and multiple targets in all possible seven combinations. To enhance diagnostic clarity, we developed a two-step CRISPR-Cas12a cascade reaction enabling an ON signal output when the target is present, a more intuitive and desirable reporting format. In this design, the first Cas12a reaction detects the target and cleaves an activator strand, preventing activation of a second Cas12a reaction that would otherwise degrade FNPs. Consequently, fluorescence is retained in the presence of the target (ON-retention) and lost in its absence, providing a clear ON signal when the target is detected, and an OFF signal when it is not. Finally, we demonstrated both ON-to-OFF and ON-retention detection modes using the whole Listeria genome amplified by isothermal recombinase polymerase amplification, with reliable detection of as few as 40 full genomic copies using fluorescent images on paper substrates. This work represents a significant advancement in Cas12a-based biosensing, uniquely demonstrating multistep biochemical reactions directly on paper support, and offers a promising platform for low-cost, scalable pathogen detection in resource-limited settings.},
}
@article {pmid41665762,
year = {2026},
author = {Farooq, M and Khan, A and Hassan, A and Shah, MM},
title = {Advances in CRISPR/Cas systems for engineering abiotic stress tolerance in plants: mechanisms and future prospects.},
journal = {Planta},
volume = {263},
number = {3},
pages = {72},
pmid = {41665762},
issn = {1432-2048},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Stress, Physiological/genetics ; *Crops, Agricultural/genetics/physiology ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Droughts ; },
abstract = {Abiotic stress factors such as drought, salinity, extreme temperatures, and oxidative stress significantly limit crop productivity and threaten global food security. Traditional breeding and transgenic approaches have been employed to enhance stress tolerance, but they are often time-consuming and face regulatory hurdles. The advent of CRISPR/Cas genome editing technology has revolutionized plant genetic engineering by enabling precise modifications to stress-responsive genes. This review explores recent advancements in CRISPR/Cas-based genome editing for improving abiotic stress resilience in crops. We discuss the mechanisms of CRISPR/Cas systems, their applications in stress tolerance, and emerging approaches such as multiplex genome editing, base editing, and AI-assisted CRISPR. Furthermore, we highlight challenges, ethical considerations, and future directions for integrating CRISPR into agricultural biotechnology. This review underscores the potential of CRISPR-based strategies in developing climate-resilient crops to ensure sustainable food production in the face of global climate change.},
}
@article {pmid41666229,
year = {2026},
author = {Fan, X and Lyu, S and Fan, W and Shu, J and Cheng, X},
title = {Precision targeting: The dawn of artificially customized disease resistance.},
journal = {PLoS pathogens},
volume = {22},
number = {2},
pages = {e1013942},
pmid = {41666229},
issn = {1553-7374},
mesh = {*Plant Diseases/immunology/prevention &amp; control/genetics ; *Disease Resistance/genetics/immunology ; *Plant Immunity/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/immunology ; *Crops, Agricultural/genetics/immunology ; },
abstract = {Advanced plant disease management strategies are essential to sustainable agriculture and global food security. Advances in plant immunity have given rise to a variety of innovative disease control strategies, such as NLR gene transfer, RNA silencing technology, and CRISPR/Cas9-based gene disruption, as well as the use of immunity inducers. Recently, several novel resistance strategies, including the bioengineering of immunoreceptors, protease-triggered resistance design, and the sentinel approach, have enabled the customized development of disease resistance traits. These new approaches envisage a new paradigm of precision-targeted, artificially engineered resistance to enhance crop protection.},
}
@article {pmid41666850,
year = {2026},
author = {Zhao, R and Chen, J and Li, Y and Jin, M and Liu, K and Liu, Y and Gao, L and Yang, G and Yuan, X and Chu, X and Wang, JG},
title = {CRISPR/Cas9-mediated knockout of the 22 kDa α-prolamin genes orchestrates the regulation of functional amino acid content in foxtail millet.},
journal = {Journal of plant physiology},
volume = {318},
number = {},
pages = {154723},
doi = {10.1016/j.jplph.2026.154723},
pmid = {41666850},
issn = {1618-1328},
mesh = {*CRISPR-Cas Systems/genetics ; *Setaria Plant/genetics/metabolism ; *Amino Acids/metabolism ; Gene Knockout Techniques ; *Prolamins/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Genes, Plant ; },
abstract = {Improving the nutritional quality of cereal crops remains a primary objective in modern breeding programs. The composition and content of prolamins directly affect the overall nutritional value. This study elucidates the role of two 22 kDa α-prolamin genes (Seita.9G301400 and Seita.9G406400) in foxtail millet using CRISPR/Cas9-mediated knockout. While simultaneous disruption of both genes in double mutants reduced prolamin content, amino acids, and soluble sugars, single-gene mutants exhibited the opposite effect. These single mutants displayed increased grain size alongside significantly enhanced levels of essential amino acids and sugars. Starch pasting properties were also improved in single mutants but compromised in double mutants. Our findings demonstrate that individual knockout of the two prolamin genes enhances nutritional and sensory quality, providing a potential strategy for developing improved foxtail millet varieties.},
}
@article {pmid41667457,
year = {2026},
author = {He, M and Wang, W and Zhou, H and Liu, C and Zhao, C and Li, J and Han, Y and Qin, Y and Chen, M},
title = {A synthetic system for RNA-responsive pyroptosis based on type III-E CRISPR nuclease-protease.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41667457},
issn = {2041-1723},
support = {//National Key R&amp;D Program of China/ ; //National Natural Science Foundation of China/ ; //Natural Science Foundation of Wuhan/ ; },
mesh = {*Pyroptosis/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA/genetics/metabolism ; Animals ; Mice ; HEK293 Cells ; },
abstract = {Pyroptosis plays a crucial role in immune defense against infections and endogenous threats by eliminating harmful cells and modulating the immune response through inflammation. However, the natural activation of pyroptosis involves intricate signaling pathways, posing significant challenges for its artificial manipulation in research and therapies. Here, we present DAMAGE (Death Manipulation Gene), an innovative system that integrates gasdermins within the type III-E CRISPR framework, enabling the specific recognition of target RNA (tgRNA) and triggering pyroptosis. This mechanism allows DAMAGE to selectively identify and eliminate virus-infected, cancerous, and senescent cells, all of which exhibit altered RNA transcriptomes. Additionally, DAMAGE exhibits considerable promise as a platform for mRNA-LNP therapy. Our study highlights the potential of this CRISPR-based system in the controllable induction of pyroptosis, offering an innovative therapeutic strategy for treating RNA-heterogeneous diseases.},
}
@article {pmid41669364,
year = {2025},
author = {Elkonin, LA and Gerashchenkov, GA and Borisenko, NV and Sarsenova, SK and Panin, VM},
title = {Study of the progeny of sorghum mutants obtained using the CRISPR/Cas9 genetic construct directed at inducing mutations in the α-kafirin k1C5 gene.},
journal = {Vavilovskii zhurnal genetiki i selektsii},
volume = {29},
number = {8},
pages = {1161-1168},
doi = {10.18699/vjgb-25-122},
pmid = {41669364},
issn = {2500-0462},
abstract = {Site-directed mutagenesis using genetic constructs carrying the CRISPR/Cas system is an effective technology that is actively used to solve a variety of problems in plant genetics and breeding. One of these problems is to improve the nutritional value of grain sorghum, a high-yielding heat- and drought-tolerant cereal crop that is becoming increasingly important in the conditions of climate aridization. The main reason for the relatively low nutritional value of sorghum grain is the resistance of its storage proteins, kafirins, to proteolytic digestion. We have previously obtained mutants with improved kafirin in vitro digestibility using the CRISPR/Cas technology in grain sorghum variety Avance. The nucleotide sequence of one of the genes (k1C5) of the gene family encoding the signal polypeptide of 22 kDa α-kafirin was used as a target. The aim of this study was to investigate the manifestation of the main agronomically-important traits in the progeny of these mutants and inheritance of high in vitro protein digestibility, and also sequencing nucleotide sequences encoding the 22 kDa α-kafirin signal polypeptide in a number of plants from the T0 generation and their T1 progeny. It was revealed that four of the six studied T0 plants, as well as their progeny, had the same mutation: a T→C substitution in the 23rd position of the nucleotide sequence of the k1C5 gene encoding the signal polypeptide, which led to a substitution of the coding triplet CTC→CCC (Leu→Pro). This mutation is located off-target, 3' from the PAM sequence. It is suggested that this mutation may have arisen as a result of Cas9 nuclease errors caused by the presence of multiple PAM sequences located close to each other. It was found that the progeny of two of the three studied mutants (T2 and T3 families), grown in the experimental field conditions, differed from the original variety by a reduced plant height (by 12.4-15.5 %). The peduncle length, 1,000-grain mass, and grain mass per panicle did not differ from the original variety, with the exception of the progeny of the 2C-1.2.5b mutant, which had a reduced grain yield per panicle. Unlike the original variety, plants from the T2 and T3 generations had kernels with a modified type of endosperm (completely floury, or floury with inclusions of vitreous endosperm, or with a thin vitreous layer). The level of grain protein digestibility in the progeny of mutants 2C-2.1.1 #13 and 2C-1.2.5a #14 varied from 77 to 84 %, significantly exceeding the original variety (63.4 ± 2.3 %, p < 0.05). The level of protein digestibility from kernels with modified endosperm was higher than that of kernels with normal vitreous endosperm (84-93 %, p <0.05). The reasons for the variation in endosperm texture in the progeny of the mutants and its relationship with the high digestibility of kafirins are discussed.},
}
@article {pmid41669806,
year = {2026},
author = {Wang, ZH and Yang, ZQ},
title = {Advances in site-specific knock-in techniques for gene editing.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {2},
pages = {128-141},
doi = {10.16288/j.yczz.25-076},
pmid = {41669806},
issn = {0253-9772},
mesh = {*Gene Editing/methods ; Humans ; CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Animals ; DNA Nucleotidyltransferases ; },
abstract = {Gene-targeted knock-in technology serves as a cornerstone tool in genetic engineering and gene therapy, designed to circumvent the unpredictability and heterogeneityassociated with conventional random integration methods. However, its practical application has long been constrained by off-target activity and low efficiency during the editing process. Recent advances in site-specific recombinase systems (e.g., Bxb1 integrase) and programmable nuclease systems (e.g., CRISPR/Cas9) have significantly enhanced the precision and efficiency of gene knock-in. Notably, the Cas9-Bxb1 integrase system enables targeted integration of large DNA fragments (5-43 kb) into genomic safe harbor (GSH) sites, offering a transformative platform for disease modeling, functional genomics, and clinical therapeutics. This review systematically summarizes the progress of site-specific recombinase and nuclease systems, discusses GSH screening strategies and the role of multi-omics data in optimizing predictive models, and compares the strengths and limitations of twinPE+Bxb1 and PASTE systems. Future research should focus on developing novel integrases with low off-target activity, refining DSB-free editing technologies, and establishing cross-species GSH databases to advance applications in precision medicine and synthetic biology.},
}
@article {pmid41669836,
year = {2026},
author = {Dai, YM and Zhao, L and Xu, T and Duan, JQ and Wang, YM and Yan, YX and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Lai, SY and Ai, YR and Huang, JB and Zhu, L and Xu, ZW},
title = {Fast and Simplified CRISPR-Cas13d Assay for Sensitive Detection of Porcine Deltacoronavirus.},
journal = {ACS synthetic biology},
volume = {15},
number = {2},
pages = {834-845},
doi = {10.1021/acssynbio.5c00909},
pmid = {41669836},
issn = {2161-5063},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems/genetics ; *Deltacoronavirus/genetics/isolation &amp; purification ; RNA, Viral/genetics/analysis ; Sensitivity and Specificity ; Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/diagnosis/virology ; },
abstract = {Rapid and portable diagnostic technologies are essential for controlling infectious diseases. Here, we describe RAPID (Rapid Automated Portable Integrated Detection), a single-step, extraction-free CRISPR-Cas13d-based assay for sensitive and specific detection of porcine deltacoronavirus (PDCoV) RNA. RAPID integrates isothermal recombinase polymerase amplification with EsCas13d-mediated collateral cleavage in a one-pot reaction, enabling sample-to-answer detection within 30 min. A brief room-temperature lysis step allows direct RNA release from unextracted samples, simplifying sample preparation and reducing equipment requirements. Lyophilized reagents enhance stability during refrigerated storage (≤4 °C) and facilitate simplified transportation using conventional cooling measures, thereby reducing reliance on strict cold-chain logistics. The assay operates optimally at 37 °C and remains functional under ambient (∼25 °C) conditions with reduced sensitivity, permitting instrument-free operation when temperature control is unavailable. Detection is achieved via in-tube fluorescence or lateral-flow readouts. Clinical validation using porcine samples showed complete concordance with RT-qPCR, achieving 100% sensitivity and specificity. RAPID provides a practical point-of-care diagnostic platform for on-farm surveillance and deployment in resource-limited settings.},
}
@article {pmid41670012,
year = {2026},
author = {Miao, Y and Li, C and Su, Y and Peng, T and Wang, J and Liu, S and Ma, C and Li, L and Wang, Y},
title = {The application of CRISPR-Cas9 system in brain diseases.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {6},
pages = {899-920},
pmid = {41670012},
issn = {1748-6963},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Brain Diseases/therapy/genetics ; *Gene Editing/methods ; Blood-Brain Barrier/metabolism ; *Genetic Therapy/methods ; Animals ; Genetic Vectors/genetics ; },
abstract = {As an efficient genome-editing technology, Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated protein9 (Cas9) system is increasingly being recognized as a significant therapeutic strategy for brain diseases. In recent years, researchers have continuously tried to regulate the expression of genes related to the nervous system through CRISPR-Cas9 system, which provides a new and efficient strategy for the treatment of brain diseases. At the same time, various delivery vectors of CRISPR-Cas9 system have been reported. Although some delivery vectors have not been applied to the research of brain diseases, they still provide valuable ideas for the brain delivery of CRISPR-Cas9 system. In this review, we summarized the principle of CRISPR-Cas9 system and its application in the nervous system, discussed the barrier of blood-brain barrier (BBB) to the treatment of brain diseases, overviewed various delivery vectors of CRISPR-Cas9 system and their applications, and highlighted advanced of CRISPR-Cas9 system applied to various brain diseases. Furthermore, we also discussed the existing obstacles and promising avenues for future investigation regarding CRISPR-Cas9-based therapeutic approaches. This article, through retrieving keyword combinations[PubMed,from Jan. 2018 to Dec. 2025], aims to elucidate the CRISPR-Cas9 system's potential for extensive future research and application as a therapeutic strategy for brain disorders.},
}
@article {pmid41670257,
year = {2026},
author = {Vollmer, SK and Stetter, MG and Hensel, G},
title = {First Successful Targeted Mutagenesis Using CRISPR/Cas9 in Stably Transformed Grain Amaranth Tissue.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70590},
pmid = {41670257},
issn = {1467-7652},
support = {EXC-2048/1 project ID 390686111//Deutsche Forschungsgemeinschaft/ ; STE 2654/4//Deutsche Forschungsgemeinschaft/ ; },
abstract = {Grain amaranth is a nutritionally rich, stress-tolerant C4 dicot with considerable potential for climate-resilient agriculture; however, efficient and reproducible protocols for stable transformation, regeneration, and CRISPR/Cas9-mediated editing have not yet been established. CRISPR/Cas-based genome editing is a cornerstone technology for accelerating the development of climate-resilient, high-yielding crops. Its effective application depends on robust, stable transformation procedures and CRISPR/Cas systems optimised for the target species. The absence of such tools remains a critical constraint for the genetic improvement of many promising yet underexplored crops. In this study, we edited key genes of the betalain biosynthesis pathway in grain amaranth (Amaranthus hypochondriacus L.) using the CasCADE modular cloning system, thereby demonstrating the feasibility of targeted mutagenesis in an orphan crop. We observed successful edits in up to 49% of transformed calli, resulting in deletions or insertions in the target genes. Our CRISPR/Cas9-mediated editing paves the way for targeted molecular research and breeding of grain amaranth.},
}
@article {pmid41670347,
year = {2026},
author = {Sullivan, JR and Ferrara, KM and Barrick, R and Romano, KP and Warrier, T and Hung, DT},
title = {An inducible CRISPRi system for phenotypic analysis of essential genes in Pseudomonas aeruginosa.},
journal = {mBio},
volume = {17},
number = {3},
pages = {e0276725},
pmid = {41670347},
issn = {2150-7511},
support = {U19 AI142780/AI/NIAID NIH HHS/United States ; U19AI142780/NH/NIH HHS/United States ; },
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/growth &amp; development ; *Genes, Essential ; Phenotype ; *CRISPR-Cas Systems ; Plasmids ; Gene Expression Regulation, Bacterial ; Rhamnose/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Promoter Regions, Genetic ; },
abstract = {Precise and tunable genetic tools are essential for high-throughput functional genomics. To address this need in the important gram-negative pathogen Pseudomonas aeruginosa, we developed and characterized a tightly regulated CRISPR-interference (CRISPRi) system that enables precise and tunable repression of essential genes. The system utilizes a rhamnose-inducible promoter to control both the Streptococcus pasteurianus-derived dCas9 and gene-specific sgRNAs, each encoded on separate plasmids for modularity and efficiency. The combination of tight regulation and high conjugation efficiency facilitated the rapid and facile construction of strains with regulated depletion of 16 essential genes spanning diverse pathways. Comparison of phenotypes across the different genetically depleted strains, including growth rate, susceptibility to antibiotics, and changes in transcriptional programs, revealed novel aspects of gene function or small-molecule mechanism of action. Finally, the rhamnose-inducible CRISPRi system supports the generation and stable maintenance of pooled mutant libraries, thereby paving the way for future genome-wide, systematic assessment of individual gene vulnerabilities, which will provide critical insights for target prioritization in antibiotic discovery efforts against this recalcitrant pathogen.IMPORTANCECRISPR-interference (CRISPRi) has become an invaluable tool for studying genetics. In particular, the ability to knockdown (KD) genes enables the study of essential genes and their role in cell survival. However, a tightly regulated gene KD system is required to gain valuable insights into these vulnerable genes by virtue of their essentiality. We report a tightly regulated CRISPRi system to study the biology of essential gene perturbations in Pseudomonas aeruginosa, an important gram-negative pathogen that causes severe infections and is increasingly resistant to current antibiotics. This system enables characterization of both chemical genetic interactions between small molecules and specific gene depletions and the impact of genetic perturbations on transcriptional networks. Genetic perturbations using CRISPRi can thus further our understanding of basic biology with translation toward future antimicrobial development.},
}
@article {pmid41671237,
year = {2026},
author = {Bagheri, N and Bertucci, A and Merlo, R and Porchetta, A},
title = {Synthetic DNA Transducers Integrate DNA Repair to CRISPR Signal Transduction.},
journal = {ACS sensors},
volume = {11},
number = {2},
pages = {1634-1644},
doi = {10.1021/acssensors.5c04118},
pmid = {41671237},
issn = {2379-3694},
mesh = {*DNA Repair ; Humans ; *CRISPR-Cas Systems/genetics ; DNA Glycosylases/metabolism ; Uracil-DNA Glycosidase/metabolism ; *Signal Transduction ; *DNA/genetics/metabolism/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins/metabolism/genetics ; },
abstract = {CRISPR-based molecular diagnostics have revolutionized nucleic acid detection, yet the integration of upstream enzyme activity into programmable CRISPR output remains largely unexplored. Here, we present a synthetic transduction platform that directly couples endogenous DNA repair activity with CRISPR-Cas12a activation. By linking base excision repair (BER) events to the structural switching of a programmable DNA transducer, we convert the activity of DNA glycosylases, such as uracil DNA glycosylase (UDG) and human 8-oxoguanine glycosylase (hOGG1), into a robust fluorescence signal via Cas12a-mediated collateral (trans-) cleavage. This one-step assay allows rapid and sensitive lysate-based detection of repair activity with high specificity. In addition, it can also be easily adapted to achieve rapid throughput screening of small molecule inhibitors. The rational modular design supports the adaptation to various glycosylase activities, establishing a general framework for transducing DNA repair activity into programmable CRISPR output. Beyond bioanalytical applications, this approach paves the way for the development of synthetic gene circuits that respond to DNA repair activity and CRISPR-based drug screening platforms.},
}
@article {pmid41671402,
year = {2026},
author = {Shi, M and Ge, W and Li, C and Liu, B and Deng, X and Liu, C and Zheng, M and Zhang, P and Li, L and Guo, Y and Han, Y and Yang, Y and Yu, YV and Jin, YN},
title = {Versatile CRISPR-Cas Tools for Gene Regulation in Zebrafish via an Enhanced Q Binary System.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e11485},
doi = {10.1002/advs.202511485},
pmid = {41671402},
issn = {2198-3844},
support = {32070832//National Natural Science Foundation of China/ ; 32150610476//National Natural Science Foundation of China/ ; 2042022dx0003//Fundamental Research Funds for the Central Universities/ ; },
abstract = {CRISPR-Cas systems revolutionize gene regulation across diverse organisms, including zebrafish. However, most zebrafish studies still rely on transient delivery of CRISPR components, with limited use of transgenic models, primarily restricted to Cas9-mediated knockouts. This limitation arises from challenges in achieving sustained, tissue-specific, and efficient expression of transgenic CRISPR effectors. To address these challenges, we introduce CRISPR-Q, a transgenic system that combines the QFvpr/QUAS binary expression platform with CRISPR-Cas technologies. CRISPR-Q overcomes the drawbacks of transient mRNA or protein delivery and circumvents the toxicity and transgene silencing issues associated with other binary systems, such as Gal4/UAS. The system enables robust and spatiotemporal expression of CasRx or dCas9vpr, allowing precise transcript knockdown (CRISPR-QKD) or gene activation (CRISPR-Qa). Using CRISPR-QKD, we achieve effective knockdown of smn1 and simultaneous knockdown of tardbp and tardbpl, modeling spinal muscular atrophy and amyotrophic lateral sclerosis, respectively. CRISPR-Qa activates endogenous lin28a and sox9b, demonstrating its functional versatility. We further validate CRISPR-Q's tissue-specific applicability in heart-specific transgenic zebrafish. Together, CRISPR-Q represents a robust and versatile platform for studying gene function and modeling human diseases in zebrafish, with broad potential for adaptation in other model organisms.},
}
@article {pmid41671414,
year = {2026},
author = {Yu, J and Park, JC and Uhm, H and Kim, YW and Im, HW and Bae, S},
title = {Evolution of Prime Editing: Enhancing Efficiency and Expanding Capacity.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {16},
pages = {e21015},
pmid = {41671414},
issn = {2198-3844},
support = {RS-2024-00332601//Korean Fund for Regenerative Medicine/ ; 25202MFDS003//Ministry of Food and Drug Safety/ ; 25B-001-0700//SNUH Lee Kun-hee Child Cancer and Rare Disease/ ; 2021M3A9H3015389//National Research Foundation of Korea/ ; RS-2024-00451880//National Research Foundation of Korea/ ; RS-2024-00455559//National Research Foundation of Korea/ ; SRC-NRF2022R1A5A102641311//National Research Foundation of Korea/ ; RS-2024-00404132//Ministry of Health &amp; Welfare/ ; },
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Genetic mutations cause approximately 80% of rare human diseases, highlighting the urgent need for precise genome editing. Since clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated 9 (Cas9) nucleases were first used for genome editing in 2012, genome editing technologies have rapidly advanced. Base editors, derived from the CRISPR-Cas system, were developed to introduce specific point mutations without requiring DNA double-strand breaks, and subsequently, prime editing (PE) technology was created to enable insertions, deletions, and all types of point mutations. The precision and versatility of PE make it a promising tool for clinical applications. However, PE has potential limitations, including low editing efficiency and limited capacity for large-scale manipulation. To overcome these limitations, research has been continuously conducted to improve PE efficiency and expand its capabilities. Therefore, this review aims to highlight current efforts and future directions for developing and improving PE-related tools.},
}
@article {pmid41671593,
year = {2026},
author = {Fattahi, M and Pirbadami, F and Motallebirad, T and Beheshti, Z and Azadi, D},
title = {Next-generation gene therapy for infectious disease: Advances, challenges, and future directions.},
journal = {Journal of infection and public health},
volume = {19},
number = {4},
pages = {103164},
doi = {10.1016/j.jiph.2026.103164},
pmid = {41671593},
issn = {1876-035X},
mesh = {Humans ; *Genetic Therapy/methods/trends ; *Communicable Diseases/therapy ; Gene Editing ; HIV Infections/therapy ; CRISPR-Cas Systems ; Bacteriophages/genetics ; },
abstract = {Infectious diseases, worsened by antimicrobial resistance and limitations of conventional treatments, demand innovative solutions. This systematic review evaluates recent advances in next-generation gene therapy for HIV, HBV, HPV, and multidrug-resistant(MDR) pathogens, while addressing key challenges and future directions. Following PRISMA guidelines, major databases were searched (2015-2025), yielding 1250 records. the114 peer-reviewed studies were included, assessed using the MMAT, and thematically synthesized for therapeutic strategies, efficacy, challenges, and prospects. Findings show CRISPR-based genome editing achieved HIV remission in preclinical and early clinical trials, engineered bacteriophages demonstrated strong efficacy against MDR bacteria, RNAi effectively silenced HBV, and phage-derived depolymerases reduced MDR biofilms. Adverse events included off-target effects, immunogenicity, and scalability issues. Efficacy varied, with CRISPR offering high specificity and phage therapies showing robust lysis. In conclusion, Gene therapy shows strong potential against resistant pathogens, but faces challenges like study heterogeneity, preclinical reliance, delivery barriers, and unequal access in low-and middle-income countries.},
}
@article {pmid41671611,
year = {2026},
author = {Raban, R and James, AA and Akbari, OS},
title = {Advances in CRISPR gene drives for mosquito population control.},
journal = {Current opinion in microbiology},
volume = {90},
number = {},
pages = {102712},
doi = {10.1016/j.mib.2026.102712},
pmid = {41671611},
issn = {1879-0364},
mesh = {Animals ; *Gene Drive Technology/methods ; *Culicidae/genetics ; *Mosquito Control/methods ; *CRISPR-Cas Systems ; *Mosquito Vectors/genetics ; },
abstract = {CRISPR-based gene drive (GD) systems bias allele inheritance during meiosis, enabling transgenes to spread at rates exceeding Mendel's law of segregation. This capability underlies their potential as powerful tools for controlling mosquito-borne diseases. GDs can be engineered either to suppress mosquito populations or to modify them by introducing traits that block pathogen transmission. Recent advances have focused on improving evolutionary stability, with modeling studies providing insights into expected population dynamics. With a focus on the most current population modification GDs, we discuss advances in GD architectures - including integral and allelic drives, combined modification-suppression systems, and both homing and non-homing toxin-antidote designs - that expand the range of possible strategies and address limitations of early homing drives. Numerous antipathogen effectors with strong pathogen-blocking activity can now be coupled to these systems, with current efforts assessing their durability against genetically diverse pathogens. Key challenges remain, including resistance evolution, ecological impacts, and long-term stability. Nonetheless, GDs offer a promising approach for reducing disease transmission, especially in regions where conventional interventions are difficult to sustain.},
}
@article {pmid41671823,
year = {2026},
author = {Wang, Z and Yan, R and Lin, P and Yao, Y and He, H and Zhuang, W and Hu, J and Chen, Y and Xu, S and Hu, WW},
title = {CRISPR/Cas12a-nanozyme visual biosensor for detection of microRNA-21.},
journal = {Talanta},
volume = {303},
number = {},
pages = {129517},
doi = {10.1016/j.talanta.2026.129517},
pmid = {41671823},
issn = {1873-3573},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; DNA, Single-Stranded/chemistry ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism ; Nucleic Acid Amplification Techniques ; Silver/chemistry ; },
abstract = {The precise diagnosis of aggressive tumors is crucial for guiding therapy, with microRNAs (miRNAs) increasingly recognized as pivotal biomarkers due to their regulatory roles in tumor development and dissemination. Herein, we report a CRISPR/Cas12a-nanozyme visual biosensor for direct and sensitive detection of tumor-associated miRNA-21. In this design, Ag/NiFe layered double hydroxide acts as a peroxidase-like nanozyme and is tethered to magnetic beads via single-stranded DNA (ssDNA) linkers. Upon recognition of miRNA-21, strand displacement amplification (SDA) can generate a DNA activator that activates CRISPR/Cas12a. The activated Cas12a then cleaves the ssDNA linkers, releasing the nanozyme to catalyze a chromogenic reaction that yields a visible color change. This approach enables sensitive detection of miRNA-21 with a detection limit of 420.0 fM, providing a direct visual readout and demonstrating strong potential for point-of-care nucleic acid diagnostics.},
}
@article {pmid41672287,
year = {2026},
author = {Chaumont, L and Peruzzi, M and Huetz, F and Raffy, C and Le Hir, J and Minke, J and Leong, JC and Boudinot, P and Collet, B},
title = {Knockout of the antiviral genes mx1 or mx3 modulates the expression of paralogous genes in a salmonid cell line.},
journal = {Fish &amp; shellfish immunology},
volume = {171},
number = {},
pages = {111204},
doi = {10.1016/j.fsi.2026.111204},
pmid = {41672287},
issn = {1095-9947},
mesh = {Animals ; *Myxovirus Resistance Proteins/genetics/immunology ; Cell Line ; *Fish Proteins/genetics/immunology ; Gene Knockout Techniques/veterinary ; *Fish Diseases/immunology ; *Immunity, Innate/genetics ; *Salmo salar/genetics/immunology ; *Gene Expression Regulation/immunology ; CRISPR-Cas Systems ; },
abstract = {Mx dynamin-like GTPases genes are Interferon Stimulated Genes (ISGs) encoding the Mx (myxovirus resistance) proteins that have an antiviral effect against a wide array of RNA viruses as well as some DNA viruses. In salmonids, the mx genes reside in four distinct chromosomal loci, that encode proteins clustering together into separate phylogenetic clades. To understand the contribution of Mx to the innate antiviral resistance, we knocked out the mx1 or mx3 gene by generating single mutant CHSE-derived cell lines by CRISPR/Cas9 genome editing. sgRNA were designed within the first coding exon of mx1 (LOC112247236), or mx3 (LOC112247235) genes, located 27 kb apart on the same chromosome LG02. Mx1, Mx2 and Mx3 proteins were quantified by western blotting. Only the Mx3 protein was found induced in the wild type EC cells after stimulation with recombinant Atlantic salmon interferon A2. mx1 gene knockout reduced the up-regulation of Mx3 protein expression, while mx3 gene knockout resulted in the induction of Mx1 and Mx2 proteins with and without additional stimulation. This was observed at the transcriptional level with the induction of mx1 gene was increased in mx3[-/-] cells compared to wild type cells. These results illustrate the complex interplay between mx1 and mx3 genes in salmonids.},
}
@article {pmid41672309,
year = {2026},
author = {Terakawa, Y and Koshi, D and Kawauchi, M and Nakazawa, T and An, GH and Honda, Y},
title = {Establishment of a CRISPR/Cas9 protocol by biolistic transformation of the filamentous basidiomycete Pleurotus ostreatus.},
journal = {Journal of microbiological methods},
volume = {243},
number = {},
pages = {107430},
doi = {10.1016/j.mimet.2026.107430},
pmid = {41672309},
issn = {1872-8359},
mesh = {*Pleurotus/genetics ; *CRISPR-Cas Systems ; *Transformation, Genetic ; *Gene Editing/methods ; *Biolistics/methods ; Genetic Vectors ; Genome, Fungal ; },
abstract = {Genome editing was successfully performed by introducing a CRISPR/Cas9 expression vector through biolistic transformation of Pleurotus ostreatus, an oyster mushroom. This method can be applied for genome editing in filamentous basidiomycetes without protoplasting. To our knowledge, this is the first report of genome editing with biolistic transformation in filamentous basidiomycetes.},
}
@article {pmid41673016,
year = {2026},
author = {Theerapanon, T and Intarak, N and Rattanapornsompong, K and Thaweesapphithak, S and Sriwattanapong, K and Prommanee, S and Kulvitit, S and Skrinjaric, T and Samaranayake, L and Pongpanich, M and Yeetong, P and Chaivoravitsakul, N and Mehl, NS and Assawapitaksakul, A and Srichomthong, C and Chetruengchai, W and Porntaveetus, T and Shotelersuk, V},
title = {A missense variant in ASCL5 leads to lobodontia.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41673016},
issn = {2041-1723},
mesh = {Animals ; Humans ; Mice ; Female ; Male ; *Basic Helix-Loop-Helix Proteins/genetics/metabolism ; *Mutation, Missense ; Pedigree ; Gene Knock-In Techniques ; Transcription Factors/genetics/metabolism ; Homeodomain Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Lobodontia, a rare dental anomaly marked by supernumerary cusps and a single pyramid-shaped molar root, has been previously linked to a variant in the CACNA1S gene without definitive evidence. This study investigates 17 patients with lobodontia from Thai and Croatian families. Microsatellite genotyping defines a 15.4 Mbp critical region encompassing CACNA1S and ASCL5 among Thai families. While genome sequencing confirms the CACNA1S variant only in the Thai patients, all 17 patients harbor the ASCL5 c.274 G > A (p.Glu92Lys) variant, which is absent in 12 unaffected members. Functional studies using CRISPR/Cas9-generated Ascl5 knock-in mutant mice demonstrate the dental anomalies resembling lobodontia in Ascl5[Mut/WT], while Ascl5[Mut/Mut] display severe defects in tooth and jaw development, underscoring the essential role of ASCL5 in craniofacial patterning. Transcriptomic analysis of E17.5 mandibular dental arches reveals differential expression of key craniofacial developmental genes in Ascl5[Mut/Mut] compared to Ascl5[WT/WT], including Dlx1as, and Dlx2. Luciferase assay shows that the p.Glu92Lys ASCL5 impairs DLX2 activation, further supporting the variant's pathogenicity. This study establishes ASCL5 as the gene responsible for lobodontia, revising its previously understood genetic basis, and highlights its crucial role in craniofacial development.},
}
@article {pmid41673027,
year = {2026},
author = {Melore, SM and McRoberts Amador, CD and Hamilton, MC and Gersbach, CA and Reddy, TE},
title = {dHyperCas12a enables multiplexed CRISPRi screens.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41673027},
issn = {2041-1723},
support = {R01 MH125236/MH/NIMH NIH HHS/United States ; UM1 HG012053/HG/NHGRI NIH HHS/United States ; U01 AI146356/AI/NIAID NIH HHS/United States ; R01 CA289574/CA/NCI NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; RM1 HG011123/HG/NHGRI NIH HHS/United States ; UM1 HG012053/HG/NHGRI NIH HHS/United States ; UM1 HG012053/HG/NHGRI NIH HHS/United States ; R01 MH125236/MH/NIMH NIH HHS/United States ; R01 MH125236/MH/NIMH NIH HHS/United States ; R01 CA289574/CA/NCI NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Induced Pluripotent Stem Cells/metabolism/cytology ; Animals ; RNA Polymerase II/genetics/metabolism ; Mice ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation ; Regulatory Sequences, Nucleic Acid/genetics ; Gene Editing/methods ; },
abstract = {Interactions between genes or cis-regulatory elements (CREs) underlie many biological processes. High-throughput CRISPR screens have allowed researchers to assess the impact of activation or repression of gene and regulatory elements on many phenotypes. However, assessment of interactions between those genes or elements remains limited. To enable efficient highly-multiplexed control of regulatory element activity, we combine a hyper-efficient version of Lachnospiraceae bacterium dCas12a (dHyperLbCas12a) with RNA Polymerase II expression of long CRISPR RNA (crRNA) arrays. We demonstrate this system with several activation and repression domains, in cultured primary immune cells, and to differentiate induced pluripotent stem cells. We also develop approaches to use dCas12a for simultaneous activation and repression. Lastly, we demonstrate that dHyperLbCas12a effectors can be used to dissect the independent and combinatorial contributions of CREs to gene expression. These tools create possibilities for highly multiplexed control of gene expression in many biological systems.},
}
@article {pmid41673416,
year = {2026},
author = {Gao, H and Li, Y and Chen, Y and Liu, X and Fang, M and Zhang, S and Ding, J and Zhu, D and Tan, A and Sheng, S},
title = {Genomic landscape and genetic manipulation of an ectoparasitoid wasp, Gregopimpla kuwanae.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41673416},
issn = {2399-3642},
support = {BK20241864//Natural Science Foundation of Jiangsu Province (Jiangsu Provincial Natural Science Foundation)/ ; 32400380//National Natural Science Foundation of China (National Science Foundation of China)/ ; 31925007//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Wasps/genetics ; *Genome, Insect ; CRISPR-Cas Systems ; RNA Interference ; Genomics ; Female ; Insect Proteins/genetics ; Pest Control, Biological ; Gene Editing ; },
abstract = {Parasitoid wasps are important biological control resources, yet their genetic manipulation has long been constrained by small body size and parasitization behavior, limiting their broader application in pest management. Here we report a chromosome-level genome assembly of the ectoparasitoid Gregopimpla kuwanae (322.87 Mb, 24 chromosomes), a relatively large species that parasitizes various lepidopteran pests. In the first part of this study, we established a foundational genomic resource and experimental platform by producing a high-quality genome and demonstrating the feasibility of functional genetics: RNA interference successfully silenced the cinnabar gene, while CRISPR/Cas9 editing generated vestigial knockout mutants, thus establishing G. kuwanae as a tractable system for gene manipulation. In the second part, we applied comparative genomics to identify lineage-specific gene-family expansions linked to parasitism, including venom-related genes, immune suppression factors, and detoxification enzymes (cytochrome P450s and UDP-glucosyltransferases), and we identified eight HGT candidates; one candidate (JSFChr12G01362) showed pre-feeding expression in females and caused increased adult mortality upon RNAi. Our study provides both the means and the candidates for mechanistic dissection of parasitoid adaptations, laying a foundation for the broader application of parasitoid wasps in sustainable biocontrol programs.},
}
@article {pmid41673492,
year = {2026},
author = {Jiang, Y and Hodgson, KJ and Segos, I and Lambie, EJ and Yang, L and Pan, M and Greig, A and Conradt, B},
title = {Tagging of C. elegans apoptosis activator EGL-1 BH3-only reveals CED-9 BCL-2-dependent mitochondrial localization and dynamic control of EGL-1 synthesis and degradation in vivo.},
journal = {Cell death and differentiation},
volume = {},
number = {},
pages = {},
pmid = {41673492},
issn = {1476-5403},
support = {BB/V007572/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/V015648/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; C0204/10-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; RSWF\R1\180008//Royal Society/ ; PhD stipend//China Scholarship Council (CSC)/ ; },
abstract = {The BH3-only protein EGL-1 is the key activator of apoptosis during C. elegans development. EGL-1 protein is thought to be synthesized predominantly in cells programmed to die and to localize to mitochondria. We used CRISPR-Cas-mediated modification of the egl-1 locus to add the coding sequence for the monomeric StayGold fluorescent protein or 18 copies of the SunTag peptide to the endogenous open reading frame. We found that tagged EGL-1 protein colocalizes with mitochondria in vivo and that mitochondrial localization is dependent on the anti-apoptotic BCL-2-like protein CED-9. Consistent with the presence of egl-1 mRNA in cells programmed to die as well as their progenitor cells ('mother' cells), EGL-1 protein is detected in both types of cells in vivo. Furthermore, real time imaging reveals that EGL-1 protein rapidly disappears from the mother cell prior to its division and that EGL-1 protein rapidly reappears specifically in the daughter cell programmed to die. Our results demonstrate CED-9 BCL-2-dependent mitochondrial localization of EGL-1 BH3-only protein and dynamic control of EGL-1 protein synthesis and degradation. Furthermore, we have identified additional levels of control of egl-1 BH3-only function that expand our understanding of apoptosis activation in vivo.},
}
@article {pmid41673558,
year = {2026},
author = {Sun, X and Wu, F and Ma, Z and Liang, G and Chen, S and Hu, X and Fan, S and Zhao, Y},
title = {Systematic synthesis of CRISPR/Cas applications for enhancing salt tolerance in crops: a decade of progress and challenges.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41673558},
issn = {1471-2229},
support = {42320104006//National Natural Science Foundation of China for International Cooperation/ ; },
mesh = {*CRISPR-Cas Systems ; *Crops, Agricultural/genetics/physiology ; *Salt Tolerance/genetics ; *Gene Editing ; Plants, Genetically Modified/genetics ; },
abstract = {Soil salinity is a major constraint on global crop productivity, driving the need for salt-tolerant varieties. While CRISPR-Cas genome editing offers targeted solutions for trait improvement, significant biological and technical bottlenecks limit its application in conferring salt stress resilience. This systematic summarizes findings from 83 peer-reviewed studies (2015-2024) employing CRISPR/Cas technologies to improve salt tolerance in five major crops (rice, wheat, maize, sorghum, barley). Our systematic review reveals that early single-gene edits achieved modest gains (30-50% Na[+] exclusion) but often showed limited yield gains in field settings, potentially due to compensatory regulation and environmental variation. The literature suggests that multiplex designs spanning ion homeostasis, osmoprotection, and ROS management can improve salt-tolerance outcomes and help maintain yield under severe salinity; however, the magnitude of benefit varies with crop, genotype, and transformation/regeneration context. Protein-protein interaction networks identified 12 hub genes and three functional modules, highlighting SOS3 and MPK6 as critical bottlenecks whose disruption risks pleiotropic effects. Spatial expression analysis underscored tissue-specific trade-offs, constitutive editing of root-dominant genes in shoots reduced yields by 15-28%, while tissue-optimized promoters minimized physiological conflicts. Persistent challenges include genotype-dependent transformation inefficiencies, epigenetic drift and environmental interactions under salt stress. Collectively, our synthesis consolidates and refines current best practices for salt-tolerance genome editing and highlights major bottlenecks-particularly regeneration/transformability, genotype dependence, and epigenetic constraints-that should be explicitly considered in experimental design and reporting.},
}
@article {pmid41673677,
year = {2026},
author = {Baena, JC and Cabrera-Salcedo, SC and Carrera Suárez, Y and Biancha-Vasco, JM and Rios-Serna, LJ and García-Mantilla, MD and Estrada-Schweineberg, M and Victoria Hincapie, JS and Toro-Pedroza, A and Garcia-Robledo, JE and Cañas, CA and Ortiz-Guzman, J and Loukanov, A},
title = {The avatar principle: exosomal dynamics guiding tumor adaptation and next-generation therapeutic strategies.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {159},
pmid = {41673677},
issn = {1477-3155},
mesh = {*Exosomes/metabolism ; Humans ; *Neoplasms/therapy/immunology ; Tumor Microenvironment ; Immunotherapy/methods ; Animals ; Receptors, Chimeric Antigen ; Avatar ; },
abstract = {Exosomes are nanoscale extracellular vesicles that transfer proteins, nucleic acids, and lipids, reflecting the state of their parent cells. A persistent scientific challenge is that tumor-derived exosomes (TDEs) facilitate immune evasion, remodel the tumor microenvironment, and create premetastatic niches, intensifying tumor aggressiveness and undermining therapeutic efficacy, ultimately narrowing treatment options to palliative strategies in advanced settings. Yet their dual roles as suppressive agents and potential therapeutic tools remain poorly integrated within current cancer immunotherapy frameworks. This review examines the molecular mechanisms underlying TDE-mediated immune suppression and therapeutic resistance, while also highlighting engineering strategies to exploit or counteract exosome biology. Exosomes derived from chimeric antigen receptor (CAR) T cells preserve antigen specificity and cytotoxic components without the risks of uncontrolled proliferation or cytokine release, offering a safer class of cell free immunotherapies. Advances in genetic engineering, hybrid vesicle design, and nanotechnology have extended exosome applications to the delivery of CRISPR/Cas systems, chemotherapeutic agents, immunoregulatory RNAs, and vaccines, with liposome or nanoparticle integration enhancing targeting and efficacy. Remaining obstacles include the lack of standardized protocols, scalability issues in production, and unresolved regulatory frameworks. Drawing on The Art of War, exosomes can be envisioned as avatars of strategy, discreet messengers capable of undermining host defenses while simultaneously carrying the potential to redirect immunity against the tumor. By embodying both deception and counterattack, they illustrate the capacity to penetrate hidden barriers and redefine the therapeutic battlefield, opening new horizons for precision cancer immunotherapy.},
}
@article {pmid41673772,
year = {2026},
author = {Gao, H and Li, B and Fengler, K and Yang, M and Schroder, M and Rahe, M and Sanyour-Doyel, N and Qi, J and LIaca, V and Beatty, M and Hu, WN and Barrett, B and Norman, B and Mo, H and Leonard, A and Wilson, B and Meeley, RB and Perugini, L and Krishnamurthy, N and Habben, JE and Tabor, G},
title = {Genome-Edited Maize Expressing Two Native Genes Confers Broad-Spectrum Resistance to Northern Corn Leaf Blight.},
journal = {Molecular plant pathology},
volume = {27},
number = {2},
pages = {e70205},
pmid = {41673772},
issn = {1364-3703},
mesh = {*Zea mays/genetics/microbiology ; *Gene Editing/methods ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; Plants, Genetically Modified ; CRISPR-Cas Systems/genetics ; *Plant Leaves/microbiology/genetics ; *Genes, Plant ; Ascomycota/pathogenicity ; },
abstract = {Northern corn leaf blight (NCLB) can result in yield losses of up to 50% in maize. The most effective strategy for managing NCLB is the deployment of resistant hybrids. Conventional breeding methods typically require 6 or 7 backcross generations to introgress a resistance locus, often bringing along undesirable traits that reduce yield. Recent advances in genome editing offer a precise alternative, enabling the targeted incorporation of resistance genes without linkage drag. In this study, we identified an NCLB resistance gene, NLB18-R, that is allelic to Htn1 and Ht2/Ht3. Using CRISPR-Cas9, we replaced the susceptible allele (NLB18-S) with NLB18-R in an elite inbred, resulting in enhanced resistance to NCLB. In a parallel experiment, we inserted both NLB18-R and the resistance gene Ht1-R into preselected, closely linked sites on chromosome 1. Through genetic crossing, we combined these edits into a stack. The resulting genome-edited plants exhibited resistance to Setosphaeria turcica races 0, 1 and 23N. Field trials under disease-free conditions showed no significant yield differences between hybrids carrying NLB18-R, Ht1-R, or the stack compared to null and wild-type controls. These findings demonstrate that CRISPR-Cas9-mediated genome editing is a powerful tool for rapidly developing commercial-grade maize hybrids with broad-spectrum resistance to NCLB, and potentially other diseases.},
}
@article {pmid41674076,
year = {2026},
author = {Arrigo, A and Rao, V and Ratan, A and Kulkarni, SS},
title = {Patient-informed CRISPR screen identifies FLNB as a congenital heart disease and ciliopathy gene.},
journal = {HGG advances},
volume = {7},
number = {2},
pages = {100580},
pmid = {41674076},
issn = {2666-2477},
mesh = {Humans ; Animals ; *Ciliopathies/genetics ; *Heart Defects, Congenital/genetics/diagnosis ; *Filamins/genetics ; *CRISPR-Cas Systems ; Male ; Female ; *Heterotaxy Syndrome/genetics/diagnosis ; Xenopus ; Exome Sequencing ; Mutation ; },
abstract = {Heterotaxy (HTX) is a congenital disorder characterized by abnormal left-right organ placement, often leading to severe congenital heart disease (CHD). Despite advances in sequencing, many CHD and HTX-associated genes remain functionally unvalidated, hindering effective clinical diagnosis and management. Here, we leveraged a high-throughput CRISPR-Cas9 screening approach in the Xenopus model to rapidly evaluate candidate genes identified from whole-exome sequencing of human CHD patients. Our screen identified Filamin B (FLNB), an actin-binding protein previously linked to skeletal disorders but not to ciliopathies or CHD. We identified 5 probands with CHD and HTX, 3 with recessive and 2 with damaging heterozygous variants in FLNB. Disrupting flnb in Xenopus reproduced key features of the human HTX phenotype, including defects in cardiac development and impaired motile cilia function. Rescue experiments confirmed the functional conservation of human FLNB, directly implicating actin cytoskeletal disruption in ciliogenesis and left-right patterning defects. Our results provide crucial evidence linking human FLNB dysfunction to ciliopathies and CHD and HTX.},
}
@article {pmid41674084,
year = {2026},
author = {Ghosh, PK and Biswas, S and Malaker, R and Pham, H and Septiningsih, EM and Ravelombola, W},
title = {Optimization of CRISPR/Cas9-mediated CtPDS knockout in guar protoplasts.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70177},
doi = {10.1002/tpg2.70177},
pmid = {41674084},
issn = {1940-3372},
support = {//Texas A&amp;M Institute for Advancing Health through Agriculture/ ; //Texas A&amp;M Advancing Discovery to Market/ ; GSC2024075//Texas Department of Agriculture/ ; 58-6046-2-008//USDA-ARS Germplasm Evaluation/ ; 58-6046-3-005//USDA-ARS Germplasm Evaluation/ ; },
mesh = {*CRISPR-Cas Systems ; *Protoplasts/metabolism ; *Gene Editing/methods ; *Cyamopsis/genetics ; *Plant Proteins/genetics ; Gene Knockout Techniques/methods ; },
abstract = {Guar (Cyamopsis tetragonoloba L. Taub.) is a climate-resilient legume with industrial and agricultural applications. Recently, gene editing has emerged as a key genetic tool for crop improvement. Despite its recent increasing value as a commodity for various uses, there is no documented report of gene editing work in guar to date. In this study, we present the first optimized protocol for protoplast-based clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing in guar. The most intact and viable protoplasts were observed in the cotyledons of 6-day-old seedlings that were isolated using the tape-sandwich method. Enzymatic digestion with 1.5% cellulase RS and 0.3% pectinase in 600 mM mannitol, followed by 10 min of vacuum infiltration, increased protoplast release and viability. Polyethylene glycol (PEG)-mediated transformation had been tailored using 40% PEG for 5 min with green fluorescent protein plasmid, and the cestrum yellow leaf curling virus promoter at room temperature showed the highest transient expression efficiency (∼57%). A multiplexed CRISPR/Cas9 construct was designed to target the Cyamopsis tetragonoloba phytoene desaturase (CtPDS) gene in guar. Polymerase chain reaction amplification and Sanger sequencing of transfected protoplasts confirmed highly efficient editing, with fragment deletions ranging from 714 to 1061 bp in CtPDS. Overall, we achieved 100% editing efficiency, as all successfully transformed samples showed CRISPR/Cas9-induced mutations. These findings establish a reliable, transient protoplast system for functional genomics and targeted trait improvement in guar, providing a key foundation for future crop improvement.},
}
@article {pmid41674223,
year = {2026},
author = {Goudar, P and Hall, A},
title = {The influence of GMO media strategies on public perceptions of CRISPR crop technologies in Southern Ontario.},
journal = {GM crops &amp; food},
volume = {17},
number = {1},
pages = {2620131},
pmid = {41674223},
issn = {2164-5701},
mesh = {Ontario ; *Public Opinion ; *Plants, Genetically Modified/genetics ; Humans ; Gene Editing ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems ; Adult ; Surveys and Questionnaires ; Male ; Female ; Middle Aged ; },
abstract = {Genetically modified organisms (GMOs) have often divided public opinion, one factor influencing perceptions of GMO technologies has been misunderstood or poorly communicated scientific messaging. However, advancements in gene editing tools like CRISPR/Cas9 offer new crop modification possibilities, prompting different regulatory frameworks than traditional GMO technologies. This research examines public understanding of GMOs, awareness of CRISPR crops, and how prior experiences with GMOs shape perceptions of new genetic technologies. A mixed-methods approach was employed, combining a public survey of adults in the Greater Toronto-Hamilton area and interviews with science journalists. Results show hesitance toward GMOs and CRISPR crops, with acceptance most impacted by consumer behavior and cost. Key interview themes include journalist's concerns about levels of public education, the role of social media, and the cost of goods. Our findings suggest increased transparency and effective communication could improve public acceptance of GMOs and CRISPR crops. While CRISPR crops do not come under the GMO regulatory framework in Canada, our findings show that the public does not recognize this distinction. Without increased transparency and more effective communication CRISPR crops may become widely associated with the negative media frames that have shaped perceptions of GMOs.},
}
@article {pmid41674382,
year = {2026},
author = {Zhang, Y and Xu, Y and Ding, Z and Cheng, Y and Gao, Y and Ye, J and Zhang, H and Li, P and Wu, H},
title = {Machine learning-optimized long single-stranded DNA synthesis technology empowers high-precision diagnostic-therapeutic integration in living cells.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41674382},
issn = {1362-4962},
support = {2021YFC2100600//National Key Research and Development Program of China/ ; 31872608//National Natural Science Foundation of China/ ; 31872608//National Natural Science Foundation of China/ ; },
mesh = {*DNA, Single-Stranded/chemistry/genetics/chemical synthesis/biosynthesis ; Humans ; *Machine Learning ; Gene Editing/methods ; Nanotechnology/methods ; DNA-Directed DNA Polymerase/metabolism ; CRISPR-Cas Systems ; },
abstract = {While DNA nanotechnology holds transformative potential across biomedical and information storage applications, current technologies face critical limitations in synthesizing long single-stranded DNA (ssDNA) with high purity and homogeneity. To address these challenges, we developed Ouroborosyn-ssDNA, a nicking enzymatic assisted replication (NEAR) platform that synergizes enzymatic engineering with computational optimization. By integrating phi29 DNA polymerase and Nb.BbvCI nickase in formate-based buffers, we achieved extended ssDNA synthesis up to 15 000 nt while preserving sequence fidelity, resulting in a 4.73-fold yield enhancement compared to commercial buffers. Notably, machine learning-guided parameter optimization identified magnesium ion dynamics and thermal modulation as pivotal determinants of enzymatic efficiency. Furthermore, solid-phase synthesis using thiol-gold immobilized templates demonstrated 86.38% purification recovery via automated magnetic bead systems, enabling scalable production. To validate functional utility, we engineered six-helix bundle DNA origami-CRISPR complexes that achieved nucleolin-targeted genome editing in cervical cancer cells, coupling GFP-based diagnostics with therapeutic E7 oncogene disruption. These advancements directly overcome key limitations in enzymatic stochasticity and product heterogeneity through buffer engineering and computational optimization, establishing a scalable pathway for applications in precision nanomedicine, synthetic biology, and molecular data storage. This integrated strategy advances DNA nanotechnology from proof-of-concept studies toward standardized biomanufacturing of sequence-defined macromolecular architectures.},
}
@article {pmid41674468,
year = {2026},
author = {Takeda, T and Aso, M and Ueda, H and Yuzawa, S},
title = {Direct Pathway Synthesis and Editing (DiPaSE): A One-Pot DNA Assembly Method for Accurate and Efficient Refactoring of High-GC Biosynthetic Gene Clusters.},
journal = {ACS synthetic biology},
volume = {15},
number = {3},
pages = {1221-1230},
doi = {10.1021/acssynbio.5c00934},
pmid = {41674468},
issn = {2161-5063},
mesh = {*Multigene Family/genetics ; *Gene Editing/methods ; Streptomyces/genetics/metabolism ; *Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; Base Composition/genetics ; Genome, Bacterial ; Escherichia coli/genetics ; Biological Products/metabolism ; },
abstract = {Natural products (NPs) produced by actinobacteria, particularly Streptomyces species, represent a rich source of bioactive compounds and have yielded many clinically important compounds. Actinobacterial genomes are characterized by high GC content and typically harbor 20-40 biosynthetic gene clusters (BGCs) per genome, which encode diverse NPs such as polyketides, peptides, and glycosides. CRISPR/Cas-based genome editing has emerged as a promising tool to activate silent BGCs and engineer NP biosynthesis. However, the efficiency of multiplex editing drastically decreases as the number of targeted sites increases. Here, we report a novel one-pot DNA assembly method, termed direct pathway synthesis and editing (DiPaSE), for the efficient synthesis and multiplex editing of long, high-GC BGCs. DiPaSE accurately assembles multiple high-GC DNA fragments up to 60 kb and enables simultaneous deletions and insertions within a target BGC without compromising the assembly efficiency. Using this approach, we identified functions of previously uncharacterized genes in the aureothin BGC and significantly enhanced the titer of the corresponding NP. The workflow employs conventional polymerase chain reaction, type IIP restriction enzymes, commercially available DNA assembly reagents, and Escherichia coli, providing a simple, cost-effective, and broadly applicable platform for genome mining, BGC refactoring, and rational design of artificial biosynthetic pathways.},
}
@article {pmid41674724,
year = {2026},
author = {Baum, R and Telugu, N and Bruyneel, AAN and Kay, M and Nair, P and Perea-Gil, I and Termglinchan, V and Bharucha, N and Lee, E and Mercola, M and Diecke, S and Karakikes, I},
title = {CRISPR-MiX: A pooled single-stranded donor strategy to enhance HDR efficiency in human iPSCs.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102820},
pmid = {41674724},
issn = {2162-2531},
abstract = {CRISPR-Cas9 is widely used to model genetic disorders by introducing or correcting disease-associated mutations in induced pluripotent stem cells (iPSCs) through homology-directed repair (HDR). However, HDR efficiency in iPSCs remains low and is highly dependent on the target locus. Here, we developed CRISPR-MiX, an improved protocol to enhance HDR efficiency in human iPSCs. Using a GFP-to-BFP reporter system, we identified key single-stranded oligodeoxynucleotide (ssODN) donor design parameters, including homology arm symmetry, CRISPR/Cas-blocking mutations, and strand complementarity, which significantly influence HDR outcomes. We applied this approach to introduce pathogenic variants into five genes related to genetic cardiomyopathies. Quantitative analysis of HDR events showed that both the target locus and ssODN design strongly affect HDR efficiency. To address the locus- and design-specific limitations, we established CRISPR-MiX, a pooled ssODN-based method for scarless genome editing using ribonucleoproteins (RNPs) that does not require selection. CRISPR-MiX consistently improved HDR efficiency across multiple loci. This strategy offers a simple, robust, and versatile approach for precise genome engineering in iPSCs, supporting broad applications in disease modeling and functional genomics.},
}
@article {pmid41674784,
year = {2025},
author = {Yashooa, RK and Nabi, AQ and Smail, SW and Azeez, SS and Nooh, WA and Mustafa, SA and Al-Farha, AA and Capitanio, N and Shekha, MS},
title = {CRISPR-Cas technologies in neurodegenerative disorders: mechanistic insights, therapeutic potential, and translational challenges.},
journal = {Frontiers in neurology},
volume = {16},
number = {},
pages = {1737468},
pmid = {41674784},
issn = {1664-2295},
abstract = {CRISPR-Cas genome-editing technologies have emerged as powerful tools for precise DNA and RNA modulation, offering promising therapeutic strategies for neurodegenerative disorders such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). This review critically evaluates current CRISPR/Cas applications in neurodegeneration, with emphasis on mechanistic insights, therapeutic outcomes, and translational feasibility. Preclinical and early translational studies demonstrate that CRISPR-Cas platforms can correct pathogenic mutations, suppress toxic gene expression, and restore neuronal function. Advanced modalities, including base and prime editing, CRISPRi/a, and RNA-targeting Cas systems, improve precision and reduce genomic damage, which is particularly advantageous in post-mitotic neurons. Emerging CRISPR-based diagnostics (e.g., SHERLOCK and DETECTR), AI-assisted sgRNA design, and machine-learning approaches for predicting off-target effects further enhance the safety, stratification, and monitoring of CRISPR therapeutics. In parallel, patient-derived brain organoids and assembloids provide scalable human-relevant platforms for mechanistic studies and preclinical validation. Despite this progress, major challenges remain, including efficient delivery across the blood-brain barrier, immune responses, long-term safety, and ethical and regulatory considerations. Overall, CRISPR-Cas technologies hold strong potential as disease-modifying interventions for neurodegenerative disorders, provided that advances in delivery systems, artificial intelligence integration, and regulatory oversight continue to evolve toward clinical translation.},
}
@article {pmid41676858,
year = {2026},
author = {Niu, Y and Wu, S and Su, J},
title = {Harnessing CRISPR/Cas systems for food safety detection: biosensor design and emerging applications for food safety detection.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {8},
pages = {1559-1582},
doi = {10.1039/d5ay01791b},
pmid = {41676858},
issn = {1759-9679},
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Food Safety/methods ; Humans ; Foodborne Diseases ; Food Microbiology/methods ; Food Contamination/analysis ; },
abstract = {Food safety has become a critical global concern, with foodborne diseases affecting approximately 600 million people annually and causing 420 000 deaths each year, posing significant risks to human health and well-being. Rapid, efficient, and reliable detection methods are essential to mitigate these risks. Traditional detection methods, such as PCR and culture-based assays, while widely used, often face challenges related to speed, accuracy, and portability. Over the past 5 years (2020-2025), the (CRISPR)/Cas system has emerged as a powerful tool for food safety detection due to its high sensitivity, specificity, and versatility. This review highlights recent advances in CRISPR/Cas-based biosensors and their applications in food safety. First, we discuss the key challenges in food safety detection and the design principles of CRISPR/Cas biosensors. Next, we comprehensively summarize their applications in detecting foodborne pathogens (viruses and bacteria), food fraud, genetically modified organisms (GMOs), toxins, heavy metals, antibiotic residues, and pesticides. Finally, we address the current limitations and future prospects of CRISPR/Cas biosensors, providing insights into their potential for next-generation food safety solutions.},
}
@article {pmid41677799,
year = {2026},
author = {Boubakri, H},
title = {CRISPR-Cas9-mediated genome editing in fungi: applications, challenges, and future directions.},
journal = {Journal of applied microbiology},
volume = {137},
number = {3},
pages = {},
doi = {10.1093/jambio/lxag046},
pmid = {41677799},
issn = {1365-2672},
mesh = {*Gene Editing/methods/trends ; *CRISPR-Cas Systems ; *Fungi/genetics ; *Genome, Fungal ; },
abstract = {The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been shown to be an effective genome-editing tool in many organisms, including fungi. It enables precise modifications to the DNA of fungal species, facilitating advancements in research, agriculture, and biotechnology. CRISPR-Cas9-edited non-pathogenic antagonists have emerged as a promising alternative for biocontrol. Several filamentous fungi have been engineered to produce secondary metabolites. Furthermore, the CRISPR-Cas9 system has been used to improve the quality of several edible fungi. However, the application of CRISPR-Cas9 technology for fungal genome editing is still facing some challenges that researchers must address. This review highlights the major approaches and applications of genome editing in fungi, as well as the associated challenges.},
}
@article {pmid41678334,
year = {2026},
author = {Wang, Y and Su, X and Chen, Y and Chen, Y and Shi, C and Liu, F and Ye, Y and Sun, P and Tan, M and Yu, M and Wang, Y and Xie, S and Liu, J and Yan, Q and Sun, Q and Neculai, D and Liu, W and Shao, J and Liu, Y and Lin, W and Lin, A},
title = {A CRISPR-based mitochondrial gene therapy tool derived by engineering guide RNAs.},
journal = {Cell reports},
volume = {45},
number = {2},
pages = {116958},
doi = {10.1016/j.celrep.2026.116958},
pmid = {41678334},
issn = {2211-1247},
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Mitochondria/genetics/metabolism ; *Genetic Therapy/methods ; DNA, Mitochondrial/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Mitochondrial Diseases/genetics/therapy ; RNA, Long Noncoding/genetics/metabolism ; Gene Editing/methods ; Mice ; RNA-Binding Proteins/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Mitochondrial genetic diseases arise from mitochondrial DNA (mtDNA) defects, which gene therapy tools may rectify. However, delivering single-guide RNAs (sgRNAs) into mitochondria remains a challenge limiting CRISPR-mediated mtDNA therapy. Here, through network analysis of mitochondrion-localized long noncoding RNAs (lncRNAs) and RNA-binding proteins (RBPs), we found that lncRNA RP11-46H11.3 translocates into mitochondria via binding mitochondria-associated RBPs using its key RNA recognition motifs (RRMs); its derived 30 nt ST2-RNA mitochondrial targeting sequence (RMTS) showed the highest mitochondrial localization efficiency. We engineered the RMTS-CRISPR tool by fusing ST2-RMTS to sgRNA, verifying its ability to target and cleave mtDNA. Strikingly, our results demonstrated that RMTS-CRISPR could achieve heteroplasmic mtDNA shifting efficiencies of up to 26.37% in m.3243A>G mutant cell models and 26.79% in vivo, offering a technological approach for the correction of heterogeneous mtDNA mutations. Taken together, our findings reveal a CRISPR-based mitochondrial gene intervention strategy that may have applications in mitochondrial disorders.},
}
@article {pmid41678341,
year = {2026},
author = {Wang, Z and Wang, Y and Ji, Q},
title = {Bacterial Cofactors for CRISPR Activation.},
journal = {Biochemistry},
volume = {65},
number = {5},
pages = {501-504},
doi = {10.1021/acs.biochem.5c00738},
pmid = {41678341},
issn = {1520-4995},
mesh = {*CRISPR-Cas Systems ; *Thioredoxins/metabolism/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacterial Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; *Coenzymes/metabolism ; *Bacteria/metabolism/genetics ; },
abstract = {Anti-CRISPR (Acr) proteins have long exemplified the viral counterattack against CRISPR-Cas immunity. By contrast, comparatively little is known about host proteins that may increase Cas effector activity. Recent work on a compact type V nuclease, Cas12p, demonstrates that this phage-associated effector depends on the bacterial thioredoxin TrxA for efficient DNA cleavage. TrxA binds a dedicated thioredoxin-binding (TB) domain on Cas12p through a redox-sensitive interaction, promoting an active conformation competent for DNA cleavage. This finding adds to a small but growing set of CRISPR activators and highlights that CRISPR-Cas systems are not static defense modules but dynamic networks shaped by auxiliary factors that can fine-tune their activity.},
}
@article {pmid41678468,
year = {2026},
author = {Mendoza-Garcia, P and Keith, B and Nordberg, M and Quist, E and Ferrás, C and Hamza, GM and Elgendy, R and Ashenden, SK and Chi, J and van Zuydam, NR and Hattersley, N and Zhang, X},
title = {Omics-aided design genome editing strategy for challenging human immortalized cell models.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0341124},
pmid = {41678468},
issn = {1932-6203},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; MCF-7 Cells ; Hep G2 Cells ; DNA End-Joining Repair ; Proliferating Cell Nuclear Antigen/genetics/metabolism ; Genomics/methods ; Gene Knock-In Techniques ; },
abstract = {CRISPR-Cas9 has become a popular genome editing tool for biomedical research and drug development due to its capability to enable precise correction or integration of genetic mutations in the genome. However, precise genome editing competency varies dramatically between cell types depending on their capabilities for DNA damage. In this proof-of-concept study, we took the example of HepG2 and MCF7 to show that omics profiling identifies bottlenecks that are associated with poor precise knock-in (KI) efficiency in hard-to-engineer cells. These bottlenecks include previously described factors such as the predominance of non-homologous end joining (NHEJ) repair and impaired homologous recombination (HR) capability, but also reveals apoptotic priming status of the cells as a limiting factor. Upon further comparative analysis between HepG2 and MCF7 cells, we pinpointed and validated the proliferating cell nuclear antigen (PCNA) as a target to overexpress to enhance precise KI efficiency in MCF7. Overall, we describe how employing a multi-omics approach to characterize cell models of interest can facilitate an in-depth understanding of their editability molecular signature, empowering us to manipulate the activity of key pathways for precise editing, and therefore increase efficiency of desired editing outcomes.},
}
@article {pmid41678638,
year = {2026},
author = {El-Brolosy, MA and Oak, A and Hoang, AT and Damergi, Y and Fischer, A and Saunders, RA and Luo, J and Balabaki, A and Guez, J and Whitfield, TW and Goldman, SR and Latifkar, A and Lu, YR and Stainier, DYR and Karczewski, KJ and Corradin, O and Weissman, JS},
title = {Mechanisms linking cytoplasmic decay of translation-defective mRNA to transcriptional adaptation.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6786},
pages = {eaea1272},
doi = {10.1126/science.aea1272},
pmid = {41678638},
issn = {1095-9203},
mesh = {Humans ; CRISPR-Cas Systems ; *Cytoplasm/metabolism ; *Protein Biosynthesis ; *RNA Stability ; *RNA, Messenger/metabolism/genetics ; *RNA-Binding Proteins/metabolism/genetics ; *Transcription, Genetic ; HEK293 Cells ; },
abstract = {Transcriptional adaptation (TA) is a genetic robustness mechanism through which mutant messenger RNA (mRNA) decay induces sequence-dependent up-regulation of so-called adapting genes. How cytoplasmically generated mRNA fragments affect nuclear transcription remains poorly understood. Using genome-wide CRISPR screens, we uncover ILF3 as an RNA binding protein connecting cytoplasmic mRNA decay and transcription during TA and show that it is required for a range of TA substrates. ILF3 is enriched at adapting genes' RNAs, and its artificial recruitment through dCas13 promotes gene expression. Using tiling oligonucleotide screens, we identify trigger RNA fragments that activate adapting genes when introduced into cells. Further functional dissection reveals a critical role for homology between trigger and target sequences. These findings enhance our molecular understanding of TA and inform the design of programmable oligonucleotides for gene expression augmentation.},
}
@article {pmid41678668,
year = {2026},
author = {Kim, J and Yoon, J and Chen, J and Lee, J and Lee, HJ and Whitworth, K and Redel, B and Prather, RS and Lee, K},
title = {Enhancing the specificity of gene editing outcomes by using Cas9 variants in porcine embryos.},
journal = {Journal of animal science},
volume = {104},
number = {},
pages = {},
pmid = {41678668},
issn = {1525-3163},
mesh = {Animals ; *Gene Editing/veterinary/methods ; Swine/embryology/genetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Embryo, Mammalian ; Female ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 technology has improved the ability to introduce targeted modifications in cells and embryos in diverse species. The use of this technology enables the establishment of genetically modified livestock models to study human diseases or improve food production. However, one of the main concerns with employing this technology is the possibility of introducing unintended genome modifications induced by the Streptococcus pyogenes Cas9 (SpCas9), a commonly used Cas9 protein. Recent advancements in CRISPR/Cas9 technology offer Cas9 variants that are designed to improve gene editing specificity. Here, three high-fidelity SpCas9 variants (eSpCas9, HiFi Cas9, and LZ3 Cas9) were employed to examine their efficacy and specificity in pig embryos. To introduce targeted modifications, mRNA coding for each Cas9 variant was mixed with IGH single guide RNA (sgRNA) and were injected into fertilized pig zygotes. The frequency of on- and off-targeting was calculated by amplifying IGH, AR, and RBFOX1 regions from genomic DNA derived from the injected embryos at the blastocyst stage and sent for Sanger sequencing. The sgRNA targeting IGH locus resulted in a 100% on-target editing rate using SpCas9. However, SpCas9 introduced off-targeting events in AR and RBFOX1 at a high frequency (> 60%) in embryos. Injecting each Cas9 variant at 20 ng/µl could modify the target gene (IGH) at 100% efficiency except for LZ3 Cas9 (59.1%). Importantly, off-target events on AR and RBFOX1 were not detected in any Cas9 variant groups. Gradually reducing the concentration of Cas9 mRNAs lowered the efficacy of on-targeting in all groups; however, the reduction was more dramatic in HiFi Cas9 and LZ3 Cas9 injected embryos. No embryonic toxicity was identified in embryo injected with Cas9 variants and more embryos reached blastocyst stage when injected with either eSpCas9 or HiFiCas9 mRNA. In vivo competency of embryos receiving eSpCas9 was examined by embryo transfer and fetuses recovered from a pregnant sow presented 100% on-target editing efficiency without any detectable off-target events. In summary, among the Cas9 variants examined, eSpCas9 presented the highest specificity with no detectable off-target events and supported the development of gene-edited fetuses. Our findings indicate that the use of Cas9 variants can advance the field of gene editing in livestock models.},
}
@article {pmid41679028,
year = {2026},
author = {Yao, F and Qi, X and Yongli, S and Xiaofen, Z},
title = {Generation of a SV2A knockout human embryonic stem cell line by CRISPR/Cas9 system.},
journal = {Stem cell research},
volume = {91},
number = {},
pages = {103924},
doi = {10.1016/j.scr.2026.103924},
pmid = {41679028},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Membrane Glycoproteins/genetics/metabolism/deficiency ; *Nerve Tissue Proteins/genetics/metabolism/deficiency ; *Gene Knockout Techniques ; Cell Line ; },
abstract = {Synaptic Vesicle Glycoprotein 2A (SV2A) is a ubiquitously expressed brain glycoprotein, localized to synaptic terminals. It regulates vesicle exocytosis, maintains neurotransmitter release, and serves as a receptor for both botulinum neurotoxins (e.g., BoNT/A) and tetanus neurotoxin (TeNT). It is a target for antiseizure drugs and implicated in epilepsy, Alzheimer's, and Parkinson's diseases. We generated a homozygous SV2A-knockout human embryonic stem cell (hESC) line WAe001-A-3F (H1-SV2A[-/-]), using CRISPR/Cas9 genome editing technology. The SV2A-knockout embryonic stem cell lines provide a precise in vitro model to dissect its roles in synaptic function and disease mechanisms.},
}
@article {pmid41679300,
year = {2026},
author = {Wang, Y and Hu, W and Xia, R and Yang, X and Gu, Y and Ning, Z and Yang, T and Yu, C and Zhang, L and Li, D and Jin, Y and Li, J and Zhang, F and Xu, Y and Xu, C and Wang, Z and Jing, N and Chen, L and Wang, G},
title = {CLIM-TIME identifies metastatic microenvironment modulators for T cell therapy response.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1555-1572.e23},
doi = {10.1016/j.cell.2025.12.042},
pmid = {41679300},
issn = {1097-4172},
mesh = {*Tumor Microenvironment/immunology/genetics ; Animals ; *T-Lymphocytes/immunology ; Mice ; Humans ; Immunotherapy/methods ; Laser Capture Microdissection/methods ; Mice, Inbred C57BL ; Lung Neoplasms/secondary/immunology/therapy ; Cell Line, Tumor ; Extracellular Matrix/metabolism ; CRISPR-Cas Systems ; Neoplasm Metastasis ; Neoplasms/immunology/therapy/pathology ; },
abstract = {The tumor microenvironment (TME) poses a major barrier to effective immunotherapy, yet high-throughput perturbation-mapping approaches to dissect TME spatial complexity and its contextual immune modulators remain lacking. Here, we introduce CRISPR-laser-captured microdissection (LCM) integration mapping of the tumor-immune microenvironment (CLIM-TIME), a scalable platform that integrates CRISPR screening with LCM of metastatic tumors for transcriptomic, deconvolution, and immunofluorescence analyses. CLIM-TIME enables spatially resolved mapping of how tumor suppressor gene (TSG) loss reshapes the TME and modulates immune responses. We identified seven distinct TME subtypes, revealing that DNA repair and Polycomb repressive complex (PRC) TSG loss is linked to immune-infiltrated TMEs sensitive to T cell therapy. In contrast, knockouts of TSGs in the Hippo pathway promoted immune evasion and therapy resistance by fostering myeloid-enriched but T cell-excluded TMEs with elevated extracellular matrix (ECM). Targeting the ECM-crosslinking enzyme LOXL2 effectively remodeled the metastatic TME, enhancing T cell infiltration and improving therapeutic efficacy in lung metastases across multiple cancers.},
}
@article {pmid41679307,
year = {2026},
author = {Zhao, J and Wang, Z and Lu, L and Bu, G and Miao, Z and Zhang, Y and Guo, Y and Yang, Z and Ma, J and Jiao, J and Ma, X},
title = {An orthogonal CRISPR/Cpf1 platform for precise spatiotemporal gene regulation and osteoporotic fracture repair.},
journal = {Cell reports methods},
volume = {6},
number = {2},
pages = {101299},
pmid = {41679307},
issn = {2667-2375},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Fracture Healing/genetics ; *Osteoporotic Fractures/genetics/therapy ; Humans ; Gene Editing/methods ; Osteoblasts/metabolism ; *Gene Expression Regulation ; Osteogenesis/genetics ; Genetic Therapy/methods ; Bone Morphogenetic Protein 2/metabolism/genetics ; Disease Models, Animal ; },
abstract = {CRISPR-Cas systems enable powerful gene editing and regulation, yet single-modality control often fails to achieve orthogonal, spatiotemporally precise regulation of multiple endogenous genes. We engineered OREC, an orthogonal platform integrating chemogenetic and optogenetic modalities for precise, reversible, multiplex gene control. OREC comprises two components: OREC[C] regulated by doxycycline (Dox) and OREC[o] controlled by light. By assembling catalytically dead Cpf1 (dCpf1), gene regulatory elements, and crRNA arrays on single transcripts, OREC enables robust simultaneous manipulation of multiple genes. We demonstrated OREC's therapeutic potential in vitro for osteoblast function modulation and in vivo for osteoporotic fracture repair. OREC effectively activated Bmp2 while inhibiting Dkk1, significantly enhancing bone formation and fracture healing in mouse models. These results establish OREC as a versatile platform for precise multiplex gene regulation, offering significant advancement for CRISPR-based gene therapy applications in complex tissues where coordinated control of multiple therapeutic targets is essential.},
}
@article {pmid41679545,
year = {2026},
author = {Bahlmann, N and Alshawabkeh, M and Tsoukas, R and Schröer, K and Schellhorn, S and Sieler, M and Dittmar, T and Ehrke-Schulz, E and Ehrhardt, A and Zhang, W},
title = {Desmoglein 2 (DSG2)-knockout human respiratory epithelial cell model to study species B adenovirus receptor usage.},
journal = {Virologica Sinica},
volume = {41},
number = {1},
pages = {172-181},
pmid = {41679545},
issn = {1995-820X},
mesh = {Humans ; *Desmoglein 2/genetics/metabolism ; *Receptors, Virus/genetics/metabolism ; Gene Knockout Techniques ; Membrane Cofactor Protein/genetics/metabolism ; *Adenoviruses, Human/genetics/physiology ; CRISPR-Cas Systems ; *Epithelial Cells/virology/metabolism ; *Coxsackie and Adenovirus Receptor-Like Membrane Protein/genetics/metabolism ; Cell Line ; Cell Proliferation ; },
abstract = {With an increasing number of human adenoviruses identified, the selection of potential therapeutic vectors broadens. For safety reasons, achieving cell-specific gene delivery is crucial to minimize off-target effects. Therefore, it is essential to gain a systematic understanding of adenovirus receptor-usage. Our aim is to establish a human-originated in vitro model for comparative analysis of human adenoviruses receptor usage. Based on our previous work of human CD46 and coxsackievirus and adenovirus receptor (CAR) knockout cell lines, we generated desmoglein 2 (DSG2) knockout cell lines using genome-engineering technology based on Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9 (CRISPR/Cas9). All together, we established a panel of cell lines that carry a single, double, or triple knockout of the three major human adenovirus receptors: CAR, CD46 and DSG2. Notably, cell proliferation speed was affected by the CAR-knockout, but not the DSG2-or CD46-knockouts. In addition, the spheroid formation ability was sharply reduced in CAR- or DSG2-knockout cells, but not the CD46-knockout cells. With this receptor-knockout model, we confirmed the receptor usage of nine species B adenoviruses. Furthermore, adenovirus vectors containing a previously identified DSG2-binding affinity-enhanced mutation showed DSG2-dependent cell entry within this cell model, which indicates that they are de-targeted from CD46 - the ubiquitously expressed receptor on all nucleated cells. Collectively, our findings show that the adenovirus major receptor knockout cell lines can serve as an in vitro model to help select adenovirus types suitable for individual applications and to better understand adenovirus infection biology.},
}
@article {pmid41679604,
year = {2026},
author = {Li, L and Wang, Y and Wang, B and Shen, L and Gao, Y and Lin, W and Li, Z},
title = {A dCas9-integrated iLight9O system enables dynamic regulation for enhanced patchoulol biosynthesis in Saccharomyces cerevisiae.},
journal = {Bioresource technology},
volume = {446},
number = {},
pages = {134195},
doi = {10.1016/j.biortech.2026.134195},
pmid = {41679604},
issn = {1873-2976},
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Optogenetics/methods ; Light ; },
abstract = {Numerous organisms have evolved the ability to utilize light through photoreceptor proteins that mediate diverse biological processes. Currently, several optogenetic sensor systems are widely used in yeast. However, when these systems are applied for gene repression to regulate endogenous yeast gene expression, they typically require the insertion of corresponding target sites near the native promoter of the gene of interest to achieve precise modulation. To address these constraints, a novel blue light-inducible optogenetic tool designated iLight9 was developed, a single-component optogenetic biosensor integrated with the CRISPR-dCas9 platform. The stability of the iLight9 system was further enhanced by employing a strategy involving the addition of a protein degradation tag. The resulting system was designated as iLight9O, which facilitated programmable regulation of distinct genes through the introduction of specific sgRNAs. Subsequently, systematic metabolic engineering strategies were employed to construct an efficient patchoulol-producing cell factory in Saccharomyces cerevisiae. Moreover, a two-step isoprenol utilization (IU) pathway was introduced into the recombinant strain to enhance its capacity for patchoulol biosynthesis. Crucially, the iLight9O system was adopted to dynamically downregulate squalene synthase, a key enzyme in the competing squalene biosynthetic pathway. This optogenetic flux control strategy increased patchoulol titers by 66 % in the IU-optimized strain and 24 % in the MVAIU2 strain, demonstrating significant improvements over static engineering approaches.},
}
@article {pmid41679990,
year = {2026},
author = {Carota, AG and Spiaggia, F and Poma, N and Palladino, P and Cuffaro, D and Vivaldi, F and Ravelet, C and Di Francesco, F and Minunni, M},
title = {Highly fluorescent copper nanoclusters as programmable reporters for CRISPR/Cas12a-based detection of bacterial DNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {300},
number = {},
pages = {118492},
doi = {10.1016/j.bios.2026.118492},
pmid = {41679990},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Copper/chemistry ; *Biosensing Techniques/methods ; *DNA, Bacterial/isolation &amp; purification/genetics ; Fluorescent Dyes/chemistry ; *Escherichia coli/genetics/isolation &amp; purification ; *Metal Nanoparticles/chemistry ; Humans ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Early and accessible pathogen detection is crucial for global health security and demands diagnostic assays that are rapid, affordable, and suitable for Point-of-Care use. This study presents a cost-effective, rapid, one-pot fluorescence assay for bacterial DNA detection that exploits the unique optical properties of DNA-templated copper nanoclusters (CuNCs). These nanoclusters offer a sustainable alternative to conventional fluorophores, thanks to their eco-friendly synthesis, high photostability, and large Stokes shift. The assay integrates CuNCs with the CRISPR/Cas12a system to achieve programmable and highly specific target recognition. Upon target binding, activation of the Cas12a/gRNA complex triggers collateral cleavage of rationally designed DNA templates that normally support CuNCs formation, resulting in a marked fluorescence decrease. A panel of hairpin and poly-thymine DNA structures was systematically evaluated to maximize both CuNCs fluorescence and responsiveness to Cas12a/gRNA trans-cleavage, ultimately identifying an AT-rich stem-loop reporter that provided strong signal intensity and complete signal shutdown upon target recognition. The final CRISPR-CuNCs assay achieved picomolar sensitivity, accurately detected E. coli DNA from reference strains, clinical isolates, and serum-spiked samples, and required no fluorophore-quencher probes or multistep procedures. Overall, this work demonstrated that combining the programmability of CRISPR/Cas12a with the versatility and low-cost of DNA-templated CuNCs enables a robust and accessible platform for molecular diagnostics, with strong potential for Point-of-Care deployment.},
}
@article {pmid41680160,
year = {2026},
author = {Wang, B and Zhou, S and Zhang, X and Wang, R and Huang, S and Li, A and He, H and Zhang, Y and Wei, Y and Yang, Z and Song, F and Li, X and Wan, X and Shen, Y and Ma, C and Mao, H and Ledesma-Amaro, R and Yin, D and Wei, Q and Deng, R and Yang, T and Liu, S and Wan, Y and Mao, C},
title = {PRICE: direct and robust detection of microRNAs at single-nucleotide resolution.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41680160},
issn = {2041-1723},
support = {14208723//Research Grants Council, University Grants Committee (RGC, UGC)/ ; },
mesh = {*MicroRNAs/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; *Polymorphism, Single Nucleotide ; *Peptide Nucleic Acids/genetics/metabolism ; },
abstract = {Accurate single-nucleotide discrimination of miRNA is clinically vital because small sequence variations can have significant phenotypic and clinical consequences, yet existing techniques can only detect single nucleotide variations (SNVs) at specific loci. Here, we present a generalized peptide nucleic acid (PNA) mediated CRISPR/Cas13a system (PRICE), enabling detection of SNVs in miRNA sequence without sacrificing the sensitivity. PRICE utilizes PNA blockers fully complementary to non-target miRNAs (e.g., miRNAs containing SNVs at loci of no interest) but not to the target miRNA. These blockers selectively hybridize with and inhibit non-target sequences in samples (serum, cells, or tissues). Only the unhybridized target miRNA then binds to crRNA within the Cas13a complex, activating Cas13a to cleave a fluorescent reporter-quencher linker, generating a detectable signal (~10 fM limit). By designing a panel of PNAs against SNVs, PRICE provides a versatile, amplification-free platform for precise miRNA analysis, advancing cancer diagnosis, prognosis, and biology.},
}
@article {pmid41680202,
year = {2026},
author = {Garmendia-Antoñana, N and Dorado-Morales, P and Gil, C and García, B and Echeverz, M and Solano, C and Penadés, JR and Lasa, I},
title = {Targeted elimination of Staphylococcus aureus mastitis infections with synthetic phage-based CRISPR-Cas delivery systems.},
journal = {NPJ biofilms and microbiomes},
volume = {12},
number = {1},
pages = {},
pmid = {41680202},
issn = {2055-5008},
support = {PRE2021-097385//Spanish Ministry of Science, Innovation and Universities/ ; PID2020-113494RB-I00/ AEI//Spanish Ministry of Science, Innovation and Universities/ ; BES-2015-07285//the Spanish Ministry of Science, Innovation and Universities/ ; },
mesh = {Animals ; *Staphylococcus aureus/genetics/virology/drug effects/physiology ; *CRISPR-Cas Systems ; *Staphylococcal Infections/therapy/microbiology ; Female ; Mice ; *Mastitis/microbiology/therapy ; Biofilms/growth &amp; development ; Disease Models, Animal ; *Bacteriophages/genetics ; Genomic Islands ; Anti-Bacterial Agents/pharmacology ; *Staphylococcus Phages/genetics ; },
abstract = {Treatment options for Staphylococcus aureus infections are increasingly limited, particularly in livestock, where S. aureus causes mastitis requiring prolonged antibiotic therapy. This study engineered Phage Inducible Chromosomal Islands (ePICIs) to deliver CRISPR-Cas9 modules targeting small RNA genes. ePICIs exhibit bactericidal activity without chromosomal integration, an expanded host range compared to their parental phages, and biofilm-dependent efficacy influenced by the extracellular matrix composition. Biofilms mediated by the Bap protein strongly protect bacteria from ePICIs, whereas PIA/PNAG-based biofilms do not. Despite Bap-mediated protection in vitro, ePICIs achieved bactericidal effects comparable to vancomycin in a mouse mastitis model caused by Bap-producing strains. These findings reveal key factors affecting phage-delivered CRISPR-Cas efficacy and highlight that antibiofilm therapies should not be dismissed based solely on in vitro performance. Non-replicative ePICIs thus represent a promising alternative for treating localized infections such as mastitis.},
}
@article {pmid41680487,
year = {2026},
author = {Kroell, AS and Hoffmann, KH and Motzkus, NA and Lemmen, N and Happ, N and Wolf, B and von Bachmann, AL and Southern, N and Vogd, F and Aschenbrenner, S and Niopek, D and Mathony, J},
title = {Modular engineering of thermoresponsive allosteric proteins.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41680487},
issn = {1552-4469},
support = {520612620//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 453202693//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
abstract = {Thermogenetics enables noninvasive spatiotemporal control over protein activity in living cells and tissues, yet its applications have largely been restricted to transcriptional regulation and membrane recruitment. Here, we present a generalizable strategy for engineering thermosensitive allosteric proteins through the insertion of optimized Avena sativa LOV2 domain variants. Applying this approach to a diverse set of structurally and functionally unrelated proteins in Escherichia coli, we generated potent, thermoswitchable chimeric variants that can be tightly controlled within narrow temperature ranges (37-41 °C). Extending this strategy to mammalian systems, we engineered CRISPR-Cas genome editors directly modulated by subtle temperature changes within the physiological range. Lastly, we showcase the incorporation of a chemoreceptor domain as an alternative thermosensing module, suggesting thermosensitivity to be a widespread feature in receptor domains. This work expands the toolkit of thermogenetics, providing a blueprint for temperature-dependent control of virtually any protein of interest.},
}
@article {pmid41680629,
year = {2026},
author = {Selhorst, P and Van Vyve, E and Falconi-Agapito, F and Mariën, J and Ariën, KK},
title = {Sensitive, flexible, and affordable serum RNA sequencing for pathogen detection on the Oxford Nanopore platform.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {188},
pmid = {41680629},
issn = {1471-2164},
support = {U01 AI151378/AI/NIAID NIH HHS/United States ; U01AI151378/NH/NIH HHS/United States ; },
abstract = {UNLABELLED: Metagenomic sequencing for pathogen detection has traditionally suffered from low sensitivity due to the overwhelming presence of host nucleic acids. Commercial host-depletion kits are often prohibitively expensive and limited to specific species, hindering adoption in resource-limited settings, where the burden of zoonotic diseases is highest. To address this, we optimized and combined Sequence-Independent Single Primer Amplification (SISPA) with Depletion of Abundant Sequences by Hybridization (DASH), establishing a low-cost metagenomic protocol on the Oxford Nanopore sequencing platform. Our approach can be adapted to any species to detect microbial RNAs in serum samples at PCR-range sensitivity, outperforming existing methods in the field.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12268-4.},
}
@article {pmid41683064,
year = {2026},
author = {Chen, Z and He, D and Yu, W and Fu, X and Zhang, L and Zhang, M and Yu, X and Ye, Z},
title = {Advancing Bongkrekic Acid Detection: From Conventional Instrumental Analysis to Advanced Biosensing for Cross-Toxin Applications.},
journal = {Foods (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {41683064},
issn = {2304-8158},
abstract = {Bongkrekic acid (BKA), a highly lethal toxin, has been implicated in frequent poisoning incidents in recent years, posing a serious threat to global food safety and creating an urgent need for rapid and sensitive detection methods. This review provides a systematic analysis of the entire BKA detection technologies, covering sample pretreatment techniques, instrumental analysis, immunoassays, and biosensing methods. It assesses the merits of key methods and also explores the strategic cross-application of detection paradigms developed for analogous toxins. This review delivers a comprehensive and critical evaluation of BKA detection technologies. First, it discusses sample pretreatment strategies, notably solid-phase extraction (SPE) and QuEChERS. Subsequently, it analyzes the principles, performance, and applications of core detection methods, including high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), high-resolution mass spectrometry (HRMS), time-resolved fluorescence immunoassay (TRFIA), dual-mode immunosensors and nanomaterial-based sensors. Instrumental methods (e.g., HRMS) offer unmatched sensitivity [with a limit of detection (LOD) as low as 0.01 μg/kg], yet remain costly and laboratory-dependent. Immunoassay and biosensor approaches (TRFIA and dual-mode sensors) enable rapid on-site detection with high sensitivity (ng/mL to pg/mL), though challenges in stability and specificity remain. Looking forward, the development of next-generation BKA detection could be accelerated by cross-applying cutting-edge strategies proven for toxins-such as Fumonisin B1 (FB1), Ochratoxin A (OTA), and Aflatoxin B1 (AFB1)-including nanobody technology, CRISPR-Cas-mediated signal amplification, and multimodal integrated platforms. To translate this potential into practical tools, future research should prioritize the synthesis of high-specificity recognition elements, innovative signal amplification strategies, and integrated portable devices, aiming to establish end-to-end biosensing systems capable of on-site rapid detection through multitechnology integration.},
}
@article {pmid41683574,
year = {2026},
author = {Sopel, J and Sarad, K and Kozinska, A and Mokrzyński, K and Szczygieł, D and Murzyn, A and Drzał, A and Słomiński, A and Szczygieł, M and Elas, M},
title = {Pmel17 Deficiency Affects Melanogenesis and Promotes Tumor Vascularization.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683574},
issn = {1422-0067},
mesh = {Animals ; *Melanins/metabolism/biosynthesis ; Mice ; *Neovascularization, Pathologic/metabolism/genetics/pathology ; *Melanoma, Experimental/metabolism/pathology/genetics/blood supply ; Reactive Oxygen Species/metabolism ; Cell Line, Tumor ; *gp100 Melanoma Antigen/genetics/metabolism/deficiency ; CRISPR-Cas Systems ; Cell Movement ; Melanosomes/metabolism ; Cell Cycle ; Melanogenesis ; },
abstract = {Premelanosomal protein (Pmel, also known as Pmel17) is the major component of melanosomal fibrils and plays a key role in melanin polymerization, making it an important factor in melanogenesis. We investigated how the absence of Pmel affects the properties of B16F10 melanoma cells. Pmel-knockout B16F10 cells were generated using CRISPR/Cas9-mediated genome editing. A viability assay revealed no significant differences between wild-type (WT) and Pmel-knockout (KO) sublines; however, melanosome maturation was impaired. In Pmel KO cells, the cell cycle was disrupted, and higher levels of reactive oxygen species (ROS) were observed compared with WT cells. Moreover, the migration capacity and tube formation of melanoma cells were increased. Tumors derived from Pmel KO cells exhibited unchanged growth kinetics but reduced melanin content, along with enhanced vascularization and oxygenation. Thus, knockout of the Pmel17 gene in melanoma cells alters pigmentation, vascularization, and oxygenation of tumors. These parameters are crucial for both tumor progression and therapeutic response.},
}
@article {pmid41683605,
year = {2026},
author = {Blazyte, A and Lee, H and Yoon, C and Jeon, S and Lee, J and Bayarsaikhan, D and Kim, J and Park, S and Cho, J and Baek, SA and Byun, G and Lee, B and Bhak, J},
title = {Neurofibromin 1 (NF1) Splicing Mutation c.61-2A&gt;G: From Aberrant mRNA Processing to Therapeutic Implications In Silico.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683605},
issn = {1422-0067},
support = {1.200047.01//Ulsan City Research Fund/ ; RS-2023-00263429//Genome Editing Research Program/ ; KEIT 20018560//Alchemist Project of the Korea Evaluation Institute of Industrial Technology/ ; RS-2024-00435468//Korea Planning &amp; Evaluation Institute of Industrial Technology with support from the Ministry of Trade, Industry and Energy/ ; },
mesh = {*Neurofibromin 1/genetics ; Humans ; *Neurofibromatosis 1/genetics/therapy ; *RNA Splicing/genetics ; *Mutation ; *RNA, Messenger/genetics ; RNA Splice Sites/genetics ; Gene Editing ; Computer Simulation ; DNA Methylation ; CRISPR-Cas Systems ; Male ; },
abstract = {The neurofibromin 1 (NF1) splice-site mutation c.61-2A>G (rs1131691100) is a rare, pathogenic, autosomal dominant variant that disrupts NF1 tumor-suppressor function, causing neurofibromatosis type 1 (NF1). Its pathogenic mechanism is poorly understood, and the potential for personalized therapeutic genome editing remains unknown due to the absence of a standard framework for investigating splicing disorders. Here, we performed a comprehensive multi-omics analysis of a de novo c.61-2A>G case from South Korea, integrating short- and long-read whole genome sequencing, whole transcriptome sequencing, and methylation profiling. We confirm that c.61-2A>G abolishes the canonical splice acceptor site, activating a cryptic splice acceptor 16 nucleotides downstream in exon 2. This splicing shift generates a 16-nucleotide deletion, causing a frameshift and premature stop codon that truncates the protein's N-terminal region. Long-read sequencing further reveals that the mutation creates a novel CpG dinucleotide, which is methylated in the majority of reads. Finally, we assessed therapeutic correction strategies, revealing that CRISPR-Cas9 prime editing is the only viable approach for in vivo correction. This study provides the first comprehensive multi-omics characterization of the NF1 c.61-2A>G mutation and establishes a minimal framework for precision therapeutic development in silico in monogenic splicing disorders.},
}
@article {pmid41683697,
year = {2026},
author = {Kowalik, S and Samoń, M and Przyborowski, M},
title = {Molecular Regulators of In Vitro Regeneration in Wheat: Roles of Morphogenic Factors in Transformation, Genome Editing, and Breeding.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683697},
issn = {1422-0067},
support = {Dotacja Celowa task 4.1//Ministry of Agriculture and Rural Development/ ; },
mesh = {*Triticum/genetics/physiology/growth &amp; development ; *Gene Editing/methods ; *Plant Breeding/methods ; *Regeneration/genetics ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; *Transformation, Genetic ; Transcription Factors/genetics/metabolism ; },
abstract = {Efficient in vitro regeneration remains a major constraint in the genetic transformation, genome editing, and molecular breeding of wheat (Triticum aestivum L.), largely due to strong genotype-dependent recalcitrance and limited activation of developmental programs required for somatic embryogenesis. Plant regeneration relies on extensive transcriptional reprogramming and epigenetic remodeling orchestrated by morphogenic regulators that modulate meristem identity, as well as cellular pluri- and totipotency. In this review, we synthesize current molecular knowledge on key transcription factors (BBM, WUS/WUS2, GRF-GIF, WOX, LAX1, SERK, WIND1/ERF115) and signaling peptides (CLE/CLV-WUS module, phytosulfokine/PSK) that regulate embryogenic competence in monocot cereals, with emphasis on their orthologs and functional relevance in wheat. We highlight how controlled expression of these morphogenic genes, promoter engineering, and transient or excisable induction systems can significantly enhance regeneration capacity, reduce chimerism in CRISPR-Cas-edited plants, and facilitate genotype-independent transformation. We also discuss epigenetic and metabolic constraints underlying wheat recalcitrance and their potential modulation to improve culture responsiveness. By integrating evidence from wheat, rice, maize, and barley, we outline conserved gene-regulatory networks that reinitiate totipotency and propose strategies to accelerate doubled haploid production and speed-breeding pipelines. Collectively, morphogenic factors emerge as central molecular tools for overcoming regeneration bottlenecks and enabling next-generation wheat improvement. The objective of this review is to synthesize and critically evaluate current molecular knowledge on morphogenic regulators controlling in vitro regeneration in wheat (Triticum aestivum L.), with particular emphasis on their roles in genetic transformation and genome editing.},
}
@article {pmid41683796,
year = {2026},
author = {Jiang, Y and Chen, Y and Huang, Z and Chen, L and Huang, X},
title = {Tyrosinase-Deficient Skin Melanophore Lineage in Xenopus tropicalis Tadpoles Shows Strong Autofluorescence.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683796},
issn = {1422-0067},
support = {LY20C120003//Zhejiang Provincial Natural Science Foundation/ ; },
mesh = {Animals ; *Monophenol Monooxygenase/genetics/deficiency/metabolism ; *Xenopus/metabolism/genetics ; Larva/metabolism/genetics ; *Melanophores/metabolism ; *Skin/metabolism ; CRISPR-Cas Systems ; Melanins ; Optical Imaging ; Xenopus Proteins/genetics/metabolism ; Skin Pigmentation ; },
abstract = {Tyrosinase, encoded by Tyr, is a key rate-limiting enzyme in melanin biosynthesis. Knockout of Tyr results in a distinct albino phenotype, making it a widely used target for evaluating gene-editing efficiency. Here, we found that the tyrosinase-deficient skin melanophore lineage of Xenopus tropicalis (X. tropicalis) tadpoles shows strong autofluorescence under the GFP filter, which may interfere with in vivo fluorescence imaging. Through spectral scanning analysis, we characterized the emission spectrum of the autofluorescence under commonly used excitation wavelengths for fluorescent proteins. Based on this, we established a reference protocol for identifying and excluding such interference in Tyr-targeted knockin studies. Furthermore, knockout of the GTP cyclohydrolase 2 gene (Gch2) using CRISPR-Cas9 significantly reduced the fluorescence intensity induced by tyrosinase deficiency, indicating an essential role of the enzyme and its mediated pterine biosynthesis in the generation of the autofluorescence. This study systematically characterized these fluorescent mutant melanophores in X. tropicalis tadpoles, providing a practical basis for avoiding fluorescent interference in experimental science and a new perspective on pigment cell development and evolution.},
}
@article {pmid41683905,
year = {2026},
author = {Ortiz-Bueno, M and Zinghirino, F and Serra, PP and Paschoudi, K and Montoliu, L and Atilla, E and Luo, Y and Cavazza, A and Lederer, CW and Benabdellah, K},
title = {From Bench to Bedside: Ethical and Clinical Best Practices for Genome Editing Applications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683905},
issn = {1422-0067},
support = {CA21113//COST (European Cooperation in Science and Technology)./ ; ProyExcel_00875//Consejer&#xed;a de Universidad, Investigaci&#xf3;n e Innovaci&#xf3;n/ ; },
mesh = {Humans ; *Gene Editing/ethics/legislation &amp; jurisprudence/methods ; *Genetic Therapy/ethics/methods ; Animals ; CRISPR-Cas Systems ; *Translational Research, Biomedical/ethics ; },
abstract = {Genome editing (GE) has transformed medicine by allowing precise changes to DNA, offering potential treatments for a range of inherited and acquired disorders. Several technologies support these advances, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, of which the latter has emerged as the most accessible, versatile, and popular. While GE holds great promise, its clinical use requires careful attention to safety, ethics and regulatory standards. Inadvertent on- and off-target DNA alterations and unintended modification of non-target cells pose major technical challenges, while bioethical considerations and the need for harmonized safety standards create regulatory challenges. The Food and Drug Administration (FDA) and European Medicines Agency (EMA), as regulatory agencies for key advanced therapy markets, provide detailed guidance on these aspects, emphasizing rigorous preclinical testing, patient monitoring, ethical consent, and compliance with legal frameworks. This concise review summarizes what is currently published in the scientific literature and recommended by regulatory agencies, providing an overview of the responsible clinical application of GE, with emphasis on patient safety, adherence to regulatory guidance, and ethical practice.},
}
@article {pmid41683941,
year = {2026},
author = {Skaliter, O and Gura, A and Livneh, Y and Cohen, R and Shklarman, E and Edelbaum, O and Masci, T and Vainstein, A},
title = {Targeted Gene Modification of HMGR Enhances Biosynthesis of Terpenoid and Phenylpropanoid Volatiles in Petunia and Lettuce.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683941},
issn = {1422-0067},
support = {1368/23//Israel Science Foundation/ ; 20-01-0209//Office of the Chief Scientist/ ; },
mesh = {*Petunia/genetics/metabolism ; *Terpenes/metabolism ; *Lactuca/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics/metabolism ; *Volatile Organic Compounds/metabolism ; },
abstract = {Terpenoids constitute the largest class of plant-specialized metabolites, playing essential roles throughout the plants' life cycle and having diverse applications for humans in nutrition, medicine, and flavor. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is a rate-limiting enzyme of the mevalonate (MVA) pathway, producing sesquiterpenes, saponins, and other terpenoids. HMGR is post-translationally regulated by downstream MVA products through its N-terminal regulatory domain, limiting terpenoid production. To overcome this bottleneck, we employed a virus-based CRISPR/Cas9 system to genetically modify the N-terminal regulatory domain of HMGR in petunia (Petunia × hybrida) and lettuce (Lactuca sativa L.). In petunia, HMGR1-edited lines exhibited vigorous growth, larger flowers, and increased production of sesquiterpenes. Interestingly, they also showed enhanced production of phenylpropanoid volatiles, revealing a connection between these pathways. Transcript analysis revealed altered expression of genes involved in terpenoid biosynthesis, pyruvate metabolism, phenylpropanoid biosynthesis, and gibberellin- and auxin-related pathways, indicating enhanced carbon flux through these metabolic networks. In lettuce, HMGR7-edited plants displayed elevated emission of sesquiterpenes, apocarotenoids, and the phenylpropanoid benzaldehyde. Together, these results establish a transgene-free strategy to enhance the production of terpenoid and phenylpropanoid volatiles, and provide a framework for developing resilient, nutrient-enriched crops.},
}
@article {pmid41683990,
year = {2026},
author = {Psaras, AM and McKay, SJ and Vasquez Vilela, J and Ospina Sanchez, E and Cintrón, MG and Elder, KK and Brooks, TA},
title = {Ovarian Cancer Susceptibility and Chemosensitivity to KRAS Modulation.},
journal = {International journal of molecular sciences},
volume = {27},
number = {3},
pages = {},
pmid = {41683990},
issn = {1422-0067},
mesh = {Humans ; Female ; *Proto-Oncogene Proteins p21(ras)/genetics/metabolism/antagonists &amp; inhibitors ; *Ovarian Neoplasms/genetics/drug therapy/pathology/metabolism ; *Drug Resistance, Neoplasm/genetics/drug effects ; Cell Line, Tumor ; Paclitaxel/pharmacology ; Cisplatin/pharmacology ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; },
abstract = {KRAS is frequently amplified or overexpressed in ovarian cancer and represents a potential therapeutic target for overcoming chemoresistance. We employed complementary approaches-CRISPR/Cas9 gene editing, Tet-ON inducible knockdown, polypurine reverse Hoogsteen hairpin (PPRH) oligonucleotides, and the pan-KRAS inhibitor BI2865-to investigate whether KRAS modulation enhances chemotherapeutic efficacy in ovarian cancer models. CRISPR-mediated KRAS knockdown in SKOV-3 cells dramatically altered three-dimensional spheroid morphology, reducing the average area six-fold, and significantly enhanced sensitivity to both cisplatin and paclitaxel in 3D cultures, where paclitaxel resistance was completely reversed. The Tet-ON system demonstrated dose-dependent chemosensitization with optimal effects at intermediate KRAS knockdown levels (~50-60%). PPRH oligonucleotides at sub-cytotoxic concentrations (50 nM) reduced cisplatin and paclitaxel IC50 values by approximately 50% in 2D cultures. Pharmacological KRAS inhibition with BI2865 produced striking synergy with paclitaxel (several hundred-fold sensitizations in 2D; complete reversal of 3D resistance), and additive effects with cisplatin. In KRAS-amplified Kuramochi cells (representing high-grade serous ovarian carcinoma), BI2865 enhanced paclitaxel efficacy, despite greater baseline chemoresistance. These findings establish KRAS as a promising chemosensitization target in ovarian cancer, with particular potential for taxane-based combination therapies.},
}
@article {pmid41685373,
year = {2025},
author = {Hanafiah, A and Sukri, A and Asmawi, MA and Yusoff, H and Mohd Puzi, S and Neoh, HM and Lopes, BS},
title = {Recent Advancements in Development and Characterization of Phages Targeting Helicobacter pylori.},
journal = {PHAGE (New Rochelle, N.Y.)},
volume = {6},
number = {4},
pages = {282-291},
pmid = {41685373},
issn = {2641-6549},
abstract = {Helicobacter pylori remains a significant global health concern, with rising antibiotic resistance posing challenges for conventional treatments. Bacteriophages, viruses that specifically target and lyse bacterial cells, present a promising alternative therapeutic approach. This review explores the advancements in phage research related to H. pylori, including the isolation, genomic and proteomic characterization, and therapeutic potential of lytic and lysogenic phages. Novel isolation techniques have identified diverse phages from clinical and environmental sources, such as sewage and wastewater, revealing unique genetic and structural adaptations that enhance their effectiveness in targeting H. pylori. Genomic analysis has highlighted the role of prophages in H. pylori evolution, while proteomic studies have uncovered structural features that enable phages to survive the acidic gastric environment. High-throughput technologies, such as next-generation sequencing, clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) systems, and microfluidic platforms, have revolutionized phage discovery and characterization. Furthermore, the potential for phage-antibiotic synergy offers new avenues for combating antibiotic resistance. Despite these advancements, challenges such as H. pylori's genetic diversity, its fastidious growth requirements, and the development of robust delivery mechanisms for gastric application persist. This review highlights the need for further research to optimize phage-based therapies as a viable alternative or adjunct to current treatments for H. pylori infections.},
}
@article {pmid41685943,
year = {2026},
author = {Wang, C and Li, D and Yu, R and Xue, J and Xie, W and Zhang, Q and Gui, X and Wang, L and Guo, S and Xie, Y and Jiang, Y and Liu, G and Wu, J},
title = {A CRISPR/Cas9-regulated dual-ring topological allosteric probe for detection of the EGFR L858R resistance mutation in CTCs.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {9},
pages = {1815-1825},
doi = {10.1039/d5ay02124c},
pmid = {41685943},
issn = {1759-9679},
mesh = {Humans ; ErbB Receptors/genetics ; *Neoplastic Cells, Circulating/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; *Lung Neoplasms/genetics/blood ; *Carcinoma, Non-Small-Cell Lung/genetics/blood ; Polymorphism, Single Nucleotide ; *Drug Resistance, Neoplasm/genetics ; Mutation ; Fluorescent Dyes/chemistry ; Cell Line, Tumor ; },
abstract = {A single-nucleotide polymorphism (SNP) is a point mutation occurring at a defined genomic locus, and its precise and rapid detection in circulating tumor cells (CTCs) is essential for early diagnosis and therapeutic monitoring of non-small cell lung cancer (NSCLC). In this study, a CRISPR/Cas9-regulated dual-ring topological allosteric probe was developed for ultrasensitive and specific detection of the EGFR L858R mutation. The recognition ring selectively hybridizes with the target sequence and is cleaved by the Cas9-sgRNA complex, triggering the release of the reporter ring. The released reporter ring then serves as a template for rolling circle amplification (RCA), generating products that hybridize with dual-labeled fluorescent probes to yield measurable signals. This assay clearly distinguished L858R from the wild-type sequence and detected mutation frequencies as low as 1.0% with high specificity against other common EGFR variants. Its robustness was further validated using clinical blood samples, enabling sensitive detection of low-abundance L858R mutations. These results demonstrate that the integration of programmable target recognition, efficient signal amplification, and fluorescence readout provides a promising platform for SNP analysis in liquid biopsy, supporting precision diagnosis and treatment monitoring in NSCLC.},
}
@article {pmid41686171,
year = {2026},
author = {Su, M and Lv, MM and Pan, MX and Zha, CJ and Nie, YG and Ying, ZM},
title = {From Self-Processing to Responsive Assembly Enabling an Autocatalytic Cas13a Circuit for Enhanced Biosensing.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2697-2706},
doi = {10.1021/acssensors.5c04673},
pmid = {41686171},
issn = {2379-3694},
mesh = {*Biosensing Techniques/methods ; Humans ; CRISPR-Cas Systems ; Survivin/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; MicroRNAs/analysis/genetics ; DNA, Viral/analysis/genetics ; RNA, Messenger/analysis/genetics ; DNA-Directed RNA Polymerases ; Viral Proteins ; },
abstract = {Despite the success of non-classical crRNA designs in Cas12a-based biosensing, application to Cas13a systems faces fundamental challenges. Our research discovers that site-specific splitting within the crRNA seed region enables effective activation of Cas13a trans-cleavage. Here, we developed an autocatalytic Cas13a circuit that transitions from self-processing to responsive assembly for enhanced biosensing (PRA-Cas13a). The system employs engineered pre-crRNA as a molecular switch, which undergoes self-processing upon target binding to assemble an active Cas13a complex and activate its trans-cleavage activity. By integrating a dual-UUU site DNA switch template and a T7 RNA polymerase-mediated signal amplification module, a "processing-assembly-amplification" cycle is constructed to enhance the detection signal. Through validation using various targets including miRNA, mRNA, and viral DNA, the PRA-Cas13a system not only achieves an attomolar (aM) level detection limit but also enables visual field detection within 10 min using a lateral flow test strip. Analysis of single-base mutations demonstrated that its sensitivity is significantly superior to conventional CRISPR-based methods. Moreover, the system successfully enabled accurate detection of survivin mRNA in different cell lines and HPV16 in clinical cervical swab samples, showing strong concordance with qPCR gold standard methods. The PRA-Cas13a strategy leverages intrinsic self-processing assembly and autocatalytic signal amplification, addresses the critical issue of off-target cleavage inherent in conventional Cas13a systems while expanding the range of applicable targets, and demonstrates high specificity and point-of-care testing potential in cancer and viral diagnostics.},
}
@article {pmid41686483,
year = {2026},
author = {Tanaka, PP and Cotta-Almeida, V and Donadi, EA and Westerberg, L and Passos, GA},
title = {Distinct mutations in the autoimmune regulator gene differentially affect transcriptional and functional properties of medullary thymic epithelial cells.},
journal = {Human molecular genetics},
volume = {35},
number = {4},
pages = {},
doi = {10.1093/hmg/ddag004},
pmid = {41686483},
issn = {1460-2083},
support = {17/10780-4//S&#xe3;o Paulo Research Foundation/ ; 311304/2021//National Council for Scientific and Technological Development/ ; 302060/2019-7//National Council for Scientific and Technological Development/ ; 88887.642780/2021-00//CAPES-STINT/ ; 001//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; },
mesh = {AIRE Protein ; Animals ; *Epithelial Cells/metabolism/pathology ; Mice ; *Transcription Factors/genetics/metabolism ; *Mutation ; *Thymus Gland/metabolism/pathology ; *Polyendocrinopathies, Autoimmune/genetics/pathology/immunology ; Humans ; CRISPR-Cas Systems ; Gene Expression Regulation ; Transcription, Genetic ; },
abstract = {Autoimmune Polyendocrine Syndrome Type 1 (APS-1) is a rare monogenic disorder caused by mutations in the autoimmune regulator (AIRE) gene. Although AIRE is essential for central immune tolerance, how distinct APS-1-associated mutations differentially affect medullary thymic epithelial cell (mTEC) biology remains incompletely understood. Here, we investigated the molecular and functional consequences of three Aire/AIRE variants using complementary murine mTEC models. To define transcriptional effects, we performed single-cell RNA sequencing (scRNA-seq) on mTECs carrying a heterozygous genomic Aire c.735delG mutation generated by CRISPR-Cas9. This analysis revealed reduced transcriptional heterogeneity, decreased expression of tissue-restricted antigens (TRAs) mRNAs (including Col4a3, Col7a1, and Neto2), and downregulation of key mTEC lineage markers (Epcam, Cldn4, Krt14). Mutant cells also displayed altered expression of mRNAs involved in chemokine-mediated migration (Ccl25, Cxcl16), extracellular matrix and cell adhesion (Fn1, Lama5, Col4a1, Nectin1, Cdh1), and actin cytoskeleton organization (Gsn, Rac1, Wasl, Actn1), indicating broad disruption of pathways governing mTEC identity and cell-cell interactions. Guided by these findings, we assessed mutation-specific functional outcomes using a CRISPR-derived Aire functional knockout and lentiviral expression of the human AIRE missense variants p.G229W and p.C313Y in wild-type mTECs. Functional assays revealed mutation-dependent alterations in mTEC morphology, thymocyte migration, and adhesion, with the p.C313Y variant exerting the strongest effects. Together, these data demonstrate that heterozygous and missense AIRE mutations exert distinct yet convergent effects on mTEC transcriptional programs and cellular behavior, providing mechanistic insight into AIRE-dependent immune tolerance failure in APS-1.},
}
@article {pmid41686830,
year = {2026},
author = {Caluianu, M and Owen, KA},
title = {A VPS33B CRISPR knockout study: In vitro evidence of an adhesion defect.},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0343240},
pmid = {41686830},
issn = {1932-6203},
mesh = {*Vesicular Transport Proteins/genetics/metabolism ; Humans ; *Cell Adhesion/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Epithelial Cells/metabolism ; Cell Line ; *Kidney Tubules, Proximal/metabolism/cytology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {VPS33B is a ubiquitously expressed regulator of vesicular membrane fusion and protein sorting involved in a broad range of cellular functions from organelle biogenesis to the establishment of apicobasal polarity. Loss-of-function mutations in VPS33B cause arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome, a rare autosomal recessive disorder with multi-organ involvement, including a characteristic proximal tubular dysfunction in the kidney. While VPS33B has been studied in several cell types, its role in proximal tubular epithelial cells remains poorly understood. To investigate its function, a proximal tubular cell line (RPTEC-TERT1) was CRISPR-edited to generate VPS33B knockout (KO) cells. These cells were characterised using brightfield imaging, immunostaining, RNA sequencing, and cell detachment assays, revealing a distinct 'peeling' phenotype and altered adhesion properties. Transcriptional profiling indicated changes in genes linked to cell adhesion. Together, these findings offer preliminary evidence that loss of VPS33B impairs cell-matrix attachment and reveal the first insights into the role of VPS33B within proximal tubular epithelial cells.},
}
@article {pmid41686849,
year = {2026},
author = {Dueñas, E and Tirado, I and Huaihua, P and Parra Del Riego, A and Cabrera-Sosa, L and Nakamoto, JA and Cruz, M and Restrepo, CM and Arévalo, J and Adaui, V},
title = {LAMP-coupled CRISPR-Cas12a assays: A promising new tool for molecular diagnosis of leishmaniasis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {2},
pages = {e0013456},
pmid = {41686849},
issn = {1935-2735},
mesh = {Humans ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; DNA, Kinetoplast/genetics ; RNA, Ribosomal, 18S/genetics ; DNA, Protozoan/genetics ; *Leishmania/genetics/isolation &amp; purification ; *Leishmaniasis/diagnosis/parasitology ; *Leishmaniasis, Cutaneous/diagnosis/parasitology ; Leishmania braziliensis/genetics/isolation &amp; purification ; },
abstract = {BACKGROUND: Tegumentary leishmaniasis is a parasitic disease endemic in the Americas. Its clinical management and control rely on early and accurate diagnosis and adequate treatment. PCR-based molecular diagnostics offer high sensitivity and specificity over microscopy or culture but are less accessible in low-resource settings. New molecular tools for detecting Leishmania infections are needed in rural endemic regions. A promising tool harnessing CRISPR-Cas technology enables highly specific and sensitive detection of nucleic acid targets, offering an exciting potential for portable molecular diagnostics. Previously, we developed CRISPR-Cas12a-based assays coupled to PCR preamplification for Leishmania detection. Here, we adapted our assays, which target the multicopy 18S rDNA and kinetoplast DNA (kDNA) minicircles, by replacing PCR with loop-mediated isothermal amplification (LAMP).<br><br>LAMP-coupled CRISPR assays were optimized for fluorescence-based and lateral flow readouts. The assays could detect as low as 0.2 genome equivalents per reaction using L. braziliensis M2904 strain genomic DNA. The kDNA assay reliably detected all tested species of the L. (Viannia) subgenus, while the 18S assay showed pan-Leishmania detection capability. There was no cross-reactivity with other protozoan (Trypanosoma cruzi and Plasmodium falciparum) and bacterial (Mycobacterium tuberculosis) pathogen DNA, nor with human DNA. When applied to 90 clinical samples (skin lesions) from the Cusco region of Peru and compared to kDNA real-time PCR, LAMP-CRISPR assays with a fluorescence readout achieved a sensitivity of 90.9% (95% CI: 80.1-97.0%) for kDNA and 72.7% (95% CI: 59.0-83.9%) for 18S rDNA, both with 100% (95% CI: 90-100%) specificity. Overall, lateral flow strip results agreed with fluorescence-based detection in 18 tested samples, with one discrepancy observed in the 18S assay associated with low parasite load.<br><br>CONCLUSIONS/SIGNIFICANCE: These new proof-of-concept LAMP-CRISPR assays, combining high sensitivity, multiple Leishmania species detection capability, and a portable lateral flow readout, hold promise as next-generation molecular tools to improve leishmaniasis diagnosis and surveillance, supporting One Health strategies for disease control.},
}
@article {pmid41686883,
year = {2026},
author = {Rathod, BU and Rajyaguru, R and Dhawale, RN and Tomar, RS and Sharma, S and Chaskar, MG and Alsaidan, OA and Hajare, ST},
title = {CRISPR/Cas9-Mediated Editing in FAD2 Gene to Enhance Oil Quality in Soybean [Glycine max (L.) Merrill].},
journal = {PloS one},
volume = {21},
number = {2},
pages = {e0342660},
pmid = {41686883},
issn = {1932-6203},
mesh = {*Glycine max/genetics/metabolism ; *Gene Editing/methods ; *Fatty Acid Desaturases/genetics ; *CRISPR-Cas Systems/genetics ; *Soybean Oil/metabolism ; Plants, Genetically Modified/genetics ; Linoleic Acid/metabolism ; *Plant Proteins/genetics ; },
abstract = {Conventional soybean oil contains high levels of linoleic acid, which reduces oxidative stability and necessitates hydrogenation, leading to trans-fat formation. In this study, 40 Indian soybean genotypes were screened for fatty acid composition, and Gujarat Junagadh Soybean-3 (GJS-3) was selected for CRISPR/Cas9-mediated editing of the fatty acid desaturase-2 (FAD2) gene. Agrobacterium-mediated transformation produced 22 regenerated plants, of which 57.1% were PCR-positive for Cas9/sgRNA. Targeted single-nucleotide substitutions were confirmed by Sanger sequencing in three edited lines (T3, T7, and T15), corresponding to an editing efficiency of 13.63%. These lines exhibited a marked increase in oleic acid content (42-45%) compared with the wild type (22%) and a concomitant reduction in linoleic acid (30-32% vs. 54%), resulting in nearly a two-fold improvement in the oleic/linoleic acid ratio. PCR analysis confirmed the absence of Cas9 and U3 sequences, indicating transgene-free edited plants. This study provides the first evidence of CRISPR/Cas9-mediated FAD2 editing in an Indian soybean cultivar and demonstrates its effectiveness in improving oil quality, oxidative stability, and processing efficiency.},
}
@article {pmid41687699,
year = {2026},
author = {García, G and Shapiro, JB and Campbell, ZT},
title = {Efficient genetic perturbation of murine sensory neurons in vivo using CRISPR/Cas9.},
journal = {The journal of pain},
volume = {41},
number = {},
pages = {106217},
pmid = {41687699},
issn = {1528-8447},
support = {R01 NS114018/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Sensory Receptor Cells/metabolism ; Ganglia, Spinal/metabolism ; *CRISPR-Cas Systems/genetics ; Mice ; *TRPV Cation Channels/genetics/metabolism ; Mice, Transgenic ; NAV1.7 Voltage-Gated Sodium Channel/genetics/metabolism ; Hyperalgesia/genetics/chemically induced ; Sciatic Nerve/metabolism ; *Gene Editing/methods ; Capsaicin ; Male ; Disease Models, Animal ; Mice, Inbred C57BL ; },
abstract = {Gene editing using CRISPR/Cas9 in vivo offers a powerful tool to investigate pain mechanisms. We generated a conditional knock-in mouse model where Streptococcus pyogenes CRISPR-associated protein 9 (Cas9) expression is restricted to cells that express SCN9A. Transgenic markers were detected in key tissues including the dorsal root ganglia (DRG) and sciatic nerve. To assess in vivo editing efficacy, RNA guides targeting TRPV1 were intrathecally administered. Two injections of guide RNAs resulted in a significant reduction of TRPV1 in both the DRG and sciatic nerve without triggering caspase-3-mediated apoptosis or motor deficits. Edited animals exhibited increased withdrawal latencies to heat and reduced nocifensive behaviors following capsaicin injection. Capsaicin-evoked thermal hyperalgesia and mechanical allodynia were diminished. This approach enables rapid and efficient sensory neuron-specific CRISPR/Cas9 gene perturbations for pain research in mice. We envisage that this method can be employed both for the exploration of molecular mechanisms underlying nociception and for the validation of therapeutic targets associated with pain. PERSPECTIVE: There are tremendous opportunities afforded by facile multi-locus genome perturbation of sensory neurons in vivo. This model and approach enables rapid and low-cost genetic depletion experiments in mice.},
}
@article {pmid41687992,
year = {2026},
author = {Bulle, M and Rahman, MM and Kota, S and Islam, MR and Keya, SS and Abbagani, S and Kirti, PB},
title = {Advancing chloroplast bioengineering: Innovations, regulatory challenges, and translational pathways for sustainable agriculture.},
journal = {International journal of biological macromolecules},
volume = {350},
number = {},
pages = {150873},
doi = {10.1016/j.ijbiomac.2026.150873},
pmid = {41687992},
issn = {1879-0003},
mesh = {*Chloroplasts/genetics/metabolism ; *Bioengineering/methods ; *Agriculture/methods ; *Crops, Agricultural/genetics ; },
abstract = {Escalating climate instability and rising global food demand necessitate the development of resilient crop systems underpinned by precise, predictable, and rapidly deployable genetic innovations. Chloroplast bioengineering has emerged as a vanguard strategy, offering a uniquely tractable platform characterized by the organelle's distinct plastome, discrete copy number, and predominantly maternal inheritance. Recent advances in plastid transformation and base editing now enable the high-fidelity, multiplex introduction of photosynthetic, osmoprotective, and redox-regulating pathways across diverse plant lineages. Controlled-environment and field-proximal trials demonstrate that chloroplast-engineered metabolic modules enhance CO2 assimilation, stabilize photochemistry under heat and drought stress, and improve osmotic buffering capacity. Beyond trait improvement, chloroplasts function as high-capacity production organelles capable of accumulating oral or mucosal biologics at levels that reduce reliance on cold-chain logistics. However, challenges such as species-level recalcitrance, prolonged timelines for achieving homoplasmy, and limited scalability of current plastid biomanufacturing pipelines continue to constrain broad agricultural and biopharmaceutical deployment. To overcome these barriers, we propose a standardized, empirically testable framework integrating optimized transformation workflows, quantitative trait benchmarking, multi-location field validation, and techno-economic analyses. This framework embeds long-term stewardship principles, including marker-free selection, proactive resistance management, and FAIR-aligned data transparency, while emphasizing equitable access pathways for resource-limited regions. Collectively, this work positions chloroplast engineering as a mechanistically grounded, field-ready platform poised to reshape crop resilience, sustainable bioproduction, and global biologic accessibility amid accelerating climate stress.},
}
@article {pmid41688137,
year = {2026},
author = {Mathew, AE and Arivarasan, VK},
title = {Cell-free systems for nanobiomaterials assembly.},
journal = {Progress in molecular biology and translational science},
volume = {219},
number = {},
pages = {189-210},
doi = {10.1016/bs.pmbts.2025.11.002},
pmid = {41688137},
issn = {1878-0814},
mesh = {Cell-Free System ; Humans ; Animals ; *Nanostructures/chemistry ; *Biocompatible Materials/chemistry ; },
abstract = {Nanobiomaterials-engineered constructs operating at the 1-100 nm scale-integrate biological macromolecules such as nucleic acids and proteins with synthetic polymers or inorganic nanocomponents to achieve programmable functionality in biomedical and industrial contexts. Representative systems, including DNA origami-based drug carriers, CRISPR-Cas delivery scaffolds, and artificial protein vesicles, demonstrate subcellular targeting precision exceeding 90 % and up to a fivefold enhancement in intratumoral drug accumulation relative to conventional nanocarriers. Despite these advances, in vivo nanomanufacturing remains constrained by cytotoxicity, intracellular metabolic load, and limited spatiotemporal control over synthetic parameters. Cell-free systems (CFS) mitigate these limitations by utilizing crude lysates from prokaryotic or eukaryotic cells that retain functional transcription-translation machinery while eliminating the constraints of cellular viability. Platforms such as the reconstituted PURE system enable high-throughput, template-directed synthesis of nanoscale architectures incorporating noncanonical elements, including synthetic polymers, fluorinated analogs, and unnatural amino acids. This open, tunable environment permits over fivefold increases in yields of cytotoxic or aggregation-prone peptides, accelerates DNA origami prototype fabrication to under 24 h, and supports assembly of hybrid enzyme-polymer conjugates with retained catalytic activity. Distinct CFS sources impart complementary advantages: bacterial extracts offer rapid, cost-effective protein expression; yeast lysates facilitate eukaryote-specific glycosylation for ligand-specific nanocapsules; and mammalian systems enable near-physiological post-translational modification essential for therapeutic nanobiomaterials. Coupling CFS with artificial intelligence-based design optimization and microfluidic automation now underpins a new paradigm of programmable, scalable nanobiomanufacturing. By decoupling molecular construction from living systems, cell-free biofabrication establishes a controllable, high-fidelity platform for the rational engineering of nano-bio hybrid systems in precision medicine, biosensing, and tissue regeneration.},
}
@article {pmid41688147,
year = {2026},
author = {Mu, M and Melms, JC and Ho, P and Izar, B},
title = {Large-scale CRISPR-Cas9 screens to define regulators of immune checkpoints.},
journal = {Methods in cell biology},
volume = {202},
number = {},
pages = {117-132},
doi = {10.1016/bs.mcb.2025.10.011},
pmid = {41688147},
issn = {0091-679X},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Flow Cytometry/methods ; *Melanoma/genetics/immunology/pathology ; *Immune Checkpoint Proteins/genetics ; Animals ; Cell Line, Tumor ; },
abstract = {Immune checkpoints, which have emerged as potent target for the treatment of a variety of cancers, are central to tumor immunobiology and deciphering their dynamic regulation will continue to enable therapeutic development. CRISPR-Cas9 screening has recently been leveraged as a powerful tool to systematically interrogate regulators of immune checkpoints. Here, we describe a framework for such screens coupled with fluorescence-activated cell sorting (FACS) as a reliable and direct method of isolating and comparing how specific CRISPR perturbations impact the expression and maintenance of immune checkpoints. This approach has provided critical insights into immune checkpoint regulation and interactions in melanoma models and can feasibly be expanded to other systems.},
}
@article {pmid41688767,
year = {2026},
author = {Mansi, M and Danai, P},
title = {The emerging impact of CRISPR and gene editing on global crop improvement.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {8},
pmid = {41688767},
issn = {1573-9368},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; Genome, Plant ; },
abstract = {The advent of CRISPR-based genome editing has revolutionized crop improvement, offering unprecedented precision and efficiency in modifying key agronomic traits. This review comprehensively examines the mechanisms, applications, and future potential of CRISPR technology in enhancing global crop production. CRISPR-Cas systems, originally identified as adaptive immune mechanisms in bacteria and archaea, have been repurposed for targeted genome editing in plants. The CRISPR-Cas9 system, in particular, has emerged as a powerful tool for introducing site-specific double-strand breaks, enabling precise genetic modifications. The three-stage process of adaptation, expression, and interference underlies the CRISPR mechanism, with guide RNAs directing Cas endonucleases to specific genomic loci. Advances in CRISPR technology have expanded its applications beyond gene knockouts, encompassing base editing, prime editing, and epigenome editing. These innovations have facilitated the development of crops with enhanced yield, stress tolerance, disease resistance, nutritional content, and post-harvest quality. However, challenges related to off-target effects, regulatory hurdles, ethical concerns, and public acceptance must be addressed to fully harness the potential of CRISPR in agriculture. Integration of CRISPR with other cutting-edge technologies, such as synthetic biology, artificial intelligence, and high-throughput phenotyping, holds immense promise for accelerating crop improvement efforts. As research continues to refine CRISPR tools and expand their applicability across diverse plant species, this transformative technology is poised to play a pivotal role in shaping a sustainable, resilient, and productive global food system for future generations.},
}
@article {pmid41689002,
year = {2026},
author = {Zhong, X and Gong, X and Zeng, N and Xie, T and Wang, S and Xia, Q},
title = {Programmable hooded DNA switches for conditional control of CRISPR/Cas12a in multiplexed biosensing.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {},
pmid = {41689002},
issn = {1477-3155},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; MicroRNAs/analysis/genetics ; *DNA/chemistry/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The CRISPR/Cas system has become an indispensable tool for programmable and accurate biosensing, with its performance critically dependent on precise activity control. While most regulatory strategies have focused on engineering Cas proteins or modifying CRISPR RNAs, relatively little attention has been given to the design of substrate probes. Here, we systematically characterize the trans-cleavage activity of split CRISPR/Cas12a on structured substrates and leverage this insight to engineer a tunable "Hooded" probe with switchable properties. This probe architecture confers protection against trans-cleavage, and its activity can be progressively modulated by varying the probe length. Utilizing this design, we constructed a multiplexed logic-gated detection platform for direct and simultaneous analysis of miRNA and PSA, which demonstrated high sensitivity and specificity. Furthermore, we validated the robust performance of this system for logic-operated imaging in diverse cellular models, confirming its reliability in complex biological settings. Overall, our Hooded probe strategy not only broadens the applicability of CRISPR/Cas12a in molecular diagnostics, but also provides a novel design principle for the multiplexed biosensing.},
}
@article {pmid41689014,
year = {2026},
author = {Lou, C and Wang, J and Dai, C and Wang, J and Yang, J and Fang, Y and Jiang, H and Pan, X and Li, H and Lan, C and Xu, G and Iqbal, S and Bao, J and Cai, L and Zheng, W},
title = {Engineered Cas9 exosome vesicles as a novel gene editing tool for targeted ASPN editing in osteoarthritis.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {165},
pmid = {41689014},
issn = {1477-3155},
support = {LQ24H060008//Basic Public Welfare Research Program of Zhejiang Province/ ; 2025HY0585//Zhejiang Medicine Health Science and Technology Program/ ; GY20250280//Wenzhou Science and Technology Plan Project/ ; },
mesh = {*Osteoarthritis/genetics/therapy ; *Gene Editing/methods ; *Exosomes/metabolism/genetics/chemistry ; Animals ; *CRISPR-Cas Systems/genetics ; Chondrocytes/metabolism ; Humans ; *CRISPR-Associated Protein 9/metabolism/genetics ; Mice ; Male ; Mesenchymal Stem Cells/metabolism ; },
abstract = {CRISPR-Cas9, an innovative genome-editing technique, holds immense promise in therapeutic applications; nevertheless, the lack of effective delivery methods for in vivo gene editing limits its utility in osteoarthritis (OA) treatment. Recently, exosomes, naturally derived nanosized vesicles secreted by cells, have attracted significant attention as potential vehicles for therapeutic cargo delivery. This study proposes a bioinspired engineered exosome-mediated CRISPR/Cas9 delivery platform for targeted editing of the Asporin (ASPN) gene as a potential precision therapy for OA. Specifically, chondrocyte affinity peptide (Cap)-modified MSC-derived exosomes were employed as natural, biocompatible carriers to deliver CRISPR/Cas9 components specifically to OA-affected chondrocytes, thereby achieving precise and efficient ASPN knockout. Flow cytometry analysis confirmed a modification efficiency of 79.1% for Cap, while the encapsulation efficiency of the ASPN-Cas9 plasmid into exosomes reached 9.5% ± 0.6%. Both in vivo and in vitro investigations revealed that this delivery approach markedly improved cellular uptake and gene-editing efficacy, achieving a substantial reduction of ASPN expression by 61.7%. This, in turn, alleviated ferroptosis, improved mitochondrial function, reduced chondrocyte senescence, inhibited inflammation, and enhanced the cartilage microenvironment. Altogether, these findings strongly suggest the promising therapeutic efficacy of this method in OA models, emphasizing its potential as a precise gene-targeting therapeutic intervention for OA.},
}
@article {pmid41690121,
year = {2026},
author = {Yin, B and Wu, X and Zhou, H and Meng, Y and Liu, J and Ding, L and Zhu, C and Tai, Z and Zhu, Q and Chen, Z},
title = {Topical ionic liquid-mediated GLUT1 gene editing ameliorates psoriasis and prevents recurrence.},
journal = {Biomaterials},
volume = {330},
number = {},
pages = {124058},
doi = {10.1016/j.biomaterials.2026.124058},
pmid = {41690121},
issn = {1878-5905},
mesh = {Animals ; *Gene Editing/methods ; *Psoriasis/therapy/genetics/pathology ; *Glucose Transporter Type 1/genetics ; Mice ; *Ionic Liquids/chemistry/administration &amp; dosage ; Humans ; CRISPR-Cas Systems/genetics ; Recurrence ; Mice, Inbred C57BL ; Keratinocytes/metabolism ; Disease Models, Animal ; Administration, Topical ; },
abstract = {Psoriasis is a chronic inflammatory skin disorder characterized by immune dysregulation and a high relapse rate. Current therapies seldom achieve lasting remission. Aberrant overexpression of glucose transporter 1 (GLUT1) in keratinocytes enhances glycolysis, fueling inflammation and immune imbalance, thus positioning GLUT1 as a promising therapeutic target. In this work, a composite ionic liquid-mediated transdermal platform was established for the delivery of CRISPR-Cas9 ribonucleoprotein (CIL-RNP), aiming to achieve efficient GLUT1 gene editing in keratinocytes. The CIL-RNP achieved 76.6% editing efficiency, downregulated pyruvate kinase M (PKM) expression, and reduced inflammatory cytokine secretion. In a psoriasis mouse model, topical administration of CIL-RNP decreased lesion severity by 50% PASI (Psoriasis Area and Severity Index) score, alleviating epidermal hyperplasia and immune infiltration. Furthermore, the treatment inhibited M1 macrophage polarization, reduced reactive oxygen species generation, rebalanced Th17/regulatory T cells (Tregs) responses, and diminished the accumulation of tissue-resident memory T cells (TRMs), thereby lowering the risk of relapse. This study establishes ionic liquid-based CRISPR-RNP transdermal editing of GLUT1 as a novel and effective strategy for restoring immune homeostasis in psoriasis, with potential for long-term remission and broader applications in cutaneous immunopathological conditions.},
}
@article {pmid41690159,
year = {2026},
author = {Ha, E and Shin, D and Ryu, S and Kong, M},
title = {Deciphering the role of endolysin LysCPD7 harboring C. perfringens spore binding domain.},
journal = {Microbiological research},
volume = {307},
number = {},
pages = {128474},
doi = {10.1016/j.micres.2026.128474},
pmid = {41690159},
issn = {1618-0623},
mesh = {*Clostridium perfringens/virology/drug effects/physiology ; *Endopeptidases/genetics/metabolism/pharmacology/chemistry ; *Spores, Bacterial/drug effects/metabolism ; *Bacteriophages/enzymology/genetics ; Animals ; Milk/microbiology ; Protein Domains ; *Viral Proteins/genetics/metabolism ; Anti-Bacterial Agents/pharmacology ; Cell Wall/metabolism ; Mutation ; },
abstract = {Due to their potent bactericidal activity, phage-derived endolysins are considered promising alternatives to conventional antibiotics. Although some endolysins from phages infecting spore-forming bacteria contain a spore binding domain (SBD), their biological function remains unclear. LysCPD7, an endolysin from the Clostridium perfringens phage CPD7, showed high antimicrobial activity, effectively reducing C. perfringens contamination in milk and beef broth. Fluorescence assays and immunogold electron microscopy showed that LysCPD7 lacks a C-terminal cell wall binding domain, but contains a SBD that localizes to the spore cortex layer. We found that an E187K mutation in the SBD resulted in reduced spore binding capacity while retaining lytic activity. Infection with the wild-type CPD7 led to a decrease in sporulation efficiency in C. perfringens, whereas the mutant CPD7 carrying the E187K substitution in the SBD had no impact on sporulation, suggesting that the SBD may play a role in the inhibition of sporulation in C. perfringens. Our findings could contribute to the rational design of effective antimicrobials or diagnostic tools for controlling C. perfringens and provide new insights into the interactions between phages and their spore-forming hosts.},
}
@article {pmid41690735,
year = {2026},
author = {Li, G and Su, Z and Li, F and Liu, J and Shi, Y and Chen, J and Wang, P and Wang, R},
title = {Advances in rapid on-site detection techniques for food safety and authenticity.},
journal = {Advances in food and nutrition research},
volume = {118},
number = {},
pages = {43-87},
doi = {10.1016/bs.afnr.2025.08.004},
pmid = {41690735},
issn = {1043-4526},
mesh = {*Food Safety/methods ; *Food Contamination/analysis ; Biosensing Techniques/methods ; *Food Analysis/methods ; Humans ; Food Microbiology ; },
abstract = {Ensuring food safety and authenticity is a pressing global concern. This chapter provides a comprehensive overview of advanced rapid, on-site detection technologies targeting foodborne hazards, including pathogens, toxins, chemical contaminants, and authenticity issues such as adulteration and species fraud. Key aspects of these technologies include signal amplification strategies like catalytic hairpin assembly (CHA) and nanozyme-enhanced systems, as well as signal output platforms encompassing colorimetric, electrochemical, and fluorescence-based methods. The chapter also explores the emerging role of nanopore-based sensing as a novel signal output platform, emphasizing its single-molecule precision and broad applicability. By integrating laboratory innovations with practical field applications, this chapter underscores the potential of these technologies to address challenges in food safety and authenticity monitoring effectively.},
}
@article {pmid41691189,
year = {2026},
author = {Lv, J and Geng, L and Shi, W and Li, C and Wang, S and Zhang, B and Yu, K and Cui, R and Liu, Y and Xiong, F and Lv, J and Dong, S and Barco, B and Xu, J},
title = {Enhancing heritable genome editing in soybean by optimizing promoter combinations for the LbCas12a system.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {},
pmid = {41691189},
issn = {1471-2229},
abstract = {UNLABELLED: The CRISPR-Cas system, adapted from prokaryotic immune mechanisms, enables programmable DNA targeting and editing. Recent advancements include base editors and prime editors, expanding genetic research applications. The LbCas12a (Cpf1) system offers unique advantages, including simplified operation and multiplex editing, yet achieving stable heritable edits in soybean remains challenging due to its complex paleo-tetraploid genome and transformation constraints. Here we optimized promoter combinations for the LbCas12a editing system to maximize both editing efficiency in the initial (E0) generation and the frequency of homozygous or biallelic mutants in the subsequent (E1) generation. We identified prAtHSP70-1 (At5G02500), prAtEF1αA4 (At5G60390), and prGmUbi1 (Glyma.10G251900) as an effective promoter set, and demonstrated that LbCas12a–crRNA accumulation needs to exceed a defined threshold to achieve efficient target cleavage. These findings provide a practical strategy for enhancing heritable genome editing in soybean and may be broadly applicable to other crops.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12870-026-08336-w.},
}
@article {pmid41691442,
year = {2026},
author = {Thevendran, R and Maheswaran, S},
title = {Molecular Genetics as the Leading-Edge Approach in Driving the Development of Live Attenuated Vaccines.},
journal = {Biotechnology journal},
volume = {21},
number = {2},
pages = {e70185},
doi = {10.1002/biot.70185},
pmid = {41691442},
issn = {1860-7314},
support = {//MOHE/ ; },
mesh = {*Vaccines, Attenuated/genetics/immunology ; Humans ; *Vaccine Development/methods ; Gene Editing ; *Molecular Biology/methods ; Animals ; CRISPR-Cas Systems ; Biotechnology ; },
abstract = {Molecular genetics has propelled advancements in scientific instrumentation, yielding transformative discoveries from CRISPR-mediated gene editing and detailed protein identification to sophisticated biosensor fabrications. This profound shift has also reshaped the landscape of live attenuated vaccine (LAV) development in contrast to conventional methods. Here we explore how modern molecular strategies have superseded previous empirical approaches, moving toward deliberate genetic modifications that both enhance and balance the aspects of LAV safety, stability, and potent immunogenicity. By detailing the transition from classical approaches to targeted, molecular-driven attenuation, our work reviews how advanced genetic methods address historical limitations and expand the potential for vaccine design. Hence, the current paper bridges the gap between foundational vaccine practices and cutting-edge biotechnology, offering a comprehensive perspective on the progression of molecular genetic strategies on LAV development and its future trajectory. The paper also elaborates on the key challenges of raising LAVs to clinical standards while describing in tandem the genetic approaches to overcome the limitations. Critical performance factors governing the LAV market and clinical deployment, alongside the pivotal role of artificial intelligence in refining LAV rational design, are also further discussed.},
}
@article {pmid41691452,
year = {2026},
author = {Li, T and Zeng, F and Zhang, J and Zhang, Y and Yin, W},
title = {Reversing Antibiotic Resistance: Strategies From Adjuvants to Innovative Therapeutics.},
journal = {MicrobiologyOpen},
volume = {15},
number = {1},
pages = {e70233},
pmid = {41691452},
issn = {2045-8827},
support = {H2024201044//Natural Science Foundation of Hebei Province/ ; 20231560//The Medical Scientific Research of Hebei Health Commission/ ; },
mesh = {*Anti-Bacterial Agents/pharmacology/therapeutic use ; Humans ; *Bacteria/drug effects/genetics ; *Drug Resistance, Bacterial/drug effects/genetics ; Gene Editing ; *Bacterial Infections/drug therapy/microbiology ; CRISPR-Cas Systems ; Photochemotherapy ; Nanotechnology ; },
abstract = {The escalating prevalence of antibiotic resistance has become a major threat to the effectiveness of conventional antibiotics. Meanwhile, the development of novel antibiotics faces substantial challenges, including lengthy research cycles, high costs, and the rapid emergence of bacterial tolerance, making it difficult for new drugs to keep pace with bacterial evolution. In this context, molecular reversal strategies targeting antibiotic resistance genes have emerged as a promising avenue to overcome this impasse. Among them, the use of antibiotic adjuvants, agents that enhance the efficacy of existing antibiotics by inhibiting resistance gene function, preventing their horizontal transfer or modulating host defense has gained considerable attention. Furthermore, innovative approaches such as CRISPR-Cas gene editing, photodynamic therapy, nanotechnology, and ecological competition strategies have shown great potential in reversing antimicrobial resistance. Collectively, these strategies offer novel insights into addressing the global crisis of antibiotic resistance, paving the way for more effective clinical interventions and ensuring the sustained efficacy of current antibiotic therapies.},
}
@article {pmid41691970,
year = {2026},
author = {Anders, M and Hoppe, S and Eberl, H and Rebs, S and Seedorf, A and Maurer, W and Schill, T and Zibat, A and Unsöld, JK and Yigit, G and Wollnik, B and Vollmann, D and Sossalla, S and Streckfuss-Bömeke, K},
title = {Generation of pluripotent stem cell line (IPWi001-A) and a corresponding CRISPR/Cas9 modified isogenic rescue control (IPWi001-A-1) from a patient with arrhythmia-induced cardiomyopathy harboring a KCNQ1 truncating mutation.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103921},
doi = {10.1016/j.scr.2026.103921},
pmid = {41691970},
issn = {1876-7753},
mesh = {Humans ; *KCNQ1 Potassium Channel/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Arrhythmias, Cardiac/genetics/complications/pathology ; *Cardiomyopathies/genetics/pathology ; *Mutation/genetics ; Cell Line ; Myocytes, Cardiac/metabolism ; Cell Differentiation ; Male ; },
abstract = {KCNQ1 functions as a slow rectifying potassium channel during the repolarization of the cardiac action potential, with mutations causing long-QT syndrome 1 and arrhythmias. A genetic link between KCNQ1 mutations and arrhythmia-induced cardiomyopathy (AIC) has not been identified, and the underlying pathways remain elusive. We generated human induced pluripotent stem cells (hiPSCs) from an AIC patient harboring the heterozygous truncating mutation p.W15* in KCNQ1 and corrected the mutation to wildtype using CRISPR/Cas9. The hiPSCs retained full pluripotency, genomic integrity, and differentiation ability. They were differentiated into hiPSC-cardiomyocytes (hiPSC-CM), establishing a patient-specific model to explore potential genetic connections to AIC.},
}
@article {pmid41693124,
year = {2026},
author = {Xin, W and Tang, Z and Wang, S and Song, ZL and Luo, X},
title = {Scaffold-Proximal DNA Extensions Enhance Cas12a Trans-cleavage for Direct and Broad-Scope Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {8},
pages = {6149-6162},
doi = {10.1021/acs.analchem.5c07125},
pmid = {41693124},
issn = {1520-6882},
mesh = {*CRISPR-Associated Proteins/metabolism/chemistry ; CRISPR-Cas Systems ; *DNA/chemistry/metabolism/genetics ; *MicroRNAs/analysis/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Polymorphism, Single Nucleotide ; *RNA, Messenger/analysis ; *Bacterial Proteins/metabolism ; },
abstract = {The CRISPR/Cas12a system has revolutionized nucleic acid diagnostics, but its direct application for RNA detection remains constrained by the requisite reverse transcription step, insufficient sensitivity, and poor compatibility across diverse RNA targets such as microRNAs (miRNAs), long mRNAs, and single nucleotide polymorphisms (SNPs). Here, we introduce a rationally engineered DNA activator architecture that markedly enhances Cas12a trans-cleavage activity and expands its analytical utility. A key mechanistic finding is the strong positional dependence of activator extensions: appending an overhang specifically at the scaffold-proximal end of the DNA activator (termed Proximal-Extended Activator, PEA) potently boosts Cas12a activation through ribonucleoprotein (RNP) stabilization, whereas distal extensions are inhibitory due to steric hindrance of essential interdomain motions. This optimized PEA system facilitates direct, amplification-free RNA detection, achieving exceptional sensitivity with detection limits of 1.3 fM for miRNA and 93 fM for mRNA, all without reverse transcription. Furthermore, a Split-PEA format confers exceptional discriminatory power for SNPs, enabling robust identification of the EGFR T790 M mutation at a 0.1% allelic frequency. This work establishes a facile and versatile platform where simple sequence modification enables highly sensitive and specific analysis of a broad range of nucleic acid targets, effectively overcoming a significant hurdle in CRISPR-based diagnostics.},
}
@article {pmid41693245,
year = {2026},
author = {Kou, S and Chua, LC and Tan, JQ and Lau, OS},
title = {Stomatal XVE: an inducible system for cell-stage-specific gene expression and editing in the stomatal lineage.},
journal = {The New phytologist},
volume = {250},
number = {2},
pages = {1330-1347},
pmid = {41693245},
issn = {1469-8137},
support = {NRF-CRP22-2019-0001//National Research Foundation Singapore/ ; },
mesh = {*Plant Stomata/genetics/cytology ; *Arabidopsis/genetics/cytology ; *Gene Expression Regulation, Plant/drug effects ; *Gene Editing/methods ; *Cell Lineage/genetics ; Arabidopsis Proteins/metabolism/genetics ; Promoter Regions, Genetic/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Phenotype ; },
abstract = {Stomatal development has emerged as a valuable model for studying developmental processes. Examining gene function along the stomatal lineage often requires gene perturbation in a controlled and cell-stage-specific manner, but this remains tedious without a dedicated genetic tool. Here, we describe Stomatal XVE, a modular, two-component XVE-based inducible system that enables user-controlled gene overexpression and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based knockout at defined stomatal cell stages in Arabidopsis thaliana. The system consists of a collection of estrogen-responsive XVE driver lines under cell-stage-specific promoters and effector vectors responsive to activated XVE. This design simplifies cloning and allows users to scale their investigation. We validated the cell-stage specificity and inducibility of the XVE driver lines and characterized key induction parameters. To test the system functionally, we employed it to study MAPKKK YODA and a pathogen effector AvrPtoB. While YODA overexpression reproduced known early- and late-stage phenotypes, stage-specific knockouts argued against its late-stage role in guard cell (GC) differentiation. Furthermore, AvrPtoB expression during later stages triggered striking disruptions in GC morphology and viability, revealing cell-type-specific effects of the pathogen protein. Overall, our Stomatal XVE system enables precise functional analysis of genes across defined stages of stomatal development and is particularly well suited for investigating genes with pleiotropic effects.},
}
@article {pmid41693565,
year = {2026},
author = {Sinnott, RW and Solanki, A and Govind, AP and Green, WN and Dickinson, BC},
title = {Engineering a human-based translational activator for targeted protein expression restoration.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41693565},
issn = {1362-4962},
support = {R01-EB035016/NH/NIH HHS/United States ; FP106237//G. Harold and Leila Y. Mathers Charitable Foundation/ ; //Bank of America/ ; DGE-2022294368//National Science Foundation/ ; //Dr. Ralph and Marian Falk Medical Research Trust/ ; R01 EB035016/EB/NIBIB NIH HHS/United States ; },
mesh = {Animals ; Humans ; Mice ; *Protein Biosynthesis ; *Epilepsies, Myoclonic/genetics/therapy ; Disease Models, Animal ; RNA, Messenger/genetics/metabolism ; *NAV1.1 Voltage-Gated Sodium Channel/genetics/metabolism ; CRISPR-Cas Systems ; *Protein Engineering/methods ; Haploinsufficiency ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate messenger RNA (mRNA) would be an advantageous approach to correct gene expression but has not been evaluated in an in vivo disease model. Here, we investigated whether a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identify and engineer human proteins capable of increasing mRNA translation using the CRISPR-Cas-inspired RNA-targeting system (CIRTS) platform to enable programmable, guide RNA-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3) that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold-key phenotypic indicators of Dravet syndrome. This work validates a strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential of targeted translational activation to address neurological haploinsufficiency.},
}
@article {pmid41695711,
year = {2026},
author = {Browne, TS and Edgell, DR and Gloor, GB},
title = {Better data for better predictions: data curation improves deep learning for sgRNA/Cas9 prediction.},
journal = {PeerJ},
volume = {14},
number = {},
pages = {e20706},
pmid = {41695711},
issn = {2167-8359},
mesh = {*Deep Learning ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Gene Editing/methods ; },
abstract = {The Cas9 enzyme along with a single guide RNA molecule is a modular tool for genetic engineering and has shown effectiveness as a species-specific antimicrobial. The ability to accurately predict on-target cleavage is critical as activity varies by target. Using the sgRNA nucleotide sequence and an activity score, predictive models have been developed with the best performance resulting from deep learning architectures. Prior work has emphasized robust and novel architectures to improve predictive performance. Here, we explore the impact of a data-centric approach through optimization of the input target site adjacent nucleotide sequence length and the use of data filtering for read counts in the control conditions to improve input data utility. Using the existing crisprHAL architecture, we develop crisprHAL Tev, a bacterial SpCas9 prediction model with performance that generalizes across related species and across data types. During this process, we also rebuilt two prior Escherichia coli Cas9 datasets, demonstrating the importance of data quality, and resulting in the production of an improved bacterial eSpCas9 prediction model. The crisprHAL models are available through GitHub: https://github.com/tbrowne5/crisprHAL.},
}
@article {pmid41696049,
year = {2026},
author = {Ahmadzadeh, M and Akbarian, F and Sanati, MH and Motaharirad, H and Farrokhi, F},
title = {Computational Optimization of CRISPR-Cas13a sgRNAs Targeting the SARS-CoV-2 Spike Gene for SHERLOCK-Based Diagnostics.},
journal = {Evolutionary bioinformatics online},
volume = {22},
number = {},
pages = {11769343251414318},
pmid = {41696049},
issn = {1176-9343},
abstract = {BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a global health crisis, emphasizing the urgent need for accurate and rapid diagnostic tools. Modern molecular biology technologies, including CRISPR-Cas systems, provide highly efficient strategies for viral detection. Bioinformatic pipelines are essential for identifying conserved genomic regions and enabling rational single-guide RNA (sgRNA) design.<br><br>METHODS: This study aimed to design specific sgRNAs targeting the spike gene of SARS-CoV-2 isolates from Iranian patients using the SHERLOCK diagnostic platform. Complete genomes of the RefSeq virus and 470 SARS-CoV-2 isolates, representing all variants of concern (VOCs) detected in Iran, were retrieved from the NCBI and GISAID databases. Multiple sequence alignment with ClustalW identified conserved sequences within the receptor-binding domain (RBD) that differ from the RBD of SARS-CoV and MERS-CoV RefSeq genomes. Based on these regions, sgRNAs and isothermal amplification primers were designed using ADAPT, OLIGO7, and the UCSC Genome Browser to maximize diagnostic sensitivity and specificity. Secondary and tertiary structures of sgRNA-target complexes were analyzed via RNAfold and RNAup to select the most efficient sgRNA-amplicon combination.<br><br>RESULTS: Twenty-two-nucleotide sgRNA candidates were initially selected based on sequence alignment, showing high similarity to the SARS-CoV-2 RefSeq and low homology to SARS-CoV and MERS-CoV genomes. Analyses of secondary structures, RNA-RNA interactions, and free energy identified 6 sgRNAs with favorable 2-dimensional conformations and strong interaction profiles. Among these, the sgRNA1-Amplicon2 sequence exhibited the most stable 3-dimensional structure and a molecular docking score of -309.67, indicating high sensitivity and specificity for viral detection.<br><br>CONCLUSION: This study successfully designed an sgRNA with high sensitivity and specificity for rapid SARS-CoV-2 detection using the CRISPR-Cas13a system, informed by genomic analysis of Iranian isolates. The proposed approach provides an efficient framework for the rapid design and deployment of CRISPR-based diagnostic tools applicable to diverse viral pathogens.},
}
@article {pmid41699287,
year = {2026},
author = {Pindi, C and Palermo, G},
title = {Computation and deep-learning-driven advances in CRISPR genome editing.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {2},
pages = {203-214},
pmid = {41699287},
issn = {1545-9985},
mesh = {*Deep Learning ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; Neural Networks, Computer ; Algorithms ; },
abstract = {Genome editing with CRISPR-Cas systems is revolutionizing medicine, molecular biology and biotechnology. In this Review, we discuss the contributions of deep learning-based structure prediction algorithms, physics-based simulations, neural networks, graph neural networks and generative models, including diffusion and large language models, in engineering and optimizing CRISPR systems and in understanding their mechanistic basis. We highlight the challenges and limitations to the transformative effects of computational modeling and tools in the context of the development of programmable genome editors for biomedicine and biotechnology.},
}
@article {pmid41699288,
year = {2026},
author = {Hu, C and Tai, H and Zhu, R and Shu, Z and Guo, G and Ma, D and Zuo, S and Tang, L and Zeng, Z},
title = {CRISPR-Cas9 screening identifies ATOX1-driven cisplatin resistance mechanisms in liver cancer and evaluates targeted inhibitor efficacy.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41699288},
issn = {2399-3642},
mesh = {*Cisplatin/pharmacology ; Humans ; *Drug Resistance, Neoplasm/genetics ; *Liver Neoplasms/drug therapy/genetics/metabolism/pathology ; *CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Animals ; Cell Line, Tumor ; Mice ; *Copper Transport Proteins/genetics/antagonists &amp; inhibitors/metabolism ; Female ; Gene Expression Regulation, Neoplastic/drug effects ; Male ; Receptor, Notch1/genetics/metabolism ; },
abstract = {Liver cancer treatment with cisplatin is often hindered by drug resistance. This study aimed to identify key genes associated with cisplatin resistance in liver cancer and develop targeted inhibitors. Using genome-wide CRISPR-Cas9 screening, ATOX1 was identified as a critical gene for cisplatin resistance. ATOX1 was highly expressed in liver cancer tissues and associated with poor prognosis. Knockdown of ATOX1 in liver cancer cells enhanced cisplatin sensitivity in vitro and in vivo. Molecular dynamics simulation and virtual screening identified compound 8 as a potent ATOX1 inhibitor with high affinity (Kd = 12.5 μM) and exhibited synergistic effects with cisplatin on liver cancer cell growth. Mechanistically, compound 8 inhibits the activity of ATOX1, leading to intracellular copper accumulation. The elevated copper levels subsequently promote increased DNA methylation at the NOTCH1 promoter, resulting in suppression of the NOTCH1/HES1 signaling pathway and enhancing the sensitivity of liver cancer cells to cisplatin. In conclusion, ATOX1 is crucial for cisplatin resistance in liver cancer and linked to poor prognosis. Targeting ATOX1 with compound 8 may be a novel therapeutic strategy for overcoming cisplatin resistance.},
}
@article {pmid41699368,
year = {2026},
author = {Khalid, M and Ishaq, A and Arshad, M and Kaul, H and Majeed, M},
title = {Multiplex CRISPR/Cas9 editing of gliotoxin biosynthesis genes in Aspergillus fumigatus reduces pathogenicity in broilers.},
journal = {Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology]},
volume = {57},
number = {1},
pages = {55},
pmid = {41699368},
issn = {1678-4405},
support = {13224/2020//Higher Education Commision, Pakistan/ ; },
mesh = {Animals ; *Aspergillus fumigatus/genetics/pathogenicity/metabolism ; *Gliotoxin/biosynthesis ; Chickens ; *CRISPR-Cas Systems ; *Gene Editing ; *Aspergillosis/veterinary/microbiology/pathology ; *Poultry Diseases/microbiology/pathology ; Virulence ; Fungal Proteins/genetics/metabolism ; Spores, Fungal/genetics ; },
abstract = {Gliotoxin of Aspergillus fumigatus has been extensively studied for its role in pathogenesis in animals and humans. It triggers pathogenesis by its immunosuppressive and cytotoxic effects. Biosynthetic gene cluster (BGC) consisting of 13 genes regulates its biosynthesis. We targeted gliZ, gliP and gliA genes of this BGC using CRISPR/Cas9 system in a multigene editing approach to check the pathogenesis in broilers. crRNAs were designed using EuPaGDT and 3 single guide RNAs (sgRNA) were commercially synthesized. Each sgRNA was combined with Cas9 to form ribonucleoprotein complexes which were then used for simultaneously transfecting fungal protoplasts. Thin-layer chromatography showed the absence of gliotoxin on silica plate and DNA sequencing showed various indels in target genes. These indels caused amino acid substitutions in all three gene products but, the gliP mutation, since it was synonymous, was likely not functionally relevant. Regenerated protoplasts were matured to form fungal hyphae and spore production was induced. These spores were inoculated intra-air sac in broiler chicks. During one-week infection trial, birds infected with the wild-type spores (group 1) showed morbidity and their mortality rate was 30%. Birds inoculated with RNP-treated spores (group 2) showed mild clinical signs and no mortality. No morbidity or mortality was recorded in birds in negative control group (group 3). Histopathological analysis of lungs showed necrosis and congestion, and presence of mixed population of inflammatory cells in wild-type infected birds, while no such lesions were seen in birds infected with RNP-treated spores. These results show that multigene editing approach was successful in creating indels simultaneously in 3 gliotoxin genes which resulted in amino acid substitution which negatively impacted gliotoxin biosynthesis and export. In vivo experiment results show that RNP-treated fungal spores were unable to cause A. fumigatus pathogenicity in broiler. Targeting gliotoxin biosynthesis could thus be a promising approach to develop antifungal therapy.},
}
@article {pmid41700332,
year = {2026},
author = {Huang, Y and Zhao, Z and Li, J and Wang, X and Qu, H and Zheng, Y},
title = {An aptamer-CRISPR/Cas12a biosensor for rapid and sensitive detection of florfenicol.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {9},
pages = {1910-1916},
doi = {10.1039/d5ay01903f},
pmid = {41700332},
issn = {1759-9679},
mesh = {*Biosensing Techniques/methods ; *Thiamphenicol/analogs &amp; derivatives/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Anti-Bacterial Agents/analysis ; Limit of Detection ; Animals ; Food Contamination/analysis ; },
abstract = {Florfenicol (FF), a broad-spectrum antibacterial agent widely used in livestock and poultry farming, has raised significant food safety concerns due to the accumulation of its residues in animal-derived products (e.g., eggs), posing potential threats to human health. Herein, we developed a novel aptamer-CRISPR/Cas12a biosensor for the rapid and sensitive detection of FF. The biosensor employs streptavidin-modified magnetic beads (SA-MBs) as a solid carrier to achieve efficient enrichment of FF-specific aptamers (APT), while integrating the dual advantages of the APT's high-specificity target recognition and the CRISPR/Cas12a system's powerful signal amplification capability. The detection mechanism is based on a competitive displacement: APT pre-hybridizes with its complementary strand (APT-c) to form stable duplexes. The presence of FF triggers the release of APT-c from APT, and the liberated APT-c then activates the trans-cleavage activity of the CRISPR/Cas12a system. This process converts the small-molecule FF into a CRISPR/Cas12a-detectable nucleic acid signal and enables quantitative FF detection. Under optimized conditions, the biosensor demonstrated a linear detection range of 10 nM to 100 µM for FF (R[2] = 0.9907) and a limit of detection (LOD) of 1.41 nM. The accuracy and practicality were confirmed through spiked recovery experiments in egg samples, yielding recoveries between 97.1% and 100.8%. Furthermore, the modular design of this platform allows its easy adaptation for detecting other antibiotics simply by replacing the specific APT and its corresponding APT-c, highlighting its considerable potential for broad applications in food safety monitoring.},
}
@article {pmid41700547,
year = {2026},
author = {Su, T and Zhu, D and Li, X and Qu, Z and Li, F and Zhao, D and Shao, G and Feng, Z and Su, S and Chao, J and Zuo, X and Fan, C and Wang, L},
title = {Accelerated CRISPR/Cas12a-Based Point-of-Care Diagnostics Through Critical Coupling Distance Control.},
journal = {Advanced materials (Deerfield Beach, Fla.)},
volume = {38},
number = {16},
pages = {e23381},
doi = {10.1002/adma.202523381},
pmid = {41700547},
issn = {1521-4095},
support = {62071251//National Natural Science Foundation of China/ ; 62235008//National Natural Science Foundation of China/ ; 62227803//National Natural Science Foundation of China/ ; 62288102//National Natural Science Foundation of China/ ; BK20212012//Natural Science Foundation&#xa0;of&#xa0;Jiangsu&#xa0;Province-Major Project/ ; BZ2022011//"Belt and Road" Innovation Cooperation Project of Jiangsu/ ; SKT2306//Project of State Key Laboratory of Transducer Technology/ ; GZR2022010026//Project of State Key Laboratory of Organic Electronics and Information Displays, Nanjing University of Posts and Telecommunications/ ; BK20243057//Basic Research Program of Jiangsu/ ; BK20253006//Basic Research Program of Jiangsu/ ; },
mesh = {*CRISPR-Cas Systems ; *Point-of-Care Systems ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism ; *CRISPR-Associated Proteins/metabolism ; DNA/chemistry ; Humans ; },
abstract = {Prompt pathogen detection in resource-limited settings remains constrained by energy-intensive instrumentation and a shortage of trained personnel. The CRISPR/Cas12a-based diagnostic technology, despite its robustness as a promising tool, is constrained by suboptimal detection speed and sensitivity. Here we designed triblock DNA-mediated spherical nucleic acids (tSNA) that acts as a spatially confined reporter with critical coupling distances between substrates, enabling Cas12a protein to rapidly identify concentrated and stretched single-stranded substrates with size-matching intervals. Precise control of distances on tSNA of varying sizes revealed a direct correlation between trans-cleavage efficiency and coupling distance, indicating that only when the distance exceeds the protein size can it offer an appropriate reaction space. It demonstrates a rapid "scooting" reaction model on tSNA, resulting in a trans-cleavage rate of 10 nm A30-tSNA 12 times faster and a sensitivity that is two orders of magnitude higher than that in bulk solution. Furthermore, tSNA can serve as a novel recognition and colorimetric element in lateral-flow strips, thereby reducing the detection time for pathogen nucleic acids to just 3 min. This "size-matching" model of tSNA offers a new perspective on the regulation of Cas12a enzymatic activity, establishing a versatile platform to advance diagnostic development through ultrafast, CRISPR-powered POC systems.},
}
@article {pmid41702027,
year = {2026},
author = {Galdikaite-Braziene, E and Krušnauskas, R and Henderson, E and Bujakowska, KM},
title = {CRISPR as a therapeutic tool for inherited retinal degenerations: Advances, challenges, and future directions.},
journal = {Molecular aspects of medicine},
volume = {108},
number = {},
pages = {101462},
doi = {10.1016/j.mam.2026.101462},
pmid = {41702027},
issn = {1872-9452},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; *Retinal Degeneration/therapy/genetics ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Inherited retinal diseases (IRDs) are a genetically diverse group of disorders characterized by progressive photoreceptor degeneration, leading to vision loss and blindness. With over 320 associated genes and significant phenotypic variability, effective treatment remains challenging. Recent advances in genome editing, particularly CRISPR/Cas-based technologies, have revolutionized therapeutic approaches by enabling precise and customizable DNA and RNA editing. This review explores the application of various CRISPR strategies-such as gene knockout via non-homologous end joining (NHEJ), exon skipping using dual-sgRNAs, homology-directed repair (HDR), base editing (BE), prime editing (PE), RNA editing with Cas13, and epigenetic modulation through CRISPRa/i-in preclinical models of IRDs. Emphasis is placed on allele-specific targeting, gene-agnostic approaches, and mutation-independent strategies to address dominant and recessive forms of disease. We also highlight recent clinical milestones, including the first human trial using CRISPR gene editing for CEP290-associated Leber congenital amaurosis. Finally, we discuss critical challenges, including delivery constraints, immune responses, and off-target effects, along with emerging solutions such as engineered Cas variants, split-intein systems, and advanced off-target detection methods. Together, these advances underscore the transformative potential of CRISPR technologies in treating IRDs and lay the foundation for future clinical translation.},
}
@article {pmid41702192,
year = {2026},
author = {Lu, L and Zhang, Y and Liu, B and Zhou, N and Yu, DJ and Wang, Y},
title = {Magnetic Fe3O4-Au@UIO-66-NH2@toehold probe mediated fluorescent sensor for detecting ovarian cancer-specific circRNA coupled with hybridization chain reaction and the CRISPR-Cas12a system.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118535},
doi = {10.1016/j.bios.2026.118535},
pmid = {41702192},
issn = {1873-4235},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *RNA, Circular/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Female ; *Ovarian Neoplasms/genetics/diagnosis ; Gold/chemistry ; Fluorescent Dyes/chemistry ; Nucleic Acid Hybridization ; Limit of Detection ; Magnetite Nanoparticles/chemistry ; Ferrosoferric Oxide/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Circular RNAs (circRNAs) represent an emerging family of noncoding transcripts defined by closed-loop architecture, which are now established as key participants in the etiology of tumorigenesis and malignant progression. While circRNAs show potential as therapeutic targets and biomarkers, the accurate detection of circRNAs remains challenging due to interference from homologous linear RNAs. In this study, an ultrasensitive method for detecting circ_0051240 based on the sulfhydrated toehold capture probe-initiator assembly-mediated hybridization chain reaction (HCR) and the CRISPR-Cas12a system was developed. The capture probe (with a toehold domain)-initiator strand duplex was ingeniously fabricated to identify the back-splice junction (BSJ) of circRNA. After magnetic enrichment and separation by the Fe3O4-Au@UIO-66-NH2 nanocomposite, the initiator strand (H0) was delivered to trigger HCR. The HCR product dsDNA concatemers contained multiply repeated CRISPR-targetable DNA sites that were readily recognized by the CRISPR RNA (crRNA). This specific recognition and binding activated the CRISPR-Cas12a system's collateral endonuclease activity, leading to cleavage of the fluorophore-quencher (FQ) reporters and fluorescence emission at a characteristic wavelength. This design eliminated linear RNA-related interference and enhanced the detection of fluorescence intensity (FI). Under optimal conditions, the proposed HCR/CRISPR-Cas12a method exhibited a wide quantitative measurement range spanning from 45 pM up to 180 nM, achieving a notable limit of detection (LOD) of 0.03 pM. In this study, a novel circRNA sensing strategy capable of accurate and highly sensitive quantification of ovarian cancer-specific circRNA was reported. The proposed method exhibits acceptable performance when compared to present approaches.},
}
@article {pmid41702403,
year = {2026},
author = {Gaizauskaite, U and Tamulaitiene, G and Silanskas, A and Gasiunas, G and Siksnys, V and Sasnauskas, G},
title = {Structural insights into Cas9-mediated prespacer selection in CRISPR-Cas adaptation.},
journal = {Molecular cell},
volume = {86},
number = {5},
pages = {791-804.e9},
doi = {10.1016/j.molcel.2026.01.022},
pmid = {41702403},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems/genetics ; Cryoelectron Microscopy ; *Streptococcus thermophilus/genetics/enzymology ; *CRISPR-Associated Protein 9/genetics/chemistry/metabolism/ultrastructure ; *Bacterial Proteins/genetics/chemistry/metabolism/ultrastructure ; Models, Molecular ; CRISPR-Associated Proteins/genetics/chemistry/metabolism ; *DNA, Bacterial/genetics/metabolism/chemistry/ultrastructure ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Conformation ; Gene Editing ; },
abstract = {During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems, selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a "supercomplex" with the Cas1-Cas2 integrase and the Csn2 protein. Here, we present cryo-electron microscopy structures of the Streptococcus thermophilus type II-A prespacer selection supercomplex in the DNA-scanning and two distinct PAM-bound configurations, providing insights into the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems. Repurposing Cas9 by the CRISPR adaptation machinery for prespacer selection, as characterized here, demonstrates Cas9 plasticity and expands our knowledge of Cas9 biology.},
}
@article {pmid41702404,
year = {2026},
author = {Li, Z and Li, Y and Kong, J and Wu, Q and Huang, P and Zhang, Y and Wu, W and Chen, M and Liu, Y and Lin, H and Hou, L and Liu, G and Zeng, T and He, Y and Hu, C and Yang, Z and Lu, M and Luo, M and Xiao, Y},
title = {Structural basis for Cas9-directed spacer acquisition in type II-A CRISPR-Cas systems.},
journal = {Molecular cell},
volume = {86},
number = {5},
pages = {805-816.e4},
doi = {10.1016/j.molcel.2026.01.024},
pmid = {41702404},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems ; Cryoelectron Microscopy ; *CRISPR-Associated Proteins/genetics/chemistry/metabolism/ultrastructure ; *Enterococcus faecalis/genetics/enzymology ; *Bacterial Proteins/genetics/chemistry/metabolism/ultrastructure ; Models, Molecular ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; Protein Binding ; *DNA, Bacterial/genetics/metabolism/chemistry/ultrastructure ; Binding Sites ; Protein Conformation ; },
abstract = {CRISPR-Cas systems confer prokaryotic immunity by integrating foreign DNA (prespacers) into host arrays. Type II-A systems employ Cas9 for protospacer-adjacent motif (PAM) recognition and coordinate with Csn2 and the Cas1-Cas2 integrase during spacer acquisition, yet their structural basis remains unresolved. Here, we report cryo-electron microscopy (cryo-EM) structures of the Enterococcus faecalis Cas9-Csn2-Cas1-Cas2 supercomplex in apo and DNA-bound states. The apo state (Cas92-Csn28-Cas18-Cas24) is a resting complex, while DNA binding forms a prespacer-catching complex threading DNA through Csn2's channel, enabling Cas9 to interrogate the PAM sequence while sliding along the DNA. Cas9 and Csn2 jointly define a 30-bp DNA segment matching the prespacer length. Cas9 dissociation triggers structural reconfiguration of the Csn2-Cas1-Cas2 assembly. This exposes the PAM-proximal DNA, allowing Cas1-Cas2 to bind the exposed site for subsequent prespacer processing and directional integration. These findings reveal how Cas9, Csn2, and Cas1-Cas2 couple PAM recognition with prespacer selection, ensuring fidelity during adaptation.},
}
@article {pmid41702797,
year = {2026},
author = {Li, C and Mei, D and Cheng, H and Pan, X and Zhang, B},
title = {CRISPR genome editing in plants without tissue culture.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.12.017},
pmid = {41702797},
issn = {1879-3096},
abstract = {Conventional plant genome editing relies on tissue culture-mediated somatic cell regeneration, a technically demanding process that limits its application across diverse species. Emerging strategies now circumvent this bottleneck by enabling direct genome editing of meristematic or germline cells. Key advances include (i) genome editing via de novo meristem induction or dormant meristem activation; (ii) germline editing facilitated by graft-mobile tRNA-like sequence systems and haploid induction technologies; and (iii) optimized viral delivery platforms that exploit mobile RNA elements and compact editors such as TnpB to achieve efficient, transgene-free, heritable modifications across a broad range of genotypes and species. The development of robust, tissue culture-free editing platforms promises to revolutionize crop improvement pipelines and accelerate trait development for sustainable agriculture.},
}
@article {pmid41702943,
year = {2026},
author = {de Menezes, MN and Chen, AXY and Kulkarni, N and Sampurno, S and Saw, NYL and Yap, KM and Pérez-Núñez, I and Roth, S and Deguit, CDT and Haugen, B and Ramsbottom, KM and Munoz, I and Beavis, PA and Parish, IA},
title = {High efficiency CRISPR knock-in demonstrates that TCF1 is insufficient to reverse T cell exhaustion.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41702943},
issn = {2041-1723},
support = {MCRF21019//Victorian Cancer Agency (VCA)/ ; MCRF20011//Victorian Cancer Agency (VCA)/ ; Lloyd J. Old STAR Grant CRI5578//Cancer Research Institute (CRI)/ ; },
mesh = {Animals ; *Hepatocyte Nuclear Factor 1-alpha/genetics/metabolism ; *Gene Knock-In Techniques/methods ; Mice ; *CD8-Positive T-Lymphocytes/immunology/metabolism/cytology ; Cell Differentiation ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; *T Cell Transcription Factor 1/genetics/metabolism ; Mice, Transgenic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; T-Cell Exhaustion ; },
abstract = {CD8[+] T cell exhaustion is a regulatory state triggered by chronic antigen stimulation in both cancer and persistent infection. The less differentiated stem-like sub-populations of exhausted T cells have been heavily studied given their importance to the efficacy of current immunotherapies. While the transcription factor TCF1 is both necessary and sufficient for formation and maintenance of these stem-like populations, it remains unclear whether TCF1 can actively de-differentiate more terminally exhausted subsets back into a stem-like state. To address this question, here we utilize and optimize a high efficiency CRISPR knock-in methodology, compatible with mouse in vivo exhaustion models, to engineer T cells that either constitutively over-express TCF1, or conditionally over-express TCF1 following differentiation of the cells into a CX3CR1[+] intermediate-exhausted state. Strikingly, we find that only constitutive, and not conditional, TCF1 over-expression can increase the size of the stem-like T cell pool. Thus, while TCF1 can slow stem-like T cell differentiation, it is insufficient to revert more differentiated cells back into a stem-like state.},
}
@article {pmid41703314,
year = {2026},
author = {Rocha, DC and Omoregbee, MO and Luo, W and Fang, H and Ye, Q and Liu, Y and Li, G and Mascoveto, J and de Souza, AA and Coleman, G and Culver, JN and Qi, Y},
title = {Transgene-free genome editing in citrus and poplar meristem tissues via biolistic ribonucleoprotein delivery of CRISPR-Cas9.},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {58},
pmid = {41703314},
issn = {1432-203X},
support = {2020/07045-3, 2021/03466-7, and 2023/09068-9//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de S&#xe3;o Paulo/ ; DE-SC0023011//U.S. Department of Energy/ ; IOS-2132693 and IOS-2428015//Directorate for Biological Sciences/ ; 2020-70029-33161, 2021-67013-34554, 2024-33522-42755//National Institute of Food and Agriculture/ ; MD-PSLA-24014//McIntire Stennis Forest Research Program/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Meristem/genetics ; *Ribonucleoproteins/genetics/metabolism ; *Populus/genetics ; *Citrus/genetics ; *Biolistics/methods ; Plants, Genetically Modified ; Transgenes/genetics ; },
abstract = {Biolistic particle bombardment was used to deliver CRISPR-Cas9 ribonucleoprotein complexes (RNP) into the shoot apical meristem tissue of citrus and axillary meristem tissue of poplar, generating directed mutations in target genes. The use of meristematic tissues offers a strategic approach to genome editing in woody species, especially those that are recalcitrant to conventional tissue culture, as these regions contain totipotent, highly regenerative cells capable of giving rise to whole plants. Here, we employed biolistic delivery of genome-editing reagents into theshoot apical meristem (SAM) of citrus and the axillary meristems (AXM) of poplar. The system was first validated using a GFP expression construct and subsequently applied for targeted genome editing. In citrus, edited plants were obtained at the CsNPR3 locus exclusively through the delivery of CRISPR/Cas9 ribonucleoproteins (RNPs), whereas plasmid-based vectors were unsuccessful. Similarly, genome editing in poplar was achieved using RNPs targeting the Pt4CL1 gene. Although chimeric events were detected, this approach provides a feasible and innovative framework for producing transgene-free edited perennial plants.},
}
@article {pmid41704216,
year = {2026},
author = {Khweis, SA and Blackburn, MA and Perdigao, CC and Pierce, MO and Lewis, CR and Dunkle, JA},
title = {Cas10 residues lining the target RNA binding channel regulate interference by distinguishing cognate target RNA from mismatched targets.},
journal = {RNA biology},
volume = {23},
number = {1},
pages = {1-18},
pmid = {41704216},
issn = {1555-8584},
support = {R35 GM142966/GM/NIGMS NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Staphylococcus epidermidis/genetics/metabolism ; *RNA, Bacterial/metabolism/genetics ; Protein Binding ; *RNA-Binding Proteins/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics/chemistry ; Models, Molecular ; *RNA/metabolism/genetics ; Binding Sites ; Mutation ; },
abstract = {Type III CRISPR systems are defined by the presence of the Cas10 protein and are among the most abundant CRISPR systems in nature. Cas10 forms a complex with crRNA and several Cas proteins that surveils prokaryotic cells for foreign RNA molecules and when they are detected it activates a cascade of interference activities. The synthesis of the cyclic oligoadenylate signalling molecule by Cas10 is a key aspect of the interference cascade. Despite structures of the Cas10 complex bound to target RNAs, the molecular mechanism by which Cas10 senses the bound state to licence interference is lacking. We identified five residues in S. epidermidis Cas10, two in the Cas10 Palm2 domain and three in domain 4, that line the target RNA binding channel. We assessed the contribution of these residues to interference in the context of a cognate or mismatched target RNA. We found that the residues regulate whether a mismatched crRNA-target RNA duplex is able to activate interference in vivo. We purified two site-directed mutants of Cas10-Csm and show with in vitro cOA synthesis assays that they demonstrate enhanced discrimination of cognate versus mismatched target RNAs.},
}
@article {pmid41704956,
year = {2026},
author = {Wang, Y and Diao, Y and Zhang, T and Zhang, F and Wang, W},
title = {Sensitive, specific, and rapid on-site detection of calf diarrhea pathogens using the RPA-CRISPR/Cas 12a assay.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1734185},
pmid = {41704956},
issn = {2235-2988},
mesh = {Animals ; Cattle ; Sensitivity and Specificity ; *Diarrhea/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Cattle Diseases/diagnosis/virology/microbiology ; *Molecular Diagnostic Techniques/methods ; Diarrhea Viruses, Bovine Viral/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Enterotoxigenic Escherichia coli/isolation &amp; purification/genetics ; },
abstract = {Calf diarrhea is a common gastrointestinal disease that usually occurs within one month of birth. The disease causes the greatest economic losses to the cattle industry. Currently, a variety of diagnostic methods have been developed for calf diarrhea infections. However, existing methods are still unsatisfactory in terms of sensitivity, specificity, simplicity, cost, and speed.To provide a more sensitive, specific, simpler, and faster detection method, we recently developed an RPA-CRISPR/Cas12a assay that can detect BVDV, BCoV, BRV, and ETEC infections in cattle on-site. Testing for each pathogen is performed in a single test tube, without the need to open the tube in the middle, and can be completed in under 50 minutes.The RPA-CRISPR/Cas12a assay can detect BVDV, BCoV, BRV, and ETEC at concentrations of at least 10 copies/μL. The RPA-CRISPR/Cas12a assay does not produce false-positive results due to the presence of other pathogens. The sensitivity of BCoV, BRV, and ETEC in the RPA-CRISPR/Cas12a quadruple assay is equivalent to that of single qPCR. The sensitivity of BVDV in the quadruple assay is slightly lower than that of the single qPCR method.Due to its sensitivity, specificity, simplicity, and rapidity, the RPA-CRISPR/Cas12a assay is more practical for on-site detection of cattle diarrhea pathogens than any existing detection method.},
}
@article {pmid41705235,
year = {2026},
author = {Li, Z and Ge, L and Yu, T and Lv, S and Fu, Q and Shi, H},
title = {Genome-wide CRISPR/Cas9 screening identifies host factors critical for antiviral defense against equine herpesvirus type 1.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1764863},
pmid = {41705235},
issn = {1664-3224},
mesh = {Animals ; *CRISPR-Cas Systems ; *Herpesvirus 1, Equid/physiology/immunology ; Virus Replication/genetics ; Cell Line ; *Host-Pathogen Interactions/genetics/immunology ; *Herpesviridae Infections/immunology/virology/genetics/veterinary ; Horses ; Gene Knockout Techniques ; *Horse Diseases/virology/immunology/genetics ; Genome-Wide Association Study ; },
abstract = {INTRODUCTION: Equine herpesvirus type 1 (EHV-1) is a major veterinary pathogen causing significant economic losses in the livestock industry. Despite its impact, effective vaccines and targeted antiviral strategies remain limited, largely due to an incomplete understanding of host factors regulating viral replication and pathogenesis.<br><br>METHODS: To systematically identify host genes essential for EHV-1 infection, we established a BHK-21 cell line stably expressing Cas9 and performed a genome-wide CRISPR/Cas9 knockout screen using a pooled lentiviral single-guide RNA library. Significantly enriched candidate genes from positive selection were validated by generating knockout cell lines. Viral replication and protein expression were assessed using quantitative polymerase chain reaction and Western blot analysis. Pathway enrichment and protein interaction network analyses were subsequently conducted.<br><br>RESULTS: Genome-wide CRISPR/Cas9 screening identified multiple host factors critical for EHV-1 replication. Pathway enrichment analysis revealed that these genes were involved in key cellular signaling and regulatory networks associated with viral infection. Functional validation demonstrated that knockout of selected host genes significantly suppressed EHV-1 replication and viral protein synthesis.<br><br>DISCUSSION: These findings highlight essential host determinants required for EHV-1 replication and suggest that targeting host factors may represent a promising strategy for antiviral intervention. This study provides a foundation for the development of host-directed immunotherapeutic and antiviral approaches against EHV-1 infection.},
}
@article {pmid41705504,
year = {2026},
author = {Sun, K and Wu, H},
title = {A highly-efficient isothermal nano-detection platform coupling CRISPR/Cas technology for detection of circRNA.},
journal = {The Analyst},
volume = {151},
number = {5},
pages = {1304-1309},
doi = {10.1039/d6an00107f},
pmid = {41705504},
issn = {1364-5528},
mesh = {Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Triple Negative Breast Neoplasms/diagnosis/genetics ; *RNA, Circular/analysis/genetics ; Limit of Detection ; *Biomarkers, Tumor/genetics/analysis ; Female ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Triple-negative breast cancer (TNBC), an aggressive molecular subtype of breast cancer with poor prognosis, is characterized by a high rate of metastasis and proliferation, which makes early detection particularly challenging. Early diagnosis of TNBC through biomarkers and prompt development of treatment methods can lower its mortality rate. This work has designed a nano-detection platform for TNBC biomarker circRNA based on the CRISPR/Cas system and isothermal amplification strategy. Specifically, this detection system uses functional nucleic acid molecules for recognition of circCD44, as well as dual signal amplification using Klenow(3'-5'exo-) and Cas9n. Furthermore, it combines Cas12a and immunomagnetic beads for an extra signal boost and output. After confirming its feasibility and optimizing the conditions, the detection system achieved a 1.73-fold enhancement in sensitivity, offering a linear detection range of 1 pM to 100 nM, with the limit of detection lowered to 95.1 fM. It also showed good specificity through testing against 5 biomarkers. Therefore, this detection system provides a novel strategy for the early diagnosis of TNBC and other diseases.},
}
@article {pmid41705810,
year = {2026},
author = {Shin, K and Kim, ET},
title = {Viral genome editing methods and applications in the CRISPR era.},
journal = {Journal of virology},
volume = {100},
number = {3},
pages = {e0204825},
pmid = {41705810},
issn = {1098-5514},
support = {RS-2025-02223595//Korea Health Industry Development Institute/ ; RS-2025-25406739//Korea Health Industry Development Institute/ ; RS-2023-00270936//National Research Foundation of Korea/ ; RS-2024-00352590//National Research Foundation of Korea/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Genome, Viral ; Herpesvirus 1, Human/genetics ; Cytomegalovirus/genetics ; *DNA Viruses/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR-Cas systems have transformed viral genetics by enabling precise and efficient manipulation of large DNA virus genomes. This review provides a practical framework for applying CRISPR technology to herpesviruses and other large DNA viruses as an alternative and complement to traditional BAC recombination. Key considerations include nuclease choice; sgRNA design that minimizes cut-to-edit distance and prevents re-cutting; donor template configuration and homology arm length; and synchronized delivery of Cas complexes and donor DNA. Strategies to promote HDR efficiency, such as the use of small-molecule modulators, are also summarized. In addition, practical workflows for clone selection, genotypic validation, and phenotypic confirmation are summarized. Case studies in herpes simplex virus type 1 and human cytomegalovirus illustrate how optimized CRISPR designs achieve reproducible, scarless knock-ins and conditional gene manipulation at essential loci without complementing cell lines. Together, these approaches establish CRISPR as a flexible, scalable platform for functional genomics, antiviral target discovery, and translational virology, enabling direct editing of clinical isolates previously inaccessible with bacterial artificial chromosome-based methods.},
}
@article {pmid41706678,
year = {2026},
author = {Verhezen, T and Van Den Eynde, A and Verstraelen, P and Gehrcken, L and Palmiotto, G and Lau, HW and De Vos, WH and Van Der Heijden, S and Brants, L and Melis, J and Van Audenaerde, J and Rodrigues Fortes, F and Le Compte, M and Roeyen, G and Prenen, H and Campillo-Davo, D and Lion, E and Argüello, RJ and Van Laere, S and Lardon, F and Deben, C and Wouters, A and Smits, E and De Waele, J},
title = {DRP1 depletion protects NK cells from hypoxia-induced dysfunction.},
journal = {Redox report : communications in free radical research},
volume = {31},
number = {1},
pages = {2626181},
pmid = {41706678},
issn = {1743-2928},
mesh = {*Killer Cells, Natural/metabolism ; Humans ; *Dynamins/metabolism/genetics ; Reactive Oxygen Species/metabolism ; Mitochondria/metabolism ; Cell Line, Tumor ; Cell Hypoxia ; Membrane Potential, Mitochondrial ; *Hypoxia/metabolism ; CRISPR-Cas Systems ; Tumor Microenvironment ; },
abstract = {OBJECTIVES: The efficacy of cellular therapies has been disappointing in solid tumors. A major barrier that contributes to the low success rate, is hypoxia within the tumor microenvironment. In this study, we investigated the influence of hypoxia on natural killer (NK) cell function and to evaluated a strategy to restore their activity in hypoxia.<br><br>METHODS: Unarmed or CAR NK cells were placed in normoxia (21% O2) or hypoxia (1% O2) prior to experimental readouts. Mitochondrial content and morphology were assessed by confocal microscopy, membrane potential and reactive oxygen species (ROS) by flow cytometry, and global transcriptional changes by RNA sequencing. Cytotoxicity was evaluated against tumor cell lines and patient-derived cancer organoids, which were characterized by RNA sequencing. DRP1 function was inhibited pharmacologically or through CRISPR-Cas9-mediated knockout.<br><br>RESULTS: Hypoxia reduced NK cell mitochondrial content and membrane potential, while increasing mitochondrial ROS and inducing broad transcriptional changes in stress response pathways. Their cytotoxic activity was drastically impaired, which could not be prevented by CD70-CAR-IL-15 engineering. Pharmacological inhibition of DRP1 restored mitochondrial content and cytotoxic function. To confirm the role of DRP1, CRISPR-Cas9-mediated DRP1 knockout (KO) NK cells preserved mitochondrial load and membrane potential under hypoxia, and DRP1[KO] CAR NK cells retained cytotoxic activity under hypoxic conditions against cancer cell lines. Patient microtumor models with distinct transcriptomic profiles exhibited divergent responses to DRP1[WT] and DRP1[KO] CAR NK cells.<br><br>CONCLUSION: These findings indicate that DRP1 inactivation supports NK cell function in hypoxia and metabolic engineering may enhance CAR-NK efficacy in solid tumors.},
}
@article {pmid41707086,
year = {2026},
author = {Banerjee, S and Banerjee, A and Ray, S and Ray, A and Paul, D and Dastidar, SG and Willard, B and Biswas, K},
title = {dCas9 Targeted Proteome Profiling Reveals p300-Mediated Reciprocal Regulation of SMAD and SP1 as a Driver of GM2-synthase Transcription in Renal Cell Carcinoma.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {4},
pages = {e71597},
doi = {10.1096/fj.202502746R},
pmid = {41707086},
issn = {1530-6860},
support = {2019-0137-CMB/adhoc/BMS//MOHFW | DHR | Indian Council of Medical Research (ICMR)/ ; EMR/2016/001983//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; CRG/2021/004623//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; },
mesh = {Humans ; *Carcinoma, Renal Cell/genetics/metabolism/pathology ; *Sp1 Transcription Factor/metabolism/genetics ; *Kidney Neoplasms/genetics/metabolism/pathology ; *Smad Proteins/metabolism/genetics ; *Sialyltransferases/genetics/metabolism ; Cell Line, Tumor ; *E1A-Associated p300 Protein/metabolism/genetics ; *Proteome/metabolism/genetics ; Gene Expression Regulation, Neoplastic ; Transcription, Genetic ; CRISPR-Cas Systems ; *p300-CBP Transcription Factors/metabolism ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore are established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2, is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion, and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of the GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasn't clear. This study demonstrated that the histone acetyl transferase p300, acts as a pivotal factor which on one hand causes acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down, and over-expression experiments. Furthermore, SP1 degradation, SMAD activation, and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD2/4 as an activator complex, p300 as a positive regulator, and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.},
}
@article {pmid41707424,
year = {2026},
author = {Zhao, L and Liu, Z and Ding, G and Zhu, Y and Wang, H and Liu, R and Qu, F and Ao, Q and Zhu, X and Zhang, Y and Yang, G and Wang, Z},
title = {Dual-readout aptasensor based on CRISPR/Cas12a and nanozyme for accurate detection of KIM-1 and its application in kidney transplant prognosis.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118533},
doi = {10.1016/j.bios.2026.118533},
pmid = {41707424},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Hepatitis A Virus Cellular Receptor 1/isolation &amp; purification ; *Aptamers, Nucleotide/chemistry ; CRISPR-Cas Systems ; *Kidney Transplantation ; Limit of Detection ; Prognosis ; Metal Nanoparticles/chemistry ; Silver/chemistry ; Colorimetry/methods ; Metal-Organic Frameworks/chemistry ; SELEX Aptamer Technique ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Kidney injury molecule-1 (KIM-1) has emerged as a pivotal prognostic biomarker for renal allograft function. However, its detection remains challenging due to sensitivity and accuracy limitations. An innovative biosensing platform synergizing aptamer recognition, CRISPR trans-cleavage and nanozyme amplification for dual-readout KIM-1 detection has been presented in this work. This platform employs a meticulously selected high-affinity aptamer with capillary electrophoresis SELEX for specific target recognition, leverages the CRISPR/Cas12a system for signal transduction and cascade amplification, and utilizes engineered FeNi MOF@AgNPs nanozyme for dual-signal output. With the presence of KIM-1, whose binding with aptamer, effectively inhibits the trans-cleavage activity of the CRISPR/Cas12a system, and uninhibited Cas12a subsequently cleaves nanozyme-conjugated magnetic probes, releasing FeNi MOF@AgNPs nanozymes that catalyze a TMB-based reaction to generate intense colorimetric and fluorescent dual-readout signals. The as developed aptasensor demonstrates satisfied sensitivity achieving detection limits of 58.7 pg/mL (colorimetric) and 34.4 pg/mL (fluorometric), and dependable accuracy achieving average relative deviation of -2.7% (colorimetric) and 3.2% (fluorometric) with commercial ELISA kit in urine samples from patients with acute kidney injury. Moreover, longitudinally track the dynamic changes in urinary KIM-1 concentrations over the first 8 days following renal transplantation was successfully realized. This work not only provides a robust analytical tool for KIM-1 detection but also establishes a generic research approach for extending CRISPR-based systems to the precise detection of proteins.},
}
@article {pmid41707427,
year = {2026},
author = {Tu, Z and Wang, Y and Qian, X and Chen, J and Li, L and Wang, T and Chen, H and Wei, H and Yang, P and Pan, J and Wang, S and Rong, Z},
title = {Plasmonic magnetic nanoparticles-enabled universal enrichment, photothermal lysis, and duplex CRISPR detection of bacteria in urine samples.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118518},
doi = {10.1016/j.bios.2026.118518},
pmid = {41707427},
issn = {1873-4235},
mesh = {Humans ; *Escherichia coli/isolation &amp; purification/genetics ; *Magnetite Nanoparticles/chemistry ; *Enterococcus faecalis/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Urinary Tract Infections/microbiology/urine/diagnosis ; CRISPR-Cas Systems ; Surface Plasmon Resonance/methods ; *Escherichia coli Infections/urine/microbiology/diagnosis ; Limit of Detection ; Boronic Acids/chemistry ; },
abstract = {Urinary tract infections (UTIs), exacerbated by antibiotic resistance and evolving pathogen diversity, demand rapid and sensitive diagnostics. This study introduces an integrated platform combining magnetic enrichment, photothermal lysis, and CRISPR-based detection (ME-CRISPR) for simultaneous identification of Escherichia coli (E. coli) and Enterococcus faecalis (E. faecalis) in UTIs. We engineered plasmonic magnetic nanoparticles functionalized with 4-mercaptophenylboronic acid for broad-spectrum bacterial capture within 10 min. Subsequent near-infrared laser irradiation leveraged localized surface plasmon resonance to lyse captured bacteria in situ. The released nucleic acids were directly analyzed in a single-step duplex RPA-CRISPR/Cas12a-Cas13a assay. This 40-min workflow achieved a limit of detection of 10 CFU/mL for both pathogens. Validation with 90 clinical samples (39 E. coli, 26 E. faecalis, and 25 negative controls) demonstrated 100% sensitivity and specificity, matching qPCR performance while significantly reducing turnaround time. The platform overcomes limitations of centrifugation and Gram-class-dependent lysis, offering a rapid and ultrasensitive point-of-care testing tool to curb antibiotic misuse.},
}
@article {pmid41707429,
year = {2026},
author = {Nong, J and Pan, Z and Li, Y and Wei, J and Gong, Y and Li, J and Zhang, K and Liao, X},
title = {COF-confined CsPbBr3 nanocomposite with CRISPR/Cas12a-driven DNA walking for ultrasensitive electrochemiluminescent detection of circulating tumor DNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118536},
doi = {10.1016/j.bios.2026.118536},
pmid = {41707429},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Humans ; *Nanocomposites/chemistry ; Luminescent Measurements/methods ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/blood/isolation &amp; purification/genetics ; Endodeoxyribonucleases/chemistry ; DNA Probes/chemistry ; Metal-Organic Frameworks/chemistry ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {A highly sensitive electrochemiluminescence biosensor was developed for circulating tumor DNA detection by integrating a covalent organic framework-confined CsPbBr3 nanocomposite with a CRISPR/Cas12a-driven amplification strategy. The covalent organic framework not only stabilizes CsPbBr3 nanocrystals in aqueous environments but also regulates interfacial charge transfer and enables programmable immobilization of DNA probes. Upon recognition of the target sequence, Cas12a is activated and progressively cleaves surface-tethered quencher-modified DNA strands, leading to stepwise restoration of electrochemiluminescence emission. This surface-confined signal amplification eliminates the need for polymerase chain reaction or isothermal preamplification. Under optimized conditions, the biosensor exhibited a linear response over a concentration range from 10 fM to 10 nM, with a detection limit of 5.4 fM. The method demonstrated good selectivity toward single-base mismatches and satisfactory performance in diluted serum and clinical plasma samples. These results highlight a synergistic material-enzyme strategy for sensitive and robust nucleic acid detection and provide a proof-of-concept platform for electrochemiluminescence-based circulating tumor DNA analysis.},
}
@article {pmid41708610,
year = {2026},
author = {Klann, M and Miura, S and Lee, SH and Vianello, SD and Ross, R and Watanabe, M and Gairin, E and Liang, Y and Hutto, HW and McCluskey, BM and Herrera, M and Solnica-Krezel, L and Besseau, L and Pigolotti, S and Parichy, DM and Kinoshita, M and Laudet, V},
title = {Cell-cell communication as underlying principle governing color pattern formation in teleost fishes.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41708610},
issn = {2041-1723},
mesh = {Animals ; *Cell Communication/genetics/physiology ; Zebrafish/genetics ; Gap Junctions/metabolism ; *Pigmentation/genetics/physiology ; Mutation, Missense ; Connexins/genetics/metabolism ; Fish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Genome-Wide Association Study ; Phenotype ; Gene Editing ; *Perciformes/genetics ; },
abstract = {The diverse pigmentation patterns of animals are crucial for predation avoidance and behavioral display. This diversity arises from interactions among distinct pigment cell types, yet mechanisms generating pattern variation across teleost fishes remain incompletely understood. In zebrafish, Turing models have been proposed to explain stripe patterns, but it is unclear if they apply to other fishes. Here, we investigate the Snowflake mutant of the anemonefish Amphiprion ocellaris, which displays enlarged white bars with irregular boundaries. Using genome-wide association mapping and targeted sequencing, we identify a missense mutation (E42K) in gja5b, encoding the gap junction protein Connexin 41.8. CRISPR/Cas9-mediated genome editing recapitulates the Snowflake phenotype, while pharmacological inhibition of gap junctions phenocopies the boundary defects, supporting a causal role for impaired intercellular communication. Expression analyses reveal that, unlike zebrafish, anemonefish gja5b is predominantly expressed in iridophores. With functional in vitro assays we demonstrate that the E42K mutation acts as a dominant negative, strongly reducing gap junctional coupling. Introducing the same mutation in zebrafish reveals context-dependent effects on pigment patterning. Taken together our findings highlighting gap junction-mediated communication as a conserved but flexible mechanism controlling pigment boundary positioning and pattern diversification.},
}
@article {pmid41708664,
year = {2026},
author = {Huo, Y and Mei, J and Zhang, D and Yan, B and Zhang, D and Dong, C and Yin, S and Liu, M and Wang, X and Chen, D and Guan, Y and Song, G and Du, B and Wang, Y and Zheng, Z and Liu, H and Li, D and Yang, L and Wang, L},
title = {Engineered Un1Cas12f1 for multiplex genome editing with enhanced activity and targeting scope.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41708664},
issn = {2041-1723},
support = {U24A20677//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32025023//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32230064//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32311530111//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24J22800400//Science and Technology Commission of Shanghai Municipality (Shanghai Municipal Science and Technology Commission)/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; Humans ; Mice ; HEK293 Cells ; Genome, Human ; Mutation ; Genetic Therapy/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {The compact CRISPR-Cas12f system is promising for AAV-delivered gene therapy, but its application has been constrained by restrictive PAM recognition (e.g., TTTR) and suboptimal editing efficiency. Through bacterial library screening and mammalian cell validation, we engineer evoCas12f, an optimized variant incorporating five key mutations, that dramatically expands PAM recognition to NTNR/NYTR. This advancement reduces median distance between two neighbouring PAM sites to 2 nucleotides in the human genome. It also demonstrates 1.4-fold enhanced activity at TTTR sites compared to wild-type Un1Cas12f1, achieving up to 91% editing efficiency. Remarkably, evoCas12f enables efficient generation of homozygous mutations in F0 generation mice, even at non-canonical PAM sites. We further adapt this system for robust transcriptional activation and precise base editing with a well-defined editing window. As a compact yet highly efficient platform, evoCas12f represents a significant advance in CRISPR technology, enabling multiplexed editing for high-resolution targeting applications and expanding possibilities for therapeutic genome engineering.},
}
@article {pmid41709648,
year = {2026},
author = {Doghish, AS and Ghaiad, HR and Elfar, N and El Said, NH and Radwan, AF and Abd-Elmawla, MA and Mohamed, HH and Mohammed, OA and Rizk, HA},
title = {Unraveling the Function of lncRNAs in Gliomas: Interaction With Signaling Pathways and Therapeutic Opportunities.},
journal = {Journal of biochemical and molecular toxicology},
volume = {40},
number = {3},
pages = {e70756},
doi = {10.1002/jbt.70756},
pmid = {41709648},
issn = {1099-0461},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Glioma/genetics/metabolism/therapy/pathology ; *Signal Transduction ; *Brain Neoplasms/metabolism/genetics/therapy/pathology ; Animals ; *Gene Expression Regulation, Neoplastic ; *RNA, Neoplasm/metabolism/genetics ; },
abstract = {Brain tumors represent some of the most formidable challenges in neuro-oncology due to their aggressive clinical course, resistance to therapy, and profound molecular heterogeneity. Among the emerging regulatory elements reshaping our understanding of tumor biology are long non-coding RNAs (lncRNAs), a diverse class of RNA transcripts that modulate gene expression and cellular behavior without encoding proteins. This review provides an in-depth and integrative examination of the biogenesis, regulatory mechanisms, and functional roles of lncRNAs in brain tumor development and progression. We systematically explore both canonical and non-canonical pathways of lncRNA biogenesis, detailing how these influence structural specificity and molecular interactions. This review synthesized evidence retrieved from PubMed/MEDLINE, Scopus, and Web of Science, covering publications from January 2010 to June 2025. This analysis highlights key gaps, such as context-dependent therapeutic effects that limit translational applicability. A major focus is placed on the interplay between lncRNAs and core oncogenic signaling pathways, including Phosphoinositide 3-kinase (PI3K)/serine/threonine kinase (AKT), Signal Transducer and Activator of Transcription 3 (STAT3), Wingless/Int-1 (Wnt)/β-catenin, and Transforming Growth Factor-Beta (TGF-β), which drive malignant transformation, invasion, stemness, and therapeutic resistance in gliomas. Furthermore, we dissect the molecular functions of lncRNAs as epigenetic regulators, competitive endogenous RNAs (ceRNAs), and structural scaffolds, and discuss their contribution to the dynamic tumor microenvironment. By synthesizing the latest findings, this review underscores the academic and translational importance of targeting lncRNA-associated networks. It also highlights emerging therapeutic approaches, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, and natural lncRNA-modulating compounds, which collectively represent a promising frontier in precision medicine for brain tumors. This work offers a critical framework for future research and therapeutic innovation in the lncRNA landscape of neuro-oncology.},
}
@article {pmid41709870,
year = {2026},
author = {Feng, L and Yu, P and He, N and Zhang, Q and Tang, Y and Geng, J and Lu, Q and Song, C and Chen, F},
title = {Label-Free MicroRNA Diagnostics: From CRISPR Nucleases to Nanomaterial-Enhanced Transducers.},
journal = {ACS synthetic biology},
volume = {15},
number = {3},
pages = {915-954},
doi = {10.1021/acssynbio.5c00868},
pmid = {41709870},
issn = {2161-5063},
mesh = {*MicroRNAs/genetics/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Nanostructures/chemistry ; },
abstract = {Label-free detection of microRNAs (miRNAs) has emerged as a pivotal approach in molecular diagnostics, integrating the programmability of CRISPR systems with the high sensitivity of nanomaterial-based transduction. miRNAs are short, noncoding RNAs that play central roles in gene regulation and disease pathogenesis, serving as valuable biomarkers for early diagnosis and prognosis. Conventional miRNA detection methods rely on labeling and multistep amplification, which hinder their adaptability for rapid and point-of-care applications. In contrast, label-free biosensing translates molecular recognition into intrinsic optical, electrochemical, or mechanical signals, enabling real-time, amplification-free analysis. This review summarizes recent advances in label-free miRNA biosensing, with emphasis on CRISPR/Cas12a, Cas13a, and Cas14a systems that couple target recognition with signal transduction, and nanomaterial-assisted platforms including gold and silver nanoparticles, carbon nanotubes, quantum dots, silica nanostructures, and magnetic composites. Particular attention is given to innovations that achieve attomolar-level sensitivity, single-nucleotide discrimination, and multiplex detection. We also discuss integration into microfluidic and wearable platforms, addressing persistent challenges in repeatability and stability, antifouling performance, and clinical translation. Emerging trends in artificial intelligence-assisted data processing, molecular logic circuits, and digital single-molecule biosensing are highlighted. These advances collectively outline the pathway toward intelligent, amplification-free, and portable miRNA diagnostics, bridging molecular biology and synthetic bioengineering for next-generation healthcare applications.},
}
@article {pmid41710881,
year = {2026},
author = {Karaoglu, IC and Odabas, A and Önder, T and Kizilel, S},
title = {Single-gene knockout of RNLS or HIVEP2 are insufficient to protect β-cell spheroids from allo- and xeno-rejection.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1759835},
pmid = {41710881},
issn = {1664-3224},
mesh = {Animals ; Mice ; *Insulin-Secreting Cells/transplantation/immunology/metabolism ; Humans ; *Graft Rejection/genetics/immunology/prevention &amp; control ; Gene Knockout Techniques ; *Spheroids, Cellular/immunology/transplantation/metabolism ; CRISPR-Cas Systems ; *Islets of Langerhans Transplantation ; Cell Line ; Gene Editing ; Transplantation, Heterologous ; },
abstract = {INTRODUCTION: β-Cell replacement therapy offers a potential cure for type 1 diabetes, but its success is limited by rapid graft rejection. While genome-wide CRISPR screens have recently identified RNLS and HIVEP2 as candidate genes capable of protecting β-cells from autoimmune destruction, their efficacy against the distinct mechanisms of allogeneic and xenogeneic rejection remains unknown. This study aimed to test the hypothesis that single-gene ablation of RNLS or HIVEP2 protects β-cell spheroids from allo- and xenorejection in immunocompetent hosts.<br><br>METHODS: Murine &#x3b2;-TC-6 and human EndoC-&#x3b2;H1 &#x3b2;-cell lines were genetically edited using CRISPR-Cas9 to knockout RNLS or HIVEP2. Editing efficiencies were confirmed via T7 endonuclease I assay and Tracking of Indels by Decomposition (TIDE) analysis. Cells were aggregated into uniform, size-controlled spheroids using an optimized agarose suspension culture. Functional integrity was assessed via glucose-stimulated insulin secretion (GSIS). To evaluate immune evasion in vivo, luciferase-labeled spheroids were transplanted subcutaneously into immunocompetent CD-1 mice, modelling allogeneic (murine-to-murine) and xenogeneic (human-to-murine) rejection, with graft survival monitored longitudinally by bioluminescence imaging.<br><br>RESULTS: Robust editing efficiencies were achieved for both targets. Functional characterization indicated that Rnls deletion modestly impaired GSIS in murine cells, whereas HIVEP2 deletion showed no functional alterations in either cell line. In vivo assessment revealed no protective effects of RNLS or HIVEP2 deletion; grafts from both knockout groups displayed rejection kinetics indistinguishable from non-targeting controls. While allogeneic grafts survived longer than xenogeneic grafts, both were ultimately cleared by the host immune system regardless of genotype.<br><br>DISCUSSION: These data indicate that single-gene deletions of RNLS or HIVEP2 are insufficient to protect &#x3b2;-cell grafts from the barriers of allo- or xenorejection. By defining the limitations of these targets in isolation, our findings highlight the necessity for combinatorial genome editing strategies or complementary integration with immunomodulatory biomaterials to achieve effective and sustained &#x3b2;-cell graft survival.},
}
@article {pmid41710969,
year = {2026},
author = {Xu, Z and Wei, M and Jiang, M and Wang, Y and He, K},
title = {Single-step duplex CRISPR coupled with lateral flow assay for point-of-care detection of human immunodeficiency virus and Treponema pallidum.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {10},
pages = {1996-2004},
doi = {10.1039/d5ay02168e},
pmid = {41710969},
issn = {1759-9679},
mesh = {Humans ; *Treponema pallidum/genetics/isolation &amp; purification ; *Point-of-Care Systems ; *HIV Infections/diagnosis/virology ; *Syphilis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; *HIV/genetics/isolation &amp; purification ; Point-of-Care Testing ; Sensitivity and Specificity ; },
abstract = {Continued acquired immunodeficiency syndrome (AIDS) caused by the human immunodeficiency virus (HIV) and syphilis caused by Treponema pallidum (TP) pose great challenges for global health, highlighting the need for rapid and sensitive diagnostics. Here, we introduced DIAL (Duplex Integrated All-in-one CRISPR Lateral flow assay), a simplified CRISPR-based diagnostic system for simultaneous detection of HIV and TP. We developed a single-step duplex CRISPR assay for dual-target recognition, which incorporated reverse transcription, multiplex recombinase polymerase amplification, transcription, CRISPR/Cas12a detection, and CRISPR/Cas13a detection in one pot. Then, we combined CRISPR detection assay with a "line-elimination" dual-line lateral flow assay for low-cost and equipment-free visual readout. We further developed reagent lyophilization and extraction-free sample lysis techniques to meet the point-of-care testing. The optimized DIAL system detected as low as 100 copies per µL of both HIV RNA and TP DNA within 45 minutes. In a clinical evaluation of 46 samples, it demonstrated 100% sensitivity and 97.5% specificity for HIV and 90% sensitivity and 100% specificity for TP, relative to quantitative PCR. The DIAL provided a scalable, accessible approach for decentralized screening of sexually transmitted infections or other pathogens in resource-limited settings.},
}
@article {pmid41711690,
year = {2026},
author = {Shi, H and Chi, H},
title = {Next-generation CRISPR screens enable causal systems immunology.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {3},
pages = {},
doi = {10.1084/jem.20241266},
pmid = {41711690},
issn = {1540-9538},
support = {//American Lebanese Syrian Associated Charities/ ; CA253188/NH/NIH HHS/United States ; CA281868/NH/NIH HHS/United States ; AI105887/NH/NIH HHS/United States ; AI131703/NH/NIH HHS/United States ; AI140761/NH/NIH HHS/United States ; AI150241/NH/NIH HHS/United States ; AI150514/NH/NIH HHS/United States ; //Lupus Research Alliance/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Systems Biology/methods ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Immunoinformatics ; },
abstract = {Mapping the causal circuits that shape the phenotypic and functional landscape of immune cells remains a formidable challenge. Recent advances in pooled CRISPR-based screens, coupled with multiplexed single-cell profiling and imaging-based spatial readouts, make this goal increasingly attainable. In this Perspective, we discuss how CRISPR-based genetic screens will fundamentally transform our understanding of immunobiology. We highlight the applications of state-of-the-art, high-throughput pooled perturbation approaches, including emerging methodologies for bulk, single-cell, and spatial CRISPR screens, to advance our understanding of immunity and in vivo biology. Additionally, we summarize new strategies to address the complexity of combinatorial perturbations to uncover genetic interactions and mechanistic drivers of immunity at unprecedented scale and resolution. By integrating CRISPR screening data with experimental insights, we advocate a new framework in immunology research that leverages perturbation-driven regulatory effects and networks to discover new therapeutic targets and establish causal systems biology and immunology for advancing immunological knowledge and therapeutic application.},
}
@article {pmid41711853,
year = {2026},
author = {Guo, Y and Zhao, J and Li, X and Zheng, X and Yuan, Y and Deng, J and Ren, Y and Xu, K},
title = {Detection of alkaline phosphatase activity based on ATP hydrolysis and CRISPR/Cas12a.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {8},
pages = {2337-2344},
pmid = {41711853},
issn = {1618-2650},
support = {2023JJ30430//Natural Science Foundation of Hunan Province/ ; 23A0075//Education Department of Hunan Province/ ; 82173572//National Natural Science Foundation of China/ ; 82574159//National Natural Science Foundation of China/ ; },
mesh = {*Alkaline Phosphatase/metabolism/analysis ; *Adenosine Triphosphate/metabolism/chemistry ; *CRISPR-Cas Systems ; Hydrolysis ; Animals ; Limit of Detection ; Milk/enzymology ; Aptamers, Nucleotide/chemistry ; *CRISPR-Associated Proteins/metabolism ; Biosensing Techniques/methods ; Cattle ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; },
abstract = {Alkaline phosphatase (ALP) activity serves as a crucial biomarker for livestock disease diagnosis, nutritional management, and the evaluation of milk pasteurization efficacy, holding significant implications for food safety and food science. In this study, we developed a novel method for detecting ALP activity in food samples, based on ATP hydrolysis coupled with a CRISPR/Cas12a system. The assay employs a DNA molecular recognition lock probe, comprising an ATP-specific aptamer and an activator strand designed to trigger the CRISPR/Cas12a trans-cleavage activity. In the absence of ALP, ATP acts as a trans-hydrolysis substrate to open the lock structure, releasing the activator strand. This subsequently activates the Cas12a protein, leading to the generation of a fluorescent signal. Conversely, when the target ALP is present, it catalyzes the hydrolysis of ATP via a dephosphorylation reaction. This prevents the opening of the "Aptamer-Activator" molecular lock, thereby inhibiting Cas12a activation and resulting in a corresponding decrease in fluorescence intensity. Under optimized conditions, the assay demonstrated a detection limit of 2.52 mU/mL for ALP activity, with a linear range of 0-18.75 mU/mL. The total detection time was 70 min. The method was successfully applied to detect ALP activity in samples from various livestock (chickens, pigs, sheep, cattle) and in milk, achieving recovery rates between 92 and 99%. In conclusion, we have developed a sensitive, cost-effective, and rapid method for ALP detection. This work provides a promising strategy for the development of point-of-care testing (POCT) devices in food safety monitoring.},
}
@article {pmid41712102,
year = {2026},
author = {Lin, J and Hazaisi, H and Guan, Y and Bai, M},
title = {Multiplex gene editing drives revolution in crop breeding: overlaid editing of multiple genes and customization of complex traits.},
journal = {Advanced biotechnology},
volume = {4},
number = {1},
pages = {5},
pmid = {41712102},
issn = {2948-2801},
support = {2023ZD040360104//Chinese Academy of Agricultural Sciences from Institute of Crop Science/ ; },
abstract = {Modern agriculture currently demands higher standards for the simultaneous improvement of crop yield, quality and stress resistance. However, traditional crop breeding methods can no longer meet the needs of modern agricultural development. Improving a single trait is no longer sufficient to meet the multifaceted demands of modern agricultural production and consumer expectations. Multiple traits breeding has increasingly become a key objective in current crop breeding. Over the past decade, CRISPR/Cas9-based multiplex genome editing (MGE) has enabled efficient pyramiding and precise regulation of multiple traits via targeted editing of multiple gene loci, revolutionizing crop breeding. In this review, we briefly describe the core CRISPR/Cas-based MGE strategies and technical workflows, and thoroughly discuss the practical outcomes of MGE applications in various fields, such as enhancing crop stress resistance, increasing yield and improving quality. This review aims to provide a summary and theoretical reference for crop breeding, as well as open up new ideas for achieving different breeding goals.},
}
@article {pmid41712626,
year = {2026},
author = {Sünderhauf, D and Ringger, JR and Payne, LJ and Pinilla-Redondo, R and Gaze, WH and Brown, SP and van Houte, S},
title = {CRISPR-Cas is beneficial in plasmid competition, but limited by competitor toxin-antitoxin activity when horizontally transferred.},
journal = {PLoS biology},
volume = {24},
number = {2},
pages = {e3003658},
pmid = {41712626},
issn = {1545-7885},
mesh = {*Plasmids/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Transfer, Horizontal ; Escherichia coli/genetics ; *Toxin-Antitoxin Systems/genetics ; },
abstract = {Bacteria can encode dozens of different immune systems that protect them from infection by mobile genetic elements (MGEs). MGEs themselves may also carry immune systems, such as CRISPR-Cas, to target competitor MGEs. It is unclear when this is favored by natural selection, and whether toxin-antitoxin (TA) systems-common competitive mechanisms carried by plasmids-can alter their efficacy. Here, we develop and test novel theory to analyze the outcome of competition between plasmids when one carries a CRISPR-Cas system that targets the other plasmid. Our mathematical model and experiments using Escherichia coli and competing IncP plasmids reveal that plasmid-borne CRISPR-Cas is beneficial to the plasmid carrying it when the plasmid has not recently transferred to a new host. However, CRISPR-Cas is selected against when the plasmid carrying it transfers horizontally, if a resident competitor plasmid encodes a TA system that elicits post-segregational killing. Consistent with a TA barrier to plasmid-borne CRISPR-Cas, a bioinformatic analysis reveals that naturally occurring CRISPR-Cas-bearing plasmids avoid targeting other plasmids with TA systems across bacterial genera. Our work shows how the benefit of plasmid-borne CRISPR-Cas is severely reduced against TA-encoding competitor plasmids, but only when plasmid-borne CRISPR-Cas is horizontally transferred. These findings have key implications for the distribution of prokaryotic defenses and our understanding of their role in competition between MGEs, and the utility of CRISPR-Cas as a tool to remove plasmids from pathogenic bacteria.},
}
@article {pmid41713039,
year = {2026},
author = {Zhang, H and Cui, C and Wang, X and Liu, S and Wang, X and Wang, Y and Ge, S and Cai, Y and Bao, J and Wang, Z},
title = {Development of a one-pot RT-RAA/CRISPR-Cas13a assay for rapid genotyping of Nipah virus in pigs.},
journal = {Diagnostic microbiology and infectious disease},
volume = {115},
number = {2},
pages = {117316},
doi = {10.1016/j.diagmicrobio.2026.117316},
pmid = {41713039},
issn = {1879-0070},
mesh = {Animals ; Swine ; *Nipah Virus/genetics/isolation &amp; purification/classification ; Sensitivity and Specificity ; *Swine Diseases/virology/diagnosis ; Genotype ; *CRISPR-Cas Systems ; *Genotyping Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Henipavirus Infections/veterinary/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; Polymorphism, Single Nucleotide ; },
abstract = {INTRODUCTION: Nipah virus (NiV) is a highly pathogenic zoonotic virus transmitted from bats to humans through pigs as a key intermediate host. Given the existence of two distinct NiV genotypes, which differ in clinical manifestations and transmission patterns in both humans and pigs, rapid and sensitive method for detection and genotyping is crucial for effective disease control. Isothermal amplification combined with CRISPR/Cas-based assay provides a promising approach to meet this need.<br><br>METHODS: Conserved regions were identified by aligning the N gene sequences from 67 NiV strains. Specific primers and probes were designed for reverse transcription recombinase-aided amplification (RT-RAA) to detect NiV. Subsequently, single nucleotide polymorphisms within the conserved region were analyzed, and corresponding crRNAs were designed to establish a one-pot RT-RAA/CRISPR-Cas13a assay for NiV genotyping. The assays were evaluated using simulated pig serums spiked with NiV pseudovirus.<br><br>RESULTS: The RT-RAA assay exhibited a detection sensitivity of 10[-2] Infection Unit/mL (IU/mL) for NiV pseudovirus, outperforming conventional qRT-PCR in simulated pig serum samples. No cross-reactivity was observed with viral RNA or DNA of PCV2, PEDV, PRRSV, PRV and SVA, confirming high specificity. The entire one-pot RT-RAA/CRISPR-Cas13a assay could be completed within 1 hour and clearly discriminated between the two NiV genotypes without requiring sophisticated instruments. Evaluation with simulated samples showed a sensitivity of 100% (95% CI, 92.87-100%) and a specificity of 94% (95% CI, 83.78-98.36%), with a detection limit of 10[-1] IU/mL for NiV pseudovirus.<br><br>CONCLUSION: The one-pot RT-RAA/CRISPR-Cas13a assay provides a rapid and sensitive platform for NiV genotyping.},
}
@article {pmid41713565,
year = {2026},
author = {Shashikala, T and Yogi, D and Akshay, K and Nagesh, SN and Manamohan, M and Venkataravanappa, V and Jha, GK and Ashok, K and Asokan, R},
title = {First report of CRISPR/Cas13a-based rapid detection of groundnut bud necrosis virus without amplification.},
journal = {Methods (San Diego, Calif.)},
volume = {249},
number = {},
pages = {9-22},
doi = {10.1016/j.ymeth.2026.02.010},
pmid = {41713565},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Plant Diseases/virology ; RNA, Viral/genetics/isolation &amp; purification ; *Tospovirus/genetics/isolation &amp; purification ; Vigna/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Globally, the groundnut bud necrosis virus (GBNV) (Bunyaviridae), pose a serious threat to solanaceous and leguminous crops causing serious crop loss. This requires a rapid and sensitive diagnostics for initially identifying at the earliest stage and further to initiate disease management. This study presents the first report of a CRISPR/Cas13a-based diagnostic assay for GBNV detection without amplification. GBNV was maintained in cowpea cv. C152 through mechanical inoculation, further viral RNA was isolated to clone the two target genes viz. nucleocapsid (NP) and movement protein (MP) genes. These genes were ligated to pTZ57R/T vector and sequenced. Similarly, the LshCas13a gene was cloned from pUC19 into pET28a, expressed in E. coli BL21, and purified using Ni-NTA affinity chromatography. Guide RNAs targeting conserved regions of NP and MP genes were synthesized by in vitro transcription and mixed with Cas13a protein to form ribonucleoprotein (RNP) complex. Target RNA, obtained either by in vitro-transcription or crude extract of infected tomato was used to detect GBNV, using a fluorescence-based reporter assay. This method found to be highly sensitive that could detect GBNV at as low as 0.01 ng. From the field perspective, GBNV could be detected from the crude extract of the GBNV infected tomato leaves using an alkaline PEG buffer. Thus CRISPR/Cas13a-based assay provides a rapid, amplification-free, and field-deployable diagnostic platform for GBNV. This lays the groundwork for a field adoptable CRISPR diagnostics for other plant RNA viruses also.},
}
@article {pmid41713574,
year = {2026},
author = {Hu, T and Hou, Z and Zhang, Y and Jing, P and Dai, X and Wang, H},
title = {Development of a one-pot integrated rapid detection method for white spot syndrome virus based on RAA-CRISPR/Cas12a technology.},
journal = {Journal of invertebrate pathology},
volume = {216},
number = {},
pages = {108569},
doi = {10.1016/j.jip.2026.108569},
pmid = {41713574},
issn = {1096-0805},
mesh = {*White spot syndrome virus 1/isolation &amp; purification ; Animals ; *Penaeidae/virology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Aquaculture ; Sensitivity and Specificity ; },
abstract = {Pathogenic microorganisms, particularly white spot syndrome virus (WSSV), pose a major threat to global shrimp aquaculture, causing mass mortalities and substantial economic losses. To address the urgent need for rapid, accurate, and field-deployable detection methods, this study developed an innovative one-pot RAA-CRISPR/Cas12a assay. The platform integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technology using sucrose-mediated density gradient phase separation in a closed-tube format: sucrose acts as a density modifier to form distinct layers, spatially isolating RAA amplification reagents from CRISPR/Cas12a detection components to avoid cross-interference and enable sequential reactions without manual intervention. Under isothermal conditions at 37℃ for 60 min, the optimized assay achieves a limit of detection as low as 1 copy/μL, validated by both fluorescence and lateral flow dipstick (LFD) readouts. High specificity was confirmed by the absence of cross-reactivity with four other prevalent shrimp pathogens: infectious hypodermal and hematopoietic necrosis virus (IHHNV), Decapod iridescent virus 1 (DIV1), Enterocytozoon hepatopenaei (EHP), and Vibrio parahaemolyticus associated with acute hepatopancreatic necrosis disease (VpAHPND). Clinical validation with 30 field samples showed concordant results with the chinese national detection standard (GB/T 28630.2-2012). This novel nucleic acid detection platform combines highly sensitive, excellent specificity, and user-friendly visual interpretation, making it highly suitable for point-of-care testing and large-scale disease surveillance in shrimp aquaculture.},
}
@article {pmid41715004,
year = {2026},
author = {Metz, P and Alves-Vasconcelos, S and Wallbank, R and Riepsaame, J and Brown, S and Hassan, AB},
title = {Variation in guide RNA library representation results in gene effect score bias in genome-wide CRISPR screens.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {},
pmid = {41715004},
issn = {1471-2164},
support = {OxPOS//F. Hoffmann-La Roche/ ; GEO//John Fell Fund, University of Oxford/ ; MPNST//Grenfell Shaw Charity/ ; Oxford Clinical Fellowships/CRUK_/Cancer Research UK/United Kingdom ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; *Gene Library ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Human ; },
abstract = {Genome wide CRISPR-based perturbation screens are powerful discovery tools enabling the identification of novel gene dependencies through either gain or loss of function. While genome wide guide RNA (gRNA) libraries have advantages when using enAsCas12a, such as multiplex single gRNAs per gene, they may be subject to similar confounding factors that can affect the interpretation of large genome-wide datasets. Here, we examine the impact of these variables in over twenty enAsCas12a multiple gRNA based perturbation screens performed using Humagne C, Humagne D and Inzolia libraries in human cells, as well as external datasets containing Cas9-based CRISPR library screens, including from DepMap. We demonstrate that the choice of CRISPR library is often the most significant factor that influences genetic perturbation results, outweighing other variables such as either target cell lines or culture media conditions. A potential contributor to this effect is gRNA representation within a given CRISPR library, where lower gRNA representation can lead to variable and more pronounced gene effect scores using either log fold change or Chronos analysis. These effects may be mitigated by using either multiple gRNA constructs per gene, by optimisation of CRISPR library production processes or by targeting with multiple independent gRNA libraries. Importantly, we also propose strategies for addressing gRNA representation bias during CRISPR screen hit prioritisation. CRISPR library gRNA representation dependent bias remains a major challenge in the interpretation of gene essentiality in perturbation screens.},
}
@article {pmid41715150,
year = {2026},
author = {Zhao, Y and Li, X and Du, Y},
title = {AI-driven CRISPR screening: optimizing gene editing through automation and intelligent decision support.},
journal = {Journal of translational medicine},
volume = {24},
number = {1},
pages = {},
pmid = {41715150},
issn = {1479-5876},
support = {No. Z04J2024E107-B-12//Bethune Charitable Foundation/ ; },
mesh = {Humans ; *Artificial Intelligence ; *Gene Editing ; *Automation ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Decision Support Techniques ; *CRISPR-Cas Systems/genetics ; Deep Learning ; *Genetic Testing/methods ; },
abstract = {BACKGROUND: CRISPR-based genetic screening has become a central methodology in functional genomics, enabling systematic interrogation of gene function, genetic interactions and context-dependent vulnerabilities at scale. However, the rapid expansion of screening modalities-including multi-condition designs, combinatorial perturbations, in vivo applications and single-cell readouts-has exposed fundamental limitations of heuristic-driven experimental design and post hoc statistical analysis.<br><br>MAIN BODY: This Review synthesizes how artificial intelligence is reshaping CRISPR screening by introducing predictive, adaptive and system-level intelligence across the experimental lifecycle. We organize recent advances into two tightly coupled modules. First, machine learning and deep learning (ML/DL) methods optimize experimental design by learning context-dependent perturbation behavior, anticipating confounding effects and enabling iterative, information-efficient screening strategies. Second, large language model-agent (LLM-agent) systems complement these advances by externalizing scientific reasoning, integrating biological knowledge at scale and coordinating analysis and decision-making in human-in-the-loop workflows.<br><br>CONCLUSIONS: Together, ML/DL and LLM-agent approaches reframe CRISPR screening from a static analytical pipeline into an intelligent experimental system, with important implications for robustness, scalability and biological discovery.},
}
@article {pmid41715936,
year = {2026},
author = {Shimizu, Y and Kataoka, K},
title = {DIPA-CRISPR Mediated Knockout of Vermilion Generates a Visible Eye Color Marker for The Band-Legged Ground Cricket Dianemobius nigrofasciatus.},
journal = {Archives of insect biochemistry and physiology},
volume = {121},
number = {2},
pages = {e70135},
doi = {10.1002/arch.70135},
pmid = {41715936},
issn = {1520-6327},
support = {JPJ009237//Bio-oriented Technology Research Advancement Institution, BRAIN/ ; 21K05614//JSPS KAKENHI Grant-in-Aid for Scientific Research (C)/ ; 21J23478/22KJ2609//Grant-in-Aid for JSPS Fellows/ ; },
mesh = {Animals ; Female ; *Gryllidae/genetics/growth &amp; development/physiology ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Eye Color/genetics ; Photoperiod ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {The molecular basis of photoperiodism, by which insects use photoperiodic cues to anticipate seasonal changes and regulate key life-history events such as development, diapause, and reproduction, remains poorly understood. Studies on the molecular mechanisms of photoperiodism in hemimetabolous insects are limited compared with those in holometabolous insects, largely due to the lack of appropriate model organisms. The band-legged ground cricket Dianemobius nigrofasciatus represents a valuable model system because it exhibits clear photoperiodic responses in the maternal induction of embryonic diapause, the wing morph, and the rate of nymphal development. With the recent availability of the D. nigrofasciatus genome sequence, the establishment of effective genome-editing methods and reliable marker genes is expected to promote functional genomic analyses. In this study, we aimed to establish a direct parental (DIPA)-CRISPR genome-editing approach and evaluate the utility of vermilion (Dn-v), a gene involved in ommochrome synthesis, as a visible eye color marker for mutant screening. Cas9 ribonucleoprotein complexes were injected into females 3-5 days after adult emergence, during the vitellogenic stage, successfully yielding Dn-v knockout mutants. These mutants had white compound eyes throughout development, with pigmentation reaching a vermilion color about 20 days after adult emergence. We further examined the photoperiodic response associated with maternal diapause induction in knockout mutants. Similar to the wild-type, knockout mutants exhibited low and high diapause incidence under long-day and short-day conditions, respectively. Our results demonstrate that DIPA-CRISPR is an effective genome-editing method in D. nigrofasciatus and that Dn-v serves as a practical and reliable marker gene. The establishment of these genomic tools provides a foundation for future functional analyses aimed at elucidating the molecular basis of photoperiodism in hemimetabolous insects.},
}
@article {pmid41717108,
year = {2026},
author = {Balasubramani, S and Li, Z and Gayathri, E and Gurusamy, D and Singh, A},
title = {Editorial: Advancing plant defense: genome editing, RNAi, and synthetic biology for sustainable pest control.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1785705},
pmid = {41717108},
issn = {1664-462X},
}
@article {pmid41717201,
year = {2026},
author = {Mittal, A and Manna, S and Nelson, V and Ladha, N},
title = {In Silico Design of gRNA for CRISPR System for Detection of Multidrug Resistant Tuberculosis Using Indian Mycobacterium tuberculosis Genomes: A Computational Study.},
journal = {Cureus},
volume = {18},
number = {1},
pages = {e101851},
pmid = {41717201},
issn = {2168-8184},
abstract = {Background Multidrug-resistant tuberculosis (MDR-TB) continues to pose a major challenge to TB elimination in India, where drug resistance and delayed diagnosis contribute significantly to ongoing transmission. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based diagnostics have emerged as versatile tools, compared to GeneXpert, capable of detecting resistance-associated mutations with rapid turnaround and high accuracy. This study aimed to design and in silico validate Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein (CRISPR-Cas)-based guide RNAs (gRNAs) targeting major drug-resistance mutations in Indian Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods Whole-genome mutation profiles were analyzed using TBProfiler, and gRNAs were designed using CHOPCHOP. Off-target evaluation was performed using Cas-OFFinder and Basic Local Alignment Search Tool (BLAST). High-confidence mutations in gyrA, rpoB, katG, rpsL, embB, and ethA were selected based on prevalence in Indian isolates and WHO-defined resistance markers. Results Numerous drug resistance-associated mutations were identified in the drug-resistant tuberculosis genome isolates. The study identified six key genetic mutations identified in MTB isolates that are associated with phenotypic drug resistance, including gyrA (Asp94Gly), rpoB (Ser450Leu), and katG (Ser315Thr). For each of the six genes, the chromosome position, locus ID, mutation type, and affected amino acids were identified, and tailored guide RNAs were designed in silico. Top-ranked gRNAs demonstrated optimal GC content, high predicted cleavage efficiency, and zero off-target activity. Each resistance locus yielded multiple candidate gRNAs suitable for CRISPR-based assays. Conclusions This computational in silico analysis provides a robust panel of mutation-targeted gRNAs tailored to Indian MDR-TB genomic profiles. These findings lay a strong foundation for developing rapid, affordable CRISPR diagnostics for point-of-care detection of drug resistance. Future laboratory validation and clinical testing are essential for translation into diagnostic practice.},
}
@article {pmid41717698,
year = {2026},
author = {Tadokoro, T and Li, H and Gan, P and Xu, Z and Tan, W and Alzhanov, D and Sánchez-Ortiz, E and McAnally, JR and Guo, L and Xu, L and Ruan, P and Liu, N and Olson, EN},
title = {Ablation of PKCα Phosphorylation by CRISPR-Cas9 Base Editing Rescues Heart Failure.},
journal = {Circulation research},
volume = {138},
number = {7},
pages = {e326738},
pmid = {41717698},
issn = {1524-4571},
support = {P01 HL160488/HL/NHLBI NIH HHS/United States ; P50 HD087351/HD/NICHD NIH HHS/United States ; R01 HL157281/HL/NHLBI NIH HHS/United States ; },
mesh = {Animals ; *Protein Kinase C-alpha/genetics/metabolism ; *Heart Failure/genetics/therapy/enzymology/physiopathology/pathology/metabolism ; Phosphorylation ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; Humans ; Myocytes, Cardiac/metabolism/enzymology ; Mice, Inbred C57BL ; Male ; Disease Models, Animal ; Induced Pluripotent Stem Cells ; },
abstract = {BACKGROUND: The prevalence of heart failure is increasing globally, with poor prognosis, highlighting the need for novel therapeutic strategies. PKCα (protein kinase C alpha), encoded by PRKCA, plays a central role in heart failure pathogenesis. Phosphorylation of PKCα at threonine 497 (T497) triggers a series of intramolecular phosphorylation events, leading to its activation. Ablation of T497 phosphorylation leads to reduced stability and activity of PKCα.<br><br>METHODS: We generated mice harboring a phospho-resistant PKC&#x3b1; (T497A) mutation in the germline using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated homology-directed repair. To assess the clinical feasibility of postnatal genome editing, we used CRISPR-Cas9 adenine base editing delivered by adeno-associated virus 9 to introduce the T497A substitution into the Prkca gene (Prkca[T497A]) in wild-type mice. Mice underwent transverse aortic constriction to model heart failure. Cardiac function, hypertrophy, fibrosis, and transcriptional changes were evaluated by echocardiography, wheat germ agglutinin staining, Masson's trichrome staining, and RNA-sequencing. The editing efficiency of Prkca[T497A] was assessed using Sanger sequencing and deep amplicon sequencing. To further explore its clinical potential, we introduced the PRKCA[T497A] mutation into human induced pluripotent stem cells by nucleofection-mediated adenine base editing. Ca[2+] homeostasis was analyzed in Fura-2-loaded human induced pluripotent stem cell-derived cardiomyocytes with PRKCA[T497A] under chronic AngII (angiotensin II) stimulation.<br><br>RESULTS: The T497A mutation in PKC&#x3b1; prevented its subsequent phosphorylation and led to PKC&#x3b1; protein degradation. Four weeks after transverse aortic constriction surgery, wild-type mice showed impaired cardiac function, cardiac remodeling, and increased lung weight. In contrast, PKC&#x3b1; phospho-resistant mice showed protection against heart failure-related aberrant changes in cardiac hypertrophy, fibrosis, and cardiac gene expression. Mice administered with adeno-associated virus 9 base editors to prevent T497 phosphorylation exhibited similar cardioprotective effects. In vitro, PKC&#x3b1;-edited induced pluripotent stem cell-derived cardiomyocytes were protected from AngII-induced impairments in contractility and Ca[2+] transients.<br><br>CONCLUSIONS: The editing of PRKCA[T497A] through adenine base editing represents a potential therapeutic approach for human cardiac diseases.},
}
@article {pmid41718462,
year = {2026},
author = {Ren, Y and Wu, R and Yang, X and Yao, B and Wang, Y},
title = {Membrane-Confined CRISPR-Cas12a Activation via Split Activator for Wash-Free Detection of Tumor-Specific Extracellular Vesicles.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2356-2365},
doi = {10.1021/acssensors.5c04166},
pmid = {41718462},
issn = {2379-3694},
mesh = {*Extracellular Vesicles/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; Aptamers, Nucleotide/chemistry/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism ; *Neoplasms/diagnosis ; },
abstract = {Current CRISPR-Cas12a biosensing systems for protein analysis rely on the release of a blocked activator from an aptamer through a competitive mechanism. However, the design of the complementary length between the activator and the aptamer involves a critical trade-off: excessively long sequences impede aptamer-target binding, whereas overly short sequences fail to activate Cas12a effectively. To overcome this limitation, we split the full-length Cas12a activator into two short single-stranded DNA fragments (S1 and S2; 10 nt each). S1 was specifically sequestered within the hairpin structure of an allosteric aptamer. The binding of the target protein to the aptamer triggered a conformational change, exposing S1 and thereby enabling its assembly with S2 to activate Cas12a. The strategy successfully preserved the binding affinity of the aptamer without compromising the efficiency of Cas12a activation. Furthermore, we anchored the Cas12a substrate to the membrane surface of extracellular vesicles (EVs) via hydrophobic interactions, localizing the cleavage reaction to the EVs interface and thereby significantly enhancing local catalytic efficiency. Finally, the strategy provided highly sensitive detection of tumor-specific EVs, not only accurately distinguishing cancer patients from healthy individuals (77.5% accuracy for early-stage and 100% for advanced-stage) but also holding potential for monitoring dynamic postoperative changes. Overall, our study provided a wash-free, one-pot detection platform for EVs analysis and established a new paradigm for CRISPR-based clinical diagnostics.},
}
@article {pmid41718886,
year = {2026},
author = {Cao, X and He, Y and Guo, H and Cao, X and Zhang, D and Lai, Y and Yang, W and Ma, Z and Yu, X and Wang, L and Li, D and Zeng, Z},
title = {Optimization of RNP-CRISPR for high-efficiency gene editing in mouse intestinal organoids.},
journal = {Molecular and cellular biochemistry},
volume = {481},
number = {4},
pages = {1817-1827},
pmid = {41718886},
issn = {1573-4919},
support = {2019YFA0110802//National Key Research and Development Program of China/ ; No.32025023//National Natural Science Foundation of China/ ; No.32200732//National Natural Science Foundation of China/ ; 20MC1920400//Shanghai Municipal Commission for Science and Technology/ ; 22YF1437700//Shanghai Municipal Commission for Science and Technology/ ; 2019-01-07-00-05-E00054//Innovation Program of Shanghai Municipal Education Commission/ ; },
mesh = {Animals ; *Organoids/metabolism/cytology ; *Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; *Intestines/cytology ; Wnt Signaling Pathway ; Mice, Knockout ; beta Catenin/metabolism ; },
abstract = {Mouse intestinal organoids are ideal models for investigating intestinal development and diseases. The full potential of these models hinges on the ability to precisely engineer their genome, yet traditional methods for CRISPR-based editing in 3D cultures often surfer from low efficiency, high cytotoxicity, and inconsistent post-editing differentiation, which limits their applications. Here, we developed an electroporation approach mediated by ribonucleoprotein (RNP)-CRISPR that achieves over 90% gene editing efficiency in mouse intestinal organoids. Using this optimized method, we generated APC-knockout organoids that exhibit Wnt pathway hyperactivation, demonstrated by R-spondin1-independent growth, increased nuclear β-catenin, and enhanced proliferation. Our method addresses a critical technical gap in murine organoid research, offering a scalable platform for intestinal disease modeling.},
}
@article {pmid41719905,
year = {2026},
author = {Yu, F and Yue, D and Wang, F and Fu, B and Qin, G and Wei, S and Zang, W and Zhang, Q and Cui, L and Wang, T},
title = {Structure-initiated CHA variant coordinating SDA for cascade amplification in CRISPR/Cas12a-based miRNA analysis.},
journal = {Talanta},
volume = {304},
number = {},
pages = {129558},
doi = {10.1016/j.talanta.2026.129558},
pmid = {41719905},
issn = {1873-3573},
mesh = {*MicroRNAs/genetics/analysis/blood ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are well-established biomarkers for tumor diagnosis and monitoring. Herein, we report a novel biosensing platform by engineering a structure-initiated variant of catalytic hairpin assembly (VCHA) that coordinates with strand displacement amplification (SDA) to drive cascade amplification for CRISPR/Cas12a-based detection. This system employs three hairpin probes which, upon recognizing the target miRNA, self-assemble into a key 5'-end dangling three-way conjugate (5'-DTC) structure. This structure serves as the exclusive trigger, simultaneously propagating the VCHA cycle and priming the SDA process through the coordinated action of polymerase and nicking enzyme. Consequently, VCHA and SDA operate synergistically within a unified circuit, generating abundant single-stranded activator DNA (acDNA) products. These acDNA molecules then activate the trans-cleavage activity of CRISPR/Cas12a, yielding a significantly amplified fluorescence readout. The VCHA-SDA/Cas12a platform demonstrated excellent performance for miRNA-155 detection, achieving a broad dynamic range from 1 pmol/L to 10 nmol/L with an ultra-low detection limit of 0.166 pmol/L. Furthermore, the platform successfully quantified miRNA levels in clinical plasma specimens and various cell lines, confirming its considerable potential as a robust tool for molecular diagnostics and clinical translation.},
}
@article {pmid41719925,
year = {2026},
author = {Park, YJ and Song, DY and Jeon, HB and Kim, DM},
title = {Nucleic acid detection via protein readout through Cas-controlled gating of cell-free protein synthesis.},
journal = {Biosensors &amp; bioelectronics},
volume = {301},
number = {},
pages = {118514},
doi = {10.1016/j.bios.2026.118514},
pmid = {41719925},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Cell-Free System ; *Protein Biosynthesis/genetics ; *Escherichia coli O157/genetics ; Bacillus anthracis/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Ribosomal, 16S/genetics/analysis ; *Nucleic Acids/analysis/genetics ; Bacterial Proteins/genetics ; },
abstract = {We present a modular platform that converts CRISPR target recognition into programmable protein outputs for nucleic acid detection. This system couples Cas-mediated collateral cleavage with cell-free protein synthesis. In the presence of a target, Cas-mediated collateral cleavage releases an extension-competent trigger DNA that gates reporter expression. Although collateral cleavage is inherently indiscriminate, we achieve deterministic fragment generation by employing chemically programmed precursors bearing backbone modifications-exemplified here by phosphorothioate linkages-that constrain cleavage to predefined sites. Using Bacillus anthracis and E. coli O157:H7 as a model, the developed CRIVER assay enables concurrent readouts of 16S rRNA together with the species-specific capB or ecf1 loci by integrating Cas13a-mediated RNA recognition and Cas12a-mediated DNA recognition into a dual-channel workflow. Taken together, proposed method establishes addressable signaling at the protein layer, supports protein-based outputs in a modular workflow, and provides a general route to sensitive, dual-channel nucleic acid detection.},
}
@article {pmid41720308,
year = {2026},
author = {Shi, Q and Huang, W and Hu, D and Zhang, P and Chen, X and Hu, H and Wang, Y and Zhou, J and Weng, R and Quan, J and Zhao, D and Du, X and Yu, Y and Jiang, Y},
title = {The nationwide genomic characteristics and phylogenetic evolution of ST23-K1 hypervirulent Klebsiella pneumoniae in relation to virulence and antimicrobial resistance acquisition.},
journal = {The Journal of infection},
volume = {92},
number = {3},
pages = {106709},
doi = {10.1016/j.jinf.2026.106709},
pmid = {41720308},
issn = {1532-2742},
mesh = {*Klebsiella pneumoniae/genetics/pathogenicity/drug effects/classification ; *Klebsiella Infections/microbiology/epidemiology ; Virulence/genetics ; Phylogeny ; Animals ; China/epidemiology ; Mice ; Humans ; Plasmids/genetics ; Whole Genome Sequencing ; Genome, Bacterial ; Evolution, Molecular ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Bacterial Proteins/genetics ; Virulence Factors/genetics ; beta-Lactamases/genetics ; Drug Resistance, Multiple, Bacterial/genetics ; },
abstract = {OBJECTIVES: Hypervirulent Klebsiella pneumoniae (hvKp) ST23-K1 poses a global health threat due to its high virulence and increasing antimicrobial resistance. This study aimed to characterise the genomic feature and phylogenetic evolution of ST23-K1 in China.<br><br>METHODS: K1 isolates from a nationwide epidemiological surveillance project underwent whole-genome sequencing. Virulence was assessed using hypermucoviscosity phenotyping and a murine infection model. For ST23-K1 carrying acquired antimicrobial resistance genes (ARGs), the CRISPR/Cas system, protospacers, anti-CRISPR (Acr) genes, and plasmidome were characterised. Time-resolved phylogenetic analysis was performed using integrated locally generated and publicly available data.<br><br>RESULTS: Among 400 K1 isolates, ST23 was the most prevalent sequence type, and its effective population size increased following CG23-I divergence. The CG23-I sub-lineage was widely distributed nationwide with limited evidence of clonal transmission. Isolates with an incomplete cps locus exhibited significantly reduced virulence compared with those carrying an intact locus. The prevalence of extended-spectrum &#x3b2;-lactamase-positive ST23-K1 isolates increased over time, whereas carbapenemase-producing isolates remained stable. Among acquired ARGs-positive ST23-K1 isolates, a conserved protospacer corresponding to a prevalent spacer was identified. This protospacer, together with AcrIE genes, was frequently co-located on IncFII-type plasmids.<br><br>CONCLUSION: ST23-K1 remains a hypervirulent lineage undergoing ongoing evolutionary expansion. The presence of acquired ARGs in ST23-K1 may be associated with AcrIE-harbouring IncFII plasmids, and functional validation is required to clarify the underlying mechanisms. Continuous genomic surveillance is essential to monitor the evolution and antimicrobial resistance trends of ST23-K1.},
}
@article {pmid41720778,
year = {2026},
author = {Lee, YJ and Zhang, D and Stolze, SC and Saridis, G and Ebert, MK and Nakagami, H and Doehlemann, G},
title = {Ustilago maydis disrupts carbohydrate signaling networks to induce hypertrophy in host cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41720778},
issn = {2041-1723},
support = {DO1421/3-3//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; //European Union&#x2019;s Horizon 2020 research and innovation program/ ; },
mesh = {*Zea mays/microbiology/metabolism/genetics ; Signal Transduction ; Plant Proteins/metabolism/genetics ; *Plant Diseases/microbiology ; Starch/metabolism/biosynthesis ; Gene Expression Regulation, Plant ; Host-Pathogen Interactions ; Fungal Proteins/metabolism/genetics ; Plant Leaves/microbiology/metabolism ; Mesophyll Cells/metabolism/microbiology ; *Carbohydrate Metabolism ; Endoreduplication ; Virulence Factors/metabolism/genetics ; Cell Cycle/genetics ; CRISPR-Cas Systems ; Basidiomycota ; },
abstract = {Ustilago maydis infection in maize causes hypertrophic leaf tumors; however, the underlying mechanisms driving this excessive cell growth are unknown. In this study, we identify Hap1 (hypertrophy-associated protein 1) as an effector and virulence factor that regulates mesophyll cell hypertrophy. Using CRISPR-Cas9 mutagenesis, we demonstrate that Hap1 contributes to endoreduplication and starch accumulation in infected tissues. Transcriptomics revealed Hap1-dependent upregulation of starch biosynthesis and cell cycle genes, as well as suppression of plant defense. This links Hap1 to metabolic and cell cycle reprogramming, and immune suppression. To identify the target of Hap1 that drives metabolic reprogramming, we investigated its interaction with ZmSnRK1α in maize. We found that Hap1 interferes with the phosphorylation of SnRK1 substrates and that two Hap1-interacting effectors, Hip1 and Hip2, enhance its protein stability. We conclude that Hap1 contributes to the reprogramming of maize metabolism and cell cycle, as well as mesophyll cell hypertrophy, by modulating the SnRK1 signaling pathway to regulate starch biosynthesis and host defense responses.},
}
@article {pmid41720881,
year = {2026},
author = {Ong, JK and Bhunia, S and Hilbert, B and Kirschner, V and Duglosz, S and Zimmermann, F and Freichel, M and Cornean, A},
title = {ABE9 fused to SpRY Cas9 nickase enables precise generation of bystander free mouse models.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41720881},
issn = {2045-2322},
mesh = {Animals ; *Gene Editing/methods ; Mice ; *CRISPR-Cas Systems ; *Deoxyribonuclease I/genetics/metabolism ; Humans ; Disease Models, Animal ; *CRISPR-Associated Protein 9/genetics/metabolism ; Point Mutation ; },
abstract = {Point mutations cause many genetic disorders, but modelling them in organisms is technically challenging. Creating mouse models that mimic these mutations is crucial for establishing a causal relationship between mutations and disease phenotype, thereby supporting the development of therapeutic strategies. Adenine base editors (ABEs) can correct single-nucleotide variants (SNVs) in disease modelling without double-stranded breaks (DSBs) or donor DNA, achieving higher product purity than traditional Cas9 methods. Earlier ABE techniques faced issues like limited targetability, bystander editing, and off-target effects. By combining two editor advancements, we introduced and tested ABE9-SpRY, an improved ABE variant fused with a PAM-flexible SpRY-Cas9 nickase. Our results show that ABE9-SpRY effectively generates three out of four targeted A-to-G mutations in mouse embryos, achieving desired editing efficiencies of up to 96% in individual adult founder mice. Furthermore, we observe fewer off-target events at predicted DNA sites in mouse embryos and in an orthogonal R-loop assay compared with ABE8e-SpRY. ABE9-SpRY also enhances product purity in mouse embryos under pooled sgRNA injections and, as a proof-of-concept, at a single endogenous locus in human induced pluripotent stem cells (hiPSCs), relative to ABE8e-SpRY. Our findings support ABE9-SpRY's precision at the loci tested and PAM-flexible versatility. Although performance remains sequence-dependent, these data support ABE9-SpRY as a PAM-flexible tool for generating precise point-mutation models where bystander editing is a concern.},
}
@article {pmid41720886,
year = {2026},
author = {Nagalakshmi, U and Rodriguez, JE and Nguyen, T and Weissman, RF and Thornton, BW and Terrace, CI and Savage, DF and Dinesh-Kumar, SP},
title = {High-efficiency, transgene-free plant genome editing by viral delivery of an engineered TnpB.},
journal = {Nature plants},
volume = {12},
number = {3},
pages = {503-511},
pmid = {41720886},
issn = {2055-0278},
support = {IOS-2303522//National Science Foundation (NSF)/ ; DGE-2146752//National Science Foundation (NSF)/ ; },
mesh = {*Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Genome, Plant/genetics ; *Plant Viruses/genetics ; CRISPR-Cas Systems ; Transgenes ; *Nicotiana/genetics ; Genetic Vectors ; *Arabidopsis/genetics ; },
abstract = {Genome editing has revolutionized plant biology research[1], yet the efficient delivery of editing reagents remains a challenge. Current methods are labour intensive, involving lengthy tissue culture and complex transformation and regeneration steps. Viral delivery can mitigate these issues[2] but CRISPR-Cas nucleases exceed viral cargo limits, restricting guide RNA (gRNA) delivery into Cas9-expressing transgenic plants[2-11]. This requires generating an initial Cas9 transgenic line. Furthermore, gRNAs delivered by plant viral vectors can induce somatic edits, although only a few produce heritable edits[3-7,9-12]. Some engineered plant negative-strand rhabdoviruses can deliver both Cas9 and gRNA, but they face other challenges, including the need for tissue regeneration or pruning infected plants, and some rhabdoviruses can be delivered only through vector transmission[13-16]. Recently, smaller editors such as TnpBs were discovered, but they are significantly less active than Cas9[17-19]. Here we optimized a tobacco rattle virus-based system to deliver recently engineered, highly active ISDra2 TnpB variants. The eTnpBc variant enables effective somatic editing in systemic leaves and achieves up to 90% editing efficiency at target loci. In addition, up to 89% of offspring exhibit a mutant phenotype, with editing efficiencies reaching 100%. The design principles outlined here could promote wider use of eTnpBc for efficient, transformation- and transgene-free plant genome editing.},
}
@article {pmid41721525,
year = {2026},
author = {Chen, N and Gao, M and Bai, Y and Wang, M and Liu, M and Xiong, W and Mo, B},
title = {5'UTR Editing of the ribosomal protein UL3Z gene unveils its critical roles in pre-rRNA processing and global mRNA translation dynamics.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
doi = {10.1093/plphys/kiag073},
pmid = {41721525},
issn = {1532-2548},
support = {32470592//National Natural Science Foundation of China/ ; 32270595//National Natural Science Foundation of China/ ; 2023B001//SZU 2035 Excellence Research Program/ ; },
mesh = {*5' Untranslated Regions/genetics ; *Ribosomal Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; *RNA Precursors/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Protein Biosynthesis/genetics ; RNA, Messenger/metabolism/genetics ; Gene Expression Regulation, Plant ; *RNA Processing, Post-Transcriptional/genetics ; Gene Editing ; CRISPR-Cas Systems ; Plants, Genetically Modified ; },
abstract = {The EMBRYO DEFECTIVE 2207 (EMB2207) gene, encoding ribosomal protein UL3Z, is critical for embryonic development in Arabidopsis, with loss of function resulting in embryo lethality. Despite its importance, the role of UL3Z in the complicated protein translation machinery in plants remains poorly understood due to the lack of viable hypomorphic alleles. In this study, we utilized CRISPR/Cas9 to edit the 5' untranslated region (5'UTR) of UL3Z, generating 5 ul3z mutants with varying degrees of reduced expression levels of UL3Z proteins. The mutant with the lowest expression exhibited the most severe developmental defects. In contrast, null mutants of its paralog UL3Y displayed no observable phenotypes. Interestingly, expression of UL3Y driven by the UL3Z/EMB2207 promoter successfully rescued the phenotypes of ul3z, demonstrating that these 2 paralogous ribosomal proteins actually possess functionally interchangeable roles. GUS staining results showed that UL3Z was constitutively expressed in all examined tissues, while UL3Y was only appreciably expressed in specific tissues. Molecular analysis further revealed the accumulation of ribosomal RNA (rRNA) maturation intermediates and increased polysome levels in ul3z mutants, indicating compromised pre-rRNA processing and disturbed global mRNA translation. Interestingly, 3' ends of many rRNA precursors in ul3z had higher frequency of non-encoded tails compared with Col-0. This study demonstrates that CRISPR/Cas9-mediated 5'UTR editing is an effective tool for generating viable hypomorphic alleles of lethal genes and uncovers the critical roles of UL3Z/EMB2207 in pre-rRNA processing and the maintenance of appropriate mRNA translation on ribosomes, underscoring its importance in plant development.},
}
@article {pmid41721881,
year = {2026},
author = {Jin, Z and Liu, M and Liu, M and Qi, X and Zhao, L and Yu, X and Guo, Y and Wen, Y},
title = {CRISPR/Cas9-mediated dual editing of VviGAI and VviFLC generates a novel early-flowering grapevine germplasm.},
journal = {Planta},
volume = {263},
number = {3},
pages = {},
pmid = {41721881},
issn = {1432-2048},
support = {No.32272670//Natural Science Foundation for Young Scientists of Shanxi Province/ ; No.31972986//the National Natural Science Foundation of China/ ; },
mesh = {*Vitis/genetics/growth &amp; development/physiology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Flowers/genetics/growth &amp; development/physiology ; *Plant Proteins/genetics/metabolism ; Phenotype ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Mutation ; },
abstract = {CRISPR/Cas9-mediated dual knockout of VviGAI1 and VviFLC in grapevine promotes early flowering and induces distinctive morphological changes, offering novel genetic resources for breeding. CRISPR/Cas9-mediated genome editing offers a transformative approach for grapevine improvement. In this study, we achieved simultaneous knockout of two central flowering regulators VviGAI1, a DELLA protein ortholog, and VviFLC, a floral repressor in Vitis vinifera 'Cabernet Sauvignon' using a dual-sgRNA vector system. Remarkably, all 15 independent edited lines exhibited biallelic mutations in both genes, primarily consisting of frameshifts that led to premature termination. The dual-mutant plants displayed a range of distinctive phenotypic alterations, including dwarfism, shortened internodes, modified leaf morphology, and disrupted tendril development. Notably, one line (EL-43) showed precocious flowering under greenhouse conditions, underscoring the synergistic role of VviGAI1 and VviFLC in repressing floral transition. Comparative analysis with previously reported gai mutants revealed both conserved and novel traits, suggesting that structural variation within the DELLA domain contributes to phenotypic diversity. Collectively, our findings establish that dual editing of VviGAI1 and VviFLC not only accelerates flowering but also introduces unique vegetative and reproductive characteristics, providing a valuable genetic resource for future grapevine domestication and precision breeding efforts.},
}
@article {pmid41722369,
year = {2026},
author = {Boumpoureka, I and Gorgogietas, V and Petkovski, E and Massart, F and Mellick, GD and Krüger, R},
title = {Generation of two isogenic control iPSC lines (LCSBi001-A-2 and LCSBi001-A-3) from a Parkinson's disease patient line (LCSBi001-A) carrying the pathogenic VPS35 p.D620N mutation.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103944},
doi = {10.1016/j.scr.2026.103944},
pmid = {41722369},
issn = {1876-7753},
mesh = {Humans ; *Parkinson Disease/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Vesicular Transport Proteins/genetics ; *Mutation/genetics ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; },
abstract = {The pathogenic mutation VPS35 p.D620N has been identified to cause autosomal dominant, late-onset Parkinson's disease (PD) in multiple individuals and families worldwide. Here, we describe the generation of two new isogenic control lines (LCSBi001-A-2 and LCSBi001-A-3) from an already established patient-derived line (LCSBi001-A) carrying the heterozygous VPS35 p.D620N mutation. The control lines were generated with CRISPR/Cas9 technology, and the correction of the mutation was verified with Sanger sequencing. Both lines express pluripotency markers, are capable of in vitro differentiation into the three germ layers, and have a normal karyotype. The mutant and control iPSC lines are available for research purposes.},
}
@article {pmid41723140,
year = {2026},
author = {Shen, K and Seow, WY and Keng, CT and Lim, MGL and Lim, DS and Guo, K and Meliani, A and Irfan Bin Hajis, M and Wang, B and Prabhakar, S and Chen, KH and Chew, WL},
title = {Spatial perturb-seq: single-cell functional genomics within intact tissue architecture.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41723140},
issn = {2041-1723},
support = {PREPARE-OC-VT-2024-008//Ministry of Health -Singapore (MOH)/ ; SC18/21-1089UI//Agency for Science, Technology and Research (A*STAR)/ ; OFIRG24jul-0096//MOH | National Medical Research Council (NMRC)/ ; OFYIRG23jul-0050//MOH | National Medical Research Council (NMRC)/ ; NRF-CRP25-2020-0001//National Research Foundation Singapore (National Research Foundation-Prime Minister's office, Republic of Singapore)/ ; },
mesh = {Animals ; *Single-Cell Analysis/methods ; Mice ; *Genomics/methods ; Brain/metabolism/cytology ; CRISPR-Cas Systems ; Neurodegenerative Diseases/genetics ; Mice, Knockout ; Neurons/metabolism ; Cell Communication/genetics ; Gene Knockout Techniques ; },
abstract = {We develop Spatial Perturb-Seq, an in vivo CRISPR technology that interrogates multiple genes within single cells of intact tissues, compatible with both sequencing-based and probe-based spatial technologies. We apply Spatial Perturb-Seq to knock out risk genes for neurodegenerative diseases in the mouse brain, uncovering cell autonomous and cell-cell microenvironmental effects within the spatially intact tissue. Spatial Perturb-Seq functionally screens multiple genes in situ and in vivo, bypasses cell processing steps that skew cell type representation, identifies intracellular and intercellular effects of knockouts, and identifies candidate genes underlying dysregulated neuronal intercellular communication pathways.},
}
@article {pmid41723978,
year = {2026},
author = {Zhen, Z and Yu, L and Zhu, Y and Lu, Z and Huang, Z},
title = {Molecular mechanism of CRISPR-SpyCas9 inhibition by AcrIIA26.},
journal = {Biochemical and biophysical research communications},
volume = {809},
number = {},
pages = {153491},
doi = {10.1016/j.bbrc.2026.153491},
pmid = {41723978},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Gene Editing ; Cryoelectron Microscopy ; *Streptococcus/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Protein Binding ; Protein Domains ; Models, Molecular ; },
abstract = {In the ongoing arms race between bacteria and phages, CRISPR-Cas systems and anti-CRISPR proteins (Acrs) have evolved to counteract one another. Recently, AcrIIA26 from Streptococcus sp. was identified as a modulator of SpyCas9 activity, although its molecular mechanism remains unclear. Here, we present the cryo-EM structure of the SpyCas9-sgRNA-AcrIIA26 complex, revealing a two-domain architecture. The 5A domain of AcrIIA26 binds the PI and WED domains of Cas9, while the 4A domain contacts the REC2 domain. This dual interaction sterically blocks target DNA binding and prevents the conformational changes required for cleavage. Notably, AcrIIA26's sgRNA-independent binding to Cas9 allows the modulation of gene editing across an extended temporal window. Our study elucidates the molecular mechanism of AcrIIA26 and provides novel strategies for precise SpyCas9 regulation.},
}
@article {pmid41724454,
year = {2026},
author = {Ghasemian, A and Al-Marzoqi, AH and Ali, ZA and Nouruzi, F and Abdollahi, A and Montaseri, Z and Memariani, M and Zarenezhad, E},
title = {Engineered Bacteria as living detectors of tumor DNA: A new diagnostic frontier.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {586},
number = {},
pages = {120914},
doi = {10.1016/j.cca.2026.120914},
pmid = {41724454},
issn = {1873-3492},
mesh = {Humans ; Animals ; *Biosensing Techniques/methods ; *Neoplasms/diagnosis/genetics ; *DNA, Neoplasm/genetics/analysis ; *Bacteria/genetics ; *Genetic Engineering ; },
abstract = {The identification of tumor-generated DNA must be accurate, minimally invasive, and precise, as it forms a fundamental aspect of effective cancer diagnosis, prognosis, and customized treatment plans. Recent advances in synthetic biology have pioneered the creation of genetically engineered bacteria as innovative biosensors capable of detecting tumor-derived DNA directly in situ. This review explores key developments in designing these microbial sentinels to pinpoint oncogenic DNA alterations, particularly emphasizing KRAS mutations that drive many cancers. By leveraging natural competence and horizontal gene transfer, in combination with CRISPR-Cas tools for selective targeting and integration of mutant DNA sequences, engineered bacteria can distinguish between tumor and wild-type DNA and produce observable reporter outputs. We further elaborate on various molecular engineering strategies using unique genetic circuits, homologous recombination, multiplexed CRISPR systems and safety circuits to improve specificity, sensitivity and biosafety. An additional perspective in the discussion incorporates diverse bacterial species and various cancer types, with a specific emphasis on colorectal and gastrointestinal cancers, while also considering possible applications to other solid tumors. Detection modalities encompass in vitro assays, organoid models, in vivo mouse models, and non-invasive stool sampling, offering an impressive range of platforms for validating biosensors. The positive aspects of these approaches, such as real-time detection, affordability, programmability, and reduced invasiveness, need to be balanced with their negative aspects concerning biosafety, colonization efficiency, and detection sensitivity limitations. Looking forward, this review delves into the translational potential of engineered bacterial biosensors for clinical cancer diagnostics, their integration with therapeutic delivery systems, and future directions that involve multiplexed detection and the incorporation of digital health. Indubitably, engineered bacterial tumor DNA biosensors represent a key fusion of microbiology, synthetic biology, and oncology, aimed at revolutionizing the diagnosis and management of cancers.},
}
@article {pmid41724570,
year = {2026},
author = {Vizoso, M},
title = {DiLiCre2.0 mouse model: An advanced genome-editing tool to induce mutagenesis in vivo with high spatio-temporal resolution.},
journal = {Methods in cell biology},
volume = {203},
number = {},
pages = {41-58},
doi = {10.1016/bs.mcb.2025.12.003},
pmid = {41724570},
issn = {0091-679X},
mesh = {Animals ; Mice ; *Mutagenesis/genetics ; Mice, Transgenic ; *Gene Editing/methods ; Disease Models, Animal ; Humans ; *Carcinogenesis/genetics ; Neoplasms/genetics/pathology ; CRISPR-Cas Systems/genetics ; },
abstract = {For decades, transgenic mouse models have been developed and utilized to study tumorigenesis in vivo, offering the ability to manipulate oncogene and tumor suppression gene expression systemically or within entire organs and tissue compartments. However, the induction of tumorigenesis in many of these experimental models contrasts sharply with the development of most human cancers, where mutations affecting gene expression occur in a spatially restricted manner and lesions generally originates from the clonal expansion of one single mutated cell. This discrepancy raises critical questions regarding the relevance of the existing transgenic mouse models in accurately replicating the mechanisms of tumor initiation observed in humans. To overcome this limitation and study tumor initiation in vivo, I developed an innovative mouse model to induce early tumorigenesis through light-targeted mutagenesis of single cells, achieving unprecedented spatio-temporal resolution. This model provides a more accurate representation of tumor initiation processes, thus enhancing our understanding of cancer mechanisms at its inception.},
}
@article {pmid41724722,
year = {2026},
author = {Limia, CG and Steffey, V and Cheng, HC and Machado, D and Hart, T and McHargue, MC and Brizzee, C and Crawford, J},
title = {Sequential, chromosome-specific glutamine synthetase double knockout with Cas-CLOVER establishes enhanced CHO platforms for cell line development.},
journal = {Biotechnology progress},
volume = {42},
number = {2},
pages = {e70113},
pmid = {41724722},
issn = {1520-6033},
mesh = {CHO Cells ; Animals ; *Glutamate-Ammonia Ligase/genetics/metabolism ; Cricetulus ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; },
abstract = {Cas-CLOVER is an emerging high-fidelity genome editing system that enables precise and efficient cell engineering. In this study, we applied Cas-CLOVER to establish a robust, gene-edited platform in suspension-adapted CHO-K1 cells supporting cell line development (CLD) for biopharmaceutical production. An attractive strategy for high-yield clone selection is the use of glutamine synthetase (GS) knockout CHO cells. The primary GS gene resides on chromosome 5 (GS5), while a recently identified GS pseudogene is located on chromosome 1 (GS1). To compare editing efficiency, we evaluated Cas-CLOVER and Cas9 at both GS loci using the Neon™ Transfection System. Cas-CLOVER achieved 84% editing at GS5 and 74% at GS1, markedly higher than Cas9. Leveraging Cas-CLOVER's dual-guide RNA design, we generated a GS5 single knockout (GS5-SKO) and subsequently a double knockout (GS-DKO) line at both the GS5 and GS1 loci, both with none detected off-target mutations analyzed in 40 predictably off-target sites. For functional validation, these cell lines were engineered with the proprietary Harbor-IN transposase system to stably express trastuzumab. Using an optimized protocol, the resulting GS-DKO platform, termed CleanCut GS CHO, enabled stringent selection and yielded high-producing clones with cell-specific productivity exceeding 100 pg/cell/day and antibody titers greater than 5 g/L in 24 deep well-plate fed-batch cultures after 14 days. The antibody titer stability analysis showed consistency over 60 generations. Collectively, these findings establish Cas-CLOVER as a versatile genome editing tool for developing high-yield CHO host platforms in CLD.},
}
@article {pmid41724841,
year = {2026},
author = {Ibrahim, R and Aranjani, JM},
title = {Bacterial defense mechanisms against bacteriophages: an evolutionary arms race.},
journal = {Archives of microbiology},
volume = {208},
number = {5},
pages = {},
pmid = {41724841},
issn = {1432-072X},
mesh = {*Bacteriophages/physiology/genetics ; *Bacteria/virology/genetics/immunology ; Biological Evolution ; CRISPR-Cas Systems ; Biofilms/growth &amp; development ; },
abstract = {Bacteria and bacteriophages are in a co-evolutionary arms race, developing intricate bacterial defense mechanisms that enable phage resistance and counterstrategies. Bacteria evolve diverse defense mechanisms to inhibit each stage of the phage infection cycle.Surface-based defenses prevent phage adsorption and infection, including receptor modifications, capsule production, and biofilm formation. Intracellular systems such as restriction-modification (R-M) and abortive infection (Abi) mechanisms degrade phage DNA or sacrifice infected cells to protect the population. Adaptive immunity, particularly through CRISPR-Cas systems, enables bacteria to recognize and neutralize recurring phage attacks. Phages counter these defenses through anti-CRISPR proteins, receptor mimicry, and depolymerization, which degrade capsules and biofilm matrices. These dynamic interactions shape microbial ecosystems, offering insights for the development of novel antimicrobial strategies. Emerging approaches, including engineered phages and combination therapies, hold promise for addressing bacterial resistance. Understanding these bacterial-phage dynamics is critical for advancing phage therapy as a powerful tool against multidrug-resistant bacterial infections. This review aims to systematically examine and integrate current knowledge on bacterial antiphage defense systems and the evolutionary adaptations employed by bacteriophages to overcome these barriers.},
}
@article {pmid41724842,
year = {2026},
author = {Jana, UK and Bawankar, P and Gupta, P and Kango, N},
title = {CRISPR/Cas systems in fungal biotechnology: advancing high-value metabolite synthesis for industrial and food security applications.},
journal = {Archives of microbiology},
volume = {208},
number = {5},
pages = {},
pmid = {41724842},
issn = {1432-072X},
}
@article {pmid41725298,
year = {2026},
author = {Chew, YP and Ferenczi, A and Dannay, M and Ponce-Lilly, C and Kovac, A and Tóth, D and Tóth, SZ and Molnar, A},
title = {Enhancing CRISPR/Cas-Mediated Gene Knockout With Short Non-Homologous Oligonucleotides.},
journal = {Plant biotechnology journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/pbi.70548},
pmid = {41725298},
issn = {1467-7652},
support = {BB/W003538/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //Darwin Trust of Edinburgh/ ; //UK Research and Innovation/ ; },
abstract = {Chlamydomonas reinhardtii is a model green microalga that has great industrial potential as a sustainable bio-factory for recombinant protein and high-value chemical production. Efficient genome editing tools are required to redesign this organism for synthetic biology applications. CRISPR-Cas editing technologies have already been adapted for gene knockout, transgene knock-in, and precise gene editing in C. reinhardtii. However, the efficacy of CRISPR/Cas-mediated gene knockout (KO) is low, which hampers pathway engineering and functional genomic studies. Here we report that co-delivery of CRISPR-Cas gene editing reagents with short double-stranded non-homologous oligodeoxynucleotides (dsNHO) increases gene knockout efficacy up to 100-fold in C. reinhardtii. This phenomenon, referred to as non-homologous oligonucleotide enhancement (NOE), is heavily affected by the length, structure, and chemical modifications of dsNHO, and is largely mediated by the DNA double-stranded break sensor KU70/80 (KU) heterodimer in a Cas nuclease-, locus-, and strain-independent manner. Our data suggest that dsNHOs disrupt the cell's double-stranded break (DSB) sensing pathways, consequently shifting the balance of DNA repair from canonical non-homologous end joining (c-NHEJ) towards the more error-prone, microhomology-mediated end joining (MMEJ), which could be harnessed as a strategy for improving gene KO efficiency in Chlamydomonas and beyond.},
}
@article {pmid41727253,
year = {2026},
author = {Meenakshi, and Komal, and Sharma, A and Prabhu, S and Awasthi, S},
title = {Modern plant stress adaptation: integrating defense, nanotechnology and genetics.},
journal = {3 Biotech},
volume = {16},
number = {3},
pages = {105},
pmid = {41727253},
issn = {2190-572X},
abstract = {This review critically analyses plant adaptive responses to biotic and abiotic stress, with a focus on recent advancements in molecular defense pathways, emerging nanotechnology approaches and CRISPR/Cas-based genome editing strategies. We critically reviewed structural, physiological, biochemical and genetic adaptations. Key regulatory processes include phytohormonal regulation, antioxidants, reactive oxygen species (ROS) signaling and stress-response gene networks are explored along with advances in nanotechnology-based strategies and CRISPR/Cas genome editing. A comparative evaluation of conventional breeding, molecular breeding, and genome-editing approaches highlights the advantages of CRISPR/Cas systems, particularly their precision, efficiency and ability to generate targeted phenotypic changes. In parallel, nanomaterials have shown promise in improved nutrient delivery, protecting cellular structures and enhancing genome-editing efficiency under stress conditions. By integrating nanotechnology and genome-editing approaches with traditional agricultural practices, it may be possible to enhance plant resilience, sustain crop productivity and reduce reliance on chemical inputs. Overall, this review provides a cohesive perspective on how these technologies can be combined to support future crop improvement efforts to tackle climate-induced agricultural challenges.},
}
@article {pmid41727334,
year = {2025},
author = {Ramezani, R and Behbahani, M and Mohabatkar, H and Sarraf Mamouri, K and Hejazi, F},
title = {Comparison of CRISPR Sequences in Archaea and Bacteria with Eukaryotic microRNAs.},
journal = {Avicenna journal of medical biotechnology},
volume = {17},
number = {4},
pages = {258-276},
pmid = {41727334},
issn = {2008-2835},
abstract = {BACKGROUND: This study explores repetitive Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) sequences from the archaea Acidianus sp. and Acidianus ambivalens (A. ambivalens), as well as from the bacterium Yersinia ruckeri (Y. ruckeri). These sequences are compared with human microRNA (miRNA) sequences to investigate potential genetic similarities and disease associations.<br><br>METHODS: CRISPR sequences were retrieved from the CRISPR/Cas[++] database, and human miRNA sequences were obtained from miRBase. Sequence alignments were performed using BLASTn with an E-value threshold of 1e-5 to identify significant similarities. Genes associated with matched human miRNAs were identified through the HGNC and GeneCards databases. Further analyses included comparison with disease-associated miRNAs reported in human and mouse datasets.<br><br>RESULTS: In Y. ruckeri, alignments revealed similarities to miRNAs linked with genes such as FOXO1, PTEN, PAX7, and DOCK3, which are associated with lung cancer and muscular dystrophies. In A. ambivalens, aligned miRNAs corresponded to loci including CHM13 and GRCh38, potentially linked to periembolic adenocarcinoma and mild pre-eclampsia. For Acidianus sp., matches were observed with miRNAs associated with genes like Irak2, NOS2, STAT1, and Numb, which have been implicated in Psoriatic arthritis, Alzheimer's disease, Hepatocellular carcinoma, and Coronary artery disease.<br><br>CONCLUSION: CRISPR sequences from these prokaryotes show notable similarities with human miRNAs, suggesting possible indirect links to genes involved in major diseases. These preliminary findings emphasize the need for further investigation into shared sequence motifs and their functional roles in host-pathogen interactions or evolutionary biology.},
}
@article {pmid41728195,
year = {2025},
author = {Zhu, F and Liu, Z and Zheng, Z},
title = {An AI-driven framework for enhancing regulatory precision and efficiency in CRISPR-Cas gene-edited crops: challenges, opportunities, and global harmonization.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1693105},
pmid = {41728195},
issn = {1664-462X},
abstract = {INTRODUCTION: The rapid advancement and adoption of CRISPR-Cas technologies in crop improvement has significantly outpaced existing regulatory frameworks, leading to inconsistencies in the global oversight of gene-edited organisms. As governments and international bodies struggle to reconcile scientific innovation with policy governance, a pressing need has emerged for methodologies that can translate biological edits into regulatory-compliant representations across jurisdictions. Traditional approaches often compartmentalize genomic and legal domains, lacking the formalism to bridge biological intent and compliance precision. These methods are typically static, unable to adapt to jurisdictional policy drift or incorporate real-time exemption logic, thereby undermining both regulatory interpretability and technical fidelity.<br><br>METHODS: To address this gap, I propose a unified computational framework built around the novel GeneRegAlignNet model and the Constraint-Aware Policy Induction (CAPI) strategy. This framework embeds regulatory semantics directly into the learning architecture, enabling the alignment of gene-editing features with heterogeneous policy descriptors in a shared latent space. GeneRegAlignNet employs symbolic gating, contrastive manifold learning, and exemption-aware vectorization to predict alignment likelihoods between edits and legal categories with high precision. CAPI extends this model with a risk-calibrated policy optimization pipeline that accounts for policy evolution, regulatory variance, and jurisdictional priorities.<br><br>RESULTS AND DISCUSSION: Empirical validation demonstrates improved performance in regulatory alignment accuracy and resilience to policy drift across a diverse set of gene-editing scenarios. By tightly integrating formal representations of molecular edits with dynamic, multi-jurisdictional policy inference, our framework offers a scalable and interpretable path forward in enhancing regulatory precision and global harmonization in the oversight of CRISPR-Cas-edited crops.},
}
@article {pmid41728946,
year = {2026},
author = {Dang, QT and Chang, CW and Chen, PY and Truong, VA and Huang, PY and Nguyen, MTT and Hu, YC},
title = {CRISPR-associated transposon for programmable viral vector engineering and prime editing.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728946},
issn = {1362-4962},
support = {NSTC 114-2223-E-007-013//National Science and Technology Council/ ; 113-2321-B-007-004//National Science and Technology Council/ ; 113-2223-E-007-010//National Science and Technology Council/ ; 113-2622-E-007-012//National Science and Technology Council/ ; 112-2622-E-007-030//National Science and Technology Council/ ; 112-2223-E-007-002//National Science and Technology Council/ ; //Veterans General Hospitals/ ; VGHUST115-G6-1-1//University System of Taiwan/ ; //National Science and Technology Council/ ; //Veterans General Hospitals/ ; VGHUST115-G6-1-1//University System of Taiwan Joint Research Program/ ; },
mesh = {*Gene Editing/methods ; Humans ; HEK293 Cells ; *Baculoviridae/genetics ; *Genetic Vectors/genetics ; *DNA Transposable Elements/genetics ; Animals ; *CRISPR-Cas Systems ; Escherichia coli/genetics ; Transgenes ; *Genetic Engineering/methods ; },
abstract = {Baculovirus, an insect virus commonly used for recombinant protein expression in insect cells and gene delivery in mammalian systems, is often generated through bacmid-based engineering. To enable flexible and programmable bacmid engineering, we developed SHOT 2.0, an optimized CRISPR-associated transposon platform that mediates RNA-guided and customized bacmid editing in Escherichia coli. The edited bacmid can be transfected into insect cells to produce recombinant baculoviruses. SHOT 2.0 supported site-specific integration of large DNA cargos (at least 14 kb) into defined loci such as v-cath and ODVe56, with integration at ODVe56 markedly improving transgene stability during serial virus passaging. The system is fully compatible with the Bac-to-Bac® workflow, enabling dual-gene insertion into the bacmid and derived baculovirus. Leveraging this platform, we constructed an all-in-one baculovirus encoding the PE5max prime editor. This vector-mediated prime editing achieves efficiencies up to 85.6% in HEK293T cells and achieves robust prime editing in hard-to-transfect cell types, including iPSCs and liver cancer cells, with efficiencies up to 37.1%. These results demonstrate that SHOT 2.0 substantially expands the baculovirus engineering toolbox, providing a flexible platform for genome editing and future gene delivery.},
}
@article {pmid41728950,
year = {2026},
author = {King, HE and O'Connell, S and Kavanagh, D and Mason, S and McCool, C and Fernandez-Chamorro, J and Chaffer, CL and Clark, SJ and Vieira, HGS and Sterne-Weiler, T and Weatheritt, RJ},
title = {Isoform-specific single-cell perturb-seq reveals distinct functions of alternative promoters in drug response.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728950},
issn = {1362-4962},
support = {DP250103133//Australian Research Council Discovery/ ; FT210100355//Future Fellowship/ ; //Scrimshaw Foundation/ ; },
mesh = {*Promoter Regions, Genetic ; Humans ; *Single-Cell Analysis/methods ; Tamoxifen/pharmacology/therapeutic use ; *Estrogen Receptor alpha/genetics ; *Breast Neoplasms/genetics/drug therapy/pathology ; CRISPR-Cas Systems ; Female ; Protein Isoforms/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic/drug effects ; },
abstract = {CRISPR interference (CRISPRi) screens have emerged as powerful tools for dissecting gene function, yet their application to genes with multiple promoters, which comprise over 60% of human genes, remains poorly understood. Here, we demonstrate that CRISPR-dCas9-based screens exhibit widespread promoter specificity, with untargeted promoters often showing compensatory upregulation to maintain gene expression. Leveraging this selective targeting of individual promoters within the same gene, we developed Isoform-Specific single-cell Perturb-Seq to systematically analyse alternative promoter function. Our analysis revealed that alternative promoters in 51.6% of targeted genes drive distinct transcriptional programs. This suggests that promoter selection represents a fundamental mechanism for generating cellular diversity rather than mere transcriptional redundancy. In breast cancer models, this promoter-specific targeting revealed differential effects on drug sensitivity, where distinct estrogen receptor (ESR1) promoters showed opposing influences on tamoxifen response and patient survival. These findings demonstrate the necessity of promoter-level analysis in functional genomics and suggest new strategies for therapeutic intervention through promoter-specific targeting.},
}
@article {pmid41728952,
year = {2026},
author = {Feng, H and Li, Z and Zhang, H and Zheng, Y and Xu, B and Zhang, Y and Zou, L and Wu, L},
title = {Characterization of gRNA-dependent and gRNA-independent off-target binding sites of PspCas13b and RfxCas13d in mammalian cells.},
journal = {Nucleic acids research},
volume = {54},
number = {4},
pages = {},
pmid = {41728952},
issn = {1362-4962},
support = {2021YFA1100201//National Key R&amp;D Program of China/ ; 2022YFA1303301//National Key R&amp;D Program of China/ ; XDB0570000//Chinese Academy of Sciences/ ; 82400181//National Natural Science Foundation of China/ ; 82270160//National Natural Science Foundation of China/ ; 2024M751998//China Postdoctoral Science Foundation/ ; 2023YFC2706401//Ministry of Science and Technology/ ; },
mesh = {Humans ; HEK293 Cells ; Binding Sites ; CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; Regulatory Factor X Transcription Factors ; Gene Editing ; Transcriptome ; },
abstract = {CRISPR-Cas13 systems, harnessed for RNA-guided transcriptome editing, hold significant promise for clinical and in vivo therapeutic applications. However, understanding their in vivo target specificity and recognition rules remains a challenge. In this study, we employed the uSpyCLIP method, which enhances sensitivity and specificity for identifying RNA-binding protein (RBP) binding sites, to map the transcriptome-wide binding sites of catalytically inactive PspCas13b (dPspCas13b) and RfxCas13d (dRfxCas13d) in HEK293T cells, using a variety of single guide RNAs (gRNAs). Surprisingly, we identified both gRNA-dependent and gRNA-independent off-target binding sites for both dCas13 complexes. These gRNA-independent off-target sites exhibited distinct RNA structural and sequence signatures: dPspCas13b's gRNA-independent binding was associated with specific RNA structural features, while dRfxCas13d's was linked to unique sequence motifs. Analysis of gRNA-dependent off-target sites revealed the crucial role of the DR-distal and middle regions of the gRNA in determining binding specificity. Further analysis demonstrated that some off-target binding events led to changes in gene expression at the messenger RNA and/or protein level. Collectively, our findings provide important insights into the characteristics of gRNA-dependent and gRNA-independent off-target binding for PspCas13b and RfxCas13d, offering valuable guidance for optimizing Cas13 and gRNA design in future applications.},
}
@article {pmid41729380,
year = {2026},
author = {Liu, YY and Lv, YR and Jia, JT and Zhang, R and Yang, B and Xue, SY and Bayaer, H and Bagen, A and Chen, RB and Tunala, S and Wang, R and Ding, YL and Zhao, L and Liu, YH},
title = {Rapid and Simple Detection of Enterocytozoon Bieneusi Using Lateral Flow Assay Based on Recombinase Polymerase Amplification or Nested PCR Combined with CRISPR-Cas12a.},
journal = {Acta parasitologica},
volume = {71},
number = {2},
pages = {},
pmid = {41729380},
issn = {1896-1851},
support = {2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 2025MS03139 and 2024LHMS03049//National Natural Science Foundation of Inner Mongolia/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; 32260887//National Natural Science Foundation of China/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; BR251303//Research and Innovation Team Building Special B-class Team/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; YLXKZX-NND-012//Inner Mongolia Autonomous Region First Class Discipline Research Special Project/ ; },
mesh = {*Enterocytozoon/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Animals ; Feces/microbiology ; *Microsporidiosis/diagnosis/veterinary/microbiology ; *Polymerase Chain Reaction/methods ; Humans ; Recombinases/genetics ; DNA, Fungal/genetics ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Enterocytozoon bieneusi is an obligate intracellular microsporidian pathogen. It primarily causes diarrhea and weight loss in infected humans and animals, resulting in substantial economic losses to the livestock industry. Therefore, establishing a highly sensitive and specific detection method for E. bieneusi is critical for its prevention and control.<br><br>METHODS: crRNA and recombinase polymerase amplification (RPA) primers were designed based on partial sequences of the 18&#xa0;S ribosomal RNA gene and the internal transcribed spacer (ITS) of E. bieneusi. DNA extracted from fecal samples was amplified using RPA or nested polymerase chain reaction (PCR). PCR amplicons were treated with a Tris-saturated phenol-chloroform-isoamyl alcohol mixture to obtain purified target DNA, which was subsequently introduced into the CRISPR-Cas12a reaction system. Post-reaction detection was performed via qPCR instrumentation, fluorescence visualization, and lateral flow strip (LFS) assays. The operational parameters for E. bieneusi detection were subsequently optimized using RPA/CRISPR-Cas12a or nested PCR/CRISPR-Cas12a platforms. The aforementioned methodology was concurrently validated using 50 clinical specimens with known E. bieneusi infection status.<br><br>RESULTS: The limits of detection were 7.13 copies/&#xb5;L for RPA/CRISPR-Cas12a and 2.35&#x2009;&#xd7;&#x2009;10[-&#x2009;2] copies/&#xb5;L for nested PCR/CRISPR-Cas12a. When the concentration of unamplified DNA in the CRISPR-Cas12a reaction system reached&#x2009;&#x2265;&#x2009;1&#x2009;&#xd7;&#x2009;10[-&#x2009;4] &#xb5;g/&#xb5;L, the single-stranded DNA reporter was efficiently cleaved, resulting in a detectable fluorescence signal. The nested PCR/CRISPR-Cas12a technology was used to analyze 50 fecal samples with confirmed E. bieneusi-positive or -negative status. The results obtained from instrument-based detection, fluorescence observation, and lateral flow test strip detection were completely consistent.<br><br>CONCLUSIONS: We established the first integration of nested PCR with CRISPR-Cas12a for the detection of E. bieneusi. and were also the first to quantitatively explore the detection limit of Cas12a using non-amplified E. bieneusi DNA. This approach offers a rapid, specific, and highly sensitive diagnostic method. Furthermore, the wide selection of appropriate visualization methods facilitates adaptation to various laboratory conditions and sample template concentrations, enabling accurate result interpretation.},
}
@article {pmid41730290,
year = {2026},
author = {Chen, L and Murillo-de-Ozores, AR and Park, E and Ou, SM and Knepper, MA},
title = {GFP reporter system reveals cell-to-cell variability in aquaporin-2 expression.},
journal = {American journal of physiology. Cell physiology},
volume = {330},
number = {4},
pages = {C782-C793},
doi = {10.1152/ajpcell.00936.2025},
pmid = {41730290},
issn = {1522-1563},
support = {ZIA-HL001285//HHS | NIH | NHLBI | Division of Intramural Research (DIR)/ ; ZIA-HL006129//HHS | NIH | NHLBI | Division of Intramural Research (DIR)/ ; },
mesh = {*Aquaporin 2/genetics/metabolism ; *Green Fluorescent Proteins/genetics/metabolism ; Animals ; Genes, Reporter ; Cell Line ; Mice ; *Kidney Tubules, Collecting/metabolism/drug effects/cytology ; Deamino Arginine Vasopressin/pharmacology ; CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {Vasopressin regulates transcription of the aquaporin-2 gene (Aqp2) in collecting duct principal cells. To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. In the absence of dDAVP (1-desamino-8-D-arginine-vasopressin), a vasopressin analog, these cells exhibited low or undetectable GFP and Aqp2 expression in all cells. dDAVP stimulation (1 nM dDAVP for 48 h) markedly increased both GFP and Aqp2 expression together with reversal upon dDAVP removal. These observations demonstrate that GFP faithfully tracks Aqp2 expression. Interestingly, fewer than 50% of cells express GFP and Aqp2 after dDAVP or forskolin, indicating significant variability even though they were clonally derived. We flow-sorted the GFP[-] cells (Aqp2[-]) and GFP[+] cells (Aqp2[+]), regrew them, and restimulated them separately with dDAVP. Cells originating from GFP[-] cells gave rise to both GFP[-] cells and GFP[+] cells, and GFP[+] cells similarly regenerated both GFP[-] and GFP[+] populations in the same proportion. Flow cytometry analysis of the DNA content showed variability in cell cycle phases, with most GFP[+] cells in G0/G1, and most GFP[-] cells in G2/S. RNA-seq analysis of the GFP[-] and GFP[+] cells revealed increased abundance of cell cycle-related transcripts in the GFP[-] cells. We conclude that: 1) heterogeneity in Aqp2 expression is related to cell cycle state and 2) the newly generated reporter cell line will likely serve as a useful tool to study Aqp2 transcriptional regulation.NEW &amp; NOTEWORTHY To investigate regulatory mechanisms in Aqp2 gene transcription, we engineered an Aqp2 reporter cell line using CRISPR/Cas9 to insert a green fluorescent protein (GFP) cassette at the endogenous Aqp2 gene locus in mpkCCD cells. We demonstrate that the GFP reporter accurately and dynamically tracks the expression and regulation of endogenous Aqp2. We reveal that Aqp2 heterogeneity in mpkCCD cells is at least partly driven by differences in cell cycle phase.},
}
@article {pmid41730595,
year = {2026},
author = {Chang, X and Han, C and Ji, H and Zeng, Z and Yang, J and Liu, Q and Jia, C and Zhao, L and Zhou, C and Chen, S and Knoll, W and Li, J and Wang, Z and Zhang, L},
title = {SPARC: A programmable molecular diagnostic platform based on a signal-triggered, self-supplied crRNA and tiered PER-transcription-CRISPR cascade for early detection of hepatocellular carcinoma.},
journal = {Analytica chimica acta},
volume = {1394},
number = {},
pages = {345209},
doi = {10.1016/j.aca.2026.345209},
pmid = {41730595},
issn = {1873-4324},
mesh = {Humans ; *Liver Neoplasms/diagnosis/genetics ; *Carcinoma, Hepatocellular/diagnosis/genetics ; *MicroRNAs/genetics/analysis ; *Early Detection of Cancer/methods ; *CRISPR-Cas Systems ; Cell Line, Tumor ; Limit of Detection ; *Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Accurate quantification of microRNAs (miRNAs) is essential for early cancer detection, yet remains challenging due to their short length, low abundance, and high sequence similarity. Existing assays often struggle to achieve sufficient sensitivity, specificity, and robustness for reliable clinical deployment.<br><br>RESULTS: We introduce SPARC, a programmable molecular diagnostic platform that integrates a signal-triggered primer exchange reaction, self-supplied crRNA generation, and a tiered PER-transcription-CRISPR/Cas12a amplification cascade. Using miRNA-21 as a model, SPARC achieves an ultralow detection limit of 1.22&#xa0;fM and a broad quantitative range from 1&#xa0;fM to 100&#xa0;nM. The system exhibits high specificity, strong analytical stability, and modular adaptability to diverse targets, including miRNA-122. Notably, the dual-directional profiling of oncogenic and tumor-suppressive miRNAs enhances diagnostic resolution. When applied to HCC cell lines and clinical tissues, SPARC accurately distinguished malignant from normal samples and showed excellent agreement with qRT-PCR measurements and histopathological assessments.<br><br>SIGNIFICANCE: This streamlined and self-amplifying cascade system provides a scalable, robust, and clinically compatible platform for ultrasensitive miRNA detection. SPARC holds strong potential for early hepatocellular carcinoma screening, molecular subtyping, and broader precision oncology applications.},
}
@article {pmid41731282,
year = {2026},
author = {du Rand, A and Buttle, B and Sheppard, H},
title = {Developing CRISPR-Based Therapies for Epidermolysis Bullosa: A Comprehensive Review of Current Strategies.},
journal = {Drugs},
volume = {86},
number = {4},
pages = {465-483},
pmid = {41731282},
issn = {1179-1950},
mesh = {Humans ; *Gene Editing/methods ; *Epidermolysis Bullosa/therapy/genetics ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Currently, there is no permanent treatment for the group of severe monogenic fragile skin conditions epidermolysis bullosa (EB). The recent US Food and Drug Administration (FDA)-approved in vivo gene replacement therapy beremagene geperpavec (Vyjuvek[®]) provides a promising solution, but it requires ongoing application and is not applicable to all forms of EB. Targeted gene editing approaches directly addressing pathogenic mutations hold great promise for the development of durable personalized therapies. Here, we comprehensively describe the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) gene editing landscape for EB, critically review the advantages and limitations of emerging therapeutic strategies, and present some future perspectives. We find that the widespread application of Cas9 nuclease is currently hindered by off-target genotoxicity, which can be mitigated using Cas9 nickases. Further, new tools including prime editing have recently emerged and hold significant potential for EB gene therapy. Ongoing developments in gene editing technologies focused on improving safety and editing precision offer significant promise for the future clinical translation of potentially lifelong treatments for people with EB.},
}
@article {pmid41732093,
year = {2026},
author = {Neupane, S and Pfrender, ME and Wang, L and Xu, S},
title = {Detection of CRISPR-Cas-induced mutations in Daphnia.},
journal = {G3 (Bethesda, Md.)},
volume = {},
number = {},
pages = {},
doi = {10.1093/g3journal/jkag050},
pmid = {41732093},
issn = {2160-1836},
support = {R35 GM133730/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas9 has established itself as a robust tool for conducting loss of function gene research in emerging model species including the freshwater zooplankton Daphnia. However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study we evaluate two of the most widely used mutation screening techniques, the T7 Endonuclease I (T7EI) assay and Fragment Analysis (FA) for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in four targeted genes, DNMT3A, DNMT3B, PERIOD2, and DMRT1 in Daphnia magna. Here, we show that T7EI, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, FA facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.},
}
@article {pmid41732321,
year = {2026},
author = {He, Y and Tu, X and Xue, Y and Chen, Y and Ye, B and Li, X and Li, D and Zhong, Z and Zhong, Q},
title = {CRISPR screening redefines therapeutic target identification and drug discovery with precision and scalability.},
journal = {Journal of pharmaceutical analysis},
volume = {16},
number = {2},
pages = {101357},
pmid = {41732321},
issn = {2214-0883},
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screening technology is redefining the landscape of drug discovery and therapeutic target identification by providing a precise and scalable platform for functional genomics. The development of extensive single-guide RNA (sgRNA) libraries enables high-throughput screening (HTS) that systematically investigates gene-drug interactions across the genome. This powerful approach has found broad applications in identifying drug targets for various diseases, including cancer, infectious diseases, metabolic disorders, and neurodegenerative conditions, playing a crucial role in elucidating drug mechanisms and facilitating drug screening. Despite challenges like off-target effects, data complexity, and ethical or regulatory concerns, ongoing advancements in CRISPR technology and bioinformatics are steadily overcoming these limitations. Additionally, by integrating with organoid models, artificial intelligence (AI), and big data technologies, CRISPR screening expands the scale, intelligence, and automation of drug discovery. This integration boosts data analysis efficiency and offers robust support for uncovering new therapeutic targets and mechanisms. This review outlines the fundamental principles and applications of CRISPR screening technology, delves into specific case studies and technical challenges, and highlights its expanding role in drug discovery and target identification. It also examines the potential for clinical translation and addresses the associated ethical and regulatory considerations.},
}
@article {pmid41732389,
year = {2026},
author = {Fanarraga, ML and García Hevia, L},
title = {Silica nanoparticles as advanced platforms for nucleic acid delivery.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102921},
pmid = {41732389},
issn = {2590-0064},
abstract = {Nucleic acid therapeutics, including siRNA, mRNA, plasmid DNA, and CRISPR/Cas systems, have demonstrated remarkable potential but continue to face translational barriers related to systemic instability, immune activation, and inefficient intracellular delivery. Conventional lipid and polymeric carriers, although clinically validated, often lack the structural resilience and versatility required for large or complex cargos. Silica-based nanoparticles, particularly mesoporous silica nanoparticles, provide a distinctive combination of mechanical rigidity, tunable porosity, and abundant surface chemistry that enables robust encapsulation, protection, and controlled release of diverse nucleic acids. This review adopts a problem-driven perspective, analyzing how specific nanoarchitectural designs, surface functionalizations, and ligand-mediated targeting strategies address key limitations in nucleic acid delivery. Emphasis is placed on overcoming systemic barriers such as premature degradation, immune recognition, and restricted biodistribution, as well as intracellular challenges including endosomal escape and nuclear access. Hybrid and biomimetic silica platforms are highlighted for their capacity to integrate combinatorial and theranostic functionalities, expanding the therapeutic scope toward complex payloads and multifunctional formulations. By linking synthesis approaches with translational requirements, an integrated roadmap is proposed that positions silica nanocarriers as advanced platforms for next-generation gene therapy. The evidence underscores the potential of silica architectures to combine structural durability with versatile chemical adaptability, thereby enabling safe, efficient, and clinically relevant delivery of nucleic acids.},
}
@article {pmid41733889,
year = {2026},
author = {Son, SH and Woo, S and Choi, A and Ahn, S and Yoo, HC},
title = {Advances in Engineered Virus-Like Particles for Genome Editing and Therapy.},
journal = {BioDrugs : clinical immunotherapeutics, biopharmaceuticals and gene therapy},
volume = {40},
number = {2},
pages = {317-333},
pmid = {41733889},
issn = {1179-190X},
support = {RS202400403169//Korea Basic Science Institute/ ; 2021R1C1C2006283//National Research Foundation/ ; RS-2024-00412879//National Research Foundation/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; Animals ; Genetic Engineering/methods ; Genetic Vectors ; CRISPR-Cas Systems ; Gene Transfer Techniques ; },
abstract = {Engineered virus-like particles (eVLPs) have emerged as a promising class of delivery systems for genome editing agents. By combining the efficient cellular entry of viral vectors with the safety advantages of nonviral platforms, eVLPs enable transient delivery of ribonucleoproteins such as Cas9, base editors, and prime editors. Successive design strategies, from rational engineering to directed optimization, have progressively improved particle assembly, cargo stability, and editing efficiency. In parallel, pseudotyping approaches have expanded the versatility of eVLPs for cell-type-specific targeting. Recent preclinical studies highlight their potential across diverse applications, ranging from monogenic disease models to complex disorders, and support their advancement toward clinical translation. This review summarizes the structural and production principles of eVLPs, surveys key advances in their development, and discusses therapeutic opportunities and future challenges for their application in genome editing.},
}
@article {pmid41735098,
year = {2026},
author = {Naruse, K and Loosli, F and Ansai, S and Birney, E and Wittbrodt, J},
title = {Medaka: a novel model for analyzing genome-environment interactions.},
journal = {Trends in genetics : TIG},
volume = {42},
number = {4},
pages = {350-361},
doi = {10.1016/j.tig.2025.12.005},
pmid = {41735098},
issn = {0168-9525},
mesh = {Animals ; *Oryzias/genetics ; *Gene-Environment Interaction ; Genome-Wide Association Study ; *Genome ; Quantitative Trait Loci ; CRISPR-Cas Systems ; },
abstract = {Medaka is an established vertebrate model system for biological and biomedical research. It possesses unique features that make it particularly suitable for studying genome-environment interactions. Endemic to habitats spanning from 4 to 40°C and varying salinities, it combines broad ecological adaptability with experimental tractability. Its exceptional tolerance to inbreeding enabled the creation of the Medaka Inbred Kiyosu-Karlsruhe panel-80 near-isogenic, fully sequenced lines derived from a single wild population. More than 100 wild-derived, fully sequenced strains, collected throughout East Asia for more than 40 years, show relatively low intra-strain variation (inbreeding coefficient of >0.75) but high inter-strain variability (SNP rates >4%). Advanced quantification methods facilitate genome-wide association studies and quantitative trait locus mapping. The system's amenability to clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 editing and emerging epigenomic profiling enables causal validation and regulatory-mechanism discovery. Collectively, medaka offers an unparalleled vertebrate framework for integrating genetics, environment, and epigenetics-bridging evolutionary, biomedical, and population-level perspectives.},
}
@article {pmid41735520,
year = {2026},
author = {Panji, JM and Germano, RFV and America, M and Lou, L and Lecordier, L and Tebabi, P and Martin, M and Vanhollebeke, B},
title = {Scalable and multimodal brain angiogenesis and blood-brain barrier genetics by somatic mutagenesis.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41735520},
issn = {2399-3642},
support = {Ctrl-BBB 865176//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Animals ; *Blood-Brain Barrier/metabolism/physiology ; Zebrafish/genetics/embryology ; *Neovascularization, Physiologic/genetics ; Mice ; CRISPR-Cas Systems ; *Mutagenesis ; *Brain/blood supply/metabolism ; Mice, Transgenic ; Angiogenesis ; },
abstract = {The endothelial blood-brain barrier (BBB) is a complex neuroprotective structure whose maturation begins during brain angiogenesis and is completed postnatally. Despite its clinical importance, our understanding of BBB ontogeny and regulation remains limited. This shortcoming results from the difficulty of modeling the BBB in vitro and the current limitation of in vivo BBB genetics to low-throughput Mendelian genetic approaches. In addition, a single animal model facilitating the study of the neurovascular events occurring during both embryogenesis and adulthood is lacking. Here, leveraging the complementary anatomical and experimental attributes of mice and zebrafish, we present an integrated somatic CRISPR-Cas9 gene disruption strategy that allows us to assess candidate genes for their role in brain angiogenesis and BBB permeability within two and six weeks, respectively. Phenotypic assessment of brain angiogenesis is conducted in the transparent embryonic zebrafish hindbrain, while BBB maintenance is evaluated at both the behavioral and the functional level by combining endothelial-specific Cas9 transgenic mice and BBB-targeting adeno-associated viruses. The quick turnaround time and multiplexing capacity of our multi-species approach enable multimodal BBB functional genomics.},
}
@article {pmid41735731,
year = {2026},
author = {Zhao, R and Chen, J and Li, Y and Chen, DY and Kang, X and Dong, S and Yuan, X and Li, X and Gao, L and Yang, G and Chu, X and Wang, JG},
title = {CRISPR/Cas9-mediated α-prolamin gene (Seita.8G190200) mutagenesis increases the content of functional amino acids in foxtail millet (Setaria italica).},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {},
pmid = {41735731},
issn = {1432-203X},
support = {2023YFD1202702-6//National Key Research and Development Program of China/ ; 2025QNLJ203//"Youth Science and Technology Leadership Talent Training Program" of Shanxi Agricultural University/ ; },
mesh = {*Setaria Plant/genetics/metabolism ; *Amino Acids/metabolism ; *CRISPR-Cas Systems/genetics ; *Prolamins/genetics/metabolism ; Gene Editing ; *Mutagenesis/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Nutritive Value ; },
abstract = {The gene editing of α-prolamin gene (Seita.8G190200) exhibited significant increases in multiple functional aminoacids. The vast majority sugar components showed significant increases in the gene editing lines. Knockout of the prolamin gene not only improves the nutritional quality but also affects its eating quality. Proteins in foxtail millet exhibits a balanced amino acid composition that meets human dietary requirements.Prolamins are notably deficient in essential amino acids such as lysine, the structure, morphology, and functional characteristics of foxtail millet prolamin have been sufficiently elucidated, but the biological roles of prolamin genesstill need further exploration. This study demonstrates that the α-prolamin gene (Seita.8G190200) plays crucial roles in regulating prolamin content, functional amino acid and sugar components levels, potentially serving as an important target for improving both nutritional and eating quality of foxtail millet. These findings provide scientific foundations for developing novel nutritionally fortified functional foxtail millet products, breeding new foxtail millet varieties enriched with functional amino acids for human health benefits.},
}
@article {pmid41736546,
year = {2026},
author = {Zhang, C and Ye, K and Shang, Y and Song, Y and Li, P and Jiang, X and Yang, C and Liang, A and Zhang, J and Meng, F and Zhang, M},
title = {Spatially concentrated adenine base editors efficiently correct PLP1 mutations in oligodendrocytes.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41736546},
issn = {1362-4962},
support = {32370853//National Natural Science Foundation of China/ ; },
mesh = {*Oligodendroglia/metabolism ; *Gene Editing/methods ; Animals ; *Myelin Proteolipid Protein/genetics/metabolism ; *Adenine/metabolism ; Mutation ; Mice ; *Pelizaeus-Merzbacher Disease/genetics/therapy/pathology ; Humans ; CRISPR-Cas Systems ; Adenosine Deaminase/metabolism/genetics ; },
abstract = {Oligodendrocytes (OLs), the myelinating cells of the central nervous system, are particularly prone to pathogenic G-to-A mutations, such as PLP1A243V, which causes Pelizaeus-Merzbacher disease (PMD), a lethal hypomyelinating disorder lacking effective therapy. Although adenine base editors (ABEs) can in principle correct such mutations, their application in OLs is limited by inefficient on-target editing. Here, we develop a spatially concentrated ABE (cABE) strategy that enhances editing by promoting nuclear translocation of tRNA adenosine deaminase (TadA*) from the cytoplasm. Using a SunTag-based multivalent recruitment system, TadA* is locally enriched at genomic targets (cABE-1.0), achieving robust editing in vitro. To enable in vivo delivery while preserving high efficiency and fidelity, SpCas9 is replaced with compact eNme2-C Cas9, generating an AAV-compatible variant (cABE-2.0). Notably, cABE-2.0 forms dynamic nuclear puncta with properties of liquid-liquid phase separation, enhancing on-target editing while substantially reducing transcriptome-wide RNA off-target effects. Functionally, cABE-2.0 efficiently corrects the PLP1A243V mutation in OLs, restores Plp subcellular localization, and rescues myelination-related phenotypes. These findings demonstrate that spatial reorganization, rather than increasing intrinsic catalytic activity of TadA*, provides a distinct principle for improving base editing in difficult-to-edit cell types, such as OLs, offering a mechanistic and technical framework for gene therapy of PMD and related myelin disorders.},
}
@article {pmid41736562,
year = {2026},
author = {Purvis, IJ and Ochoa Olmos, OE and Park, KU and Kaufman, ML and Henry, CM and Schaaf, C and Clise, OJ and Tesdahl, CD and Haas, A and Brzezinski, JA},
title = {A robust cis-regulatory network ensures Otx2 expression during retinal development.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {6},
pages = {},
doi = {10.1242/dev.204881},
pmid = {41736562},
issn = {1477-9129},
support = {R01-EY024272/NH/NIH HHS/United States ; //Research to Prevent Blindness/ ; R01 EY024272/EY/NEI NIH HHS/United States ; F31-EY034794/NH/NIH HHS/United States ; F31 EY034794/EY/NEI NIH HHS/United States ; R01-EY024272/NH/NIH HHS/United States ; F31EY034794/NH/NIH HHS/United States ; //Gates Institute/ ; //Anschutz Medical Campus, University of Colorado/ ; //University of Colorado/ ; },
mesh = {*Otx Transcription Factors/genetics/metabolism ; Animals ; *Retina/metabolism/embryology ; *Gene Expression Regulation, Developmental ; Enhancer Elements, Genetic/genetics ; Mice ; *Gene Regulatory Networks/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {The transcription factor Otx2 is essential for photoreceptor and bipolar cell formation during retinal development. Otx2 expression is complex and underlies multiple cell fate decisions during development. To understand how Otx2 expression is regulated, we explored the activity and function of three of its enhancers (DHS2, DHS4 and DHS15). Enhancer reporter assays and lineage tracing show that DHS4 initiates Otx2 expression while DHS2 and DHS15 maintain expression in photoreceptors. Matched CRISPR/Cas9 and CRISPR interference systems were used to mutate or epigenetically silence enhancers, respectively. CRISPR reduced OTX2 expression acutely, but failed to significantly alter cell fate choice over the long term. In contrast, CRISPR interference of these enhancers caused permanent OTX2 loss and corresponding cell fate changes. While these data suggest that each enhancer is needed for normal Otx2 expression, it also highlights that the enhancers can interact and substitute for each other during development. This cis-regulatory element flexibility likely promotes Otx2 expression robustness. Such robustness may enable complex genes, like Otx2, to resist environmental stressors and regulatory disruptions to promote reproducible developmental outcomes.},
}
@article {pmid41738557,
year = {2026},
author = {Girard, V and Sorge, S and Kurth, J and Alexandre, C and Gould, AP},
title = {ShineGAL4 drivers for tissue and cell-type specific optogenetics in Drosophila.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {4},
pages = {},
pmid = {41738557},
issn = {1477-9129},
support = {FC001088/CRUK_/Cancer Research UK/United Kingdom ; FC001088/MRC_/Medical Research Council/United Kingdom ; FC001088/WT_/Wellcome Trust/United Kingdom ; 223760/WT_/Wellcome Trust/United Kingdom ; 104566/WT_/Wellcome Trust/United Kingdom ; 543-2022//EMBO/ ; //Francis Crick Institute/ ; },
mesh = {Animals ; *Optogenetics/methods ; *Drosophila Proteins/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *Drosophila melanogaster/genetics ; Organ Specificity/genetics ; CRISPR-Cas Systems/genetics ; Neurons/metabolism ; *Drosophila/genetics ; },
abstract = {An optogenetic split-GAL4 system, ShineGAL4, allows genes to be manipulated with unprecedented spatiotemporal precision. Here, we convert a panel of 14 GAL4 drivers widely used in Drosophila research into their ShineGAL4 counterparts. Homology assisted CRISPR knock-in (HACK) is used to replace GAL4 with the GAL4 DNA binding domain fused to a Magnet photoswitch. We show that the resulting ShineGAL4 drivers enable gene expression to be rapidly induced by light specifically in fat body, muscles, enterocytes, oenocytes, Malpighian tubules, neurons, neuroblast lineages, glial subtypes or in all glia. We also develop an optogenetic cassette for photoactivation of GAL4 in 'silent' FLP-out clones. This panel of optogenetic tools will enable precise spatiotemporal control of gene expression in a wide range of different Drosophila tissues and cell-types.},
}
@article {pmid41738762,
year = {2026},
author = {Irfan, M and Duran-Pinedo, A and Solbiati, J and Rocha, FG and Gibson, FC and Frias-Lopez, J},
title = {A CRISPR array orchestrates virulence and host response in Porphyromonas gingivalis.},
journal = {Microbiology spectrum},
volume = {14},
number = {4},
pages = {e0283425},
pmid = {41738762},
issn = {2165-0497},
support = {DE029775/DE/NIDCR NIH HHS/United States ; },
mesh = {*Porphyromonas gingivalis/genetics/pathogenicity ; Animals ; Virulence/genetics ; Biofilms/growth &amp; development ; Macrophages/microbiology/immunology ; Humans ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Moths/microbiology ; Bacteroidaceae Infections/microbiology/immunology ; Cytokines/metabolism ; Host-Pathogen Interactions ; THP-1 Cells ; Larva/microbiology ; },
abstract = {CRISPR-Cas systems are primarily recognized for their defensive role against foreign DNA. Recent studies, however, indicate involvement in regulatory functions. The persistence of a large spacer array without apparent phage targets in Porphyromonas gingivalis ATCC 33277 remains unexplained. This study demonstrates that deletion of the non-coding CRISPR array 30.1 in P. gingivalis ATCC 33277 results in increased biofilm formation, elevated virulence in a Galleria mellonella infection model, and significant alterations in the macrophage transcriptomic response. The ΔCRISPR-30.1 mutant forms twice as much biofilm as the wild type, induces 50% mortality in Galleria larvae within 130 h compared to 200 h for the wild type (P < 0.0001), and elicits a cytokine profile characterized by increased IL-6, CXCL1, CXCL2, and CXCL9 secretion. Dual RNA sequencing of THP-1 macrophages infected with wild-type and ΔCRISPR 30.1 strains reveals that the loss of the array activates bacterial metabolic and secretion pathways while suppressing host innate and adaptive immune signaling. Single-primer amplification (SPA) identifies numerous self-genome loci bound by individual 30.1 spacers, supporting a direct, spacer-mediated regulatory mechanism. These findings establish CRISPR array 30.1 as a previously unrecognized regulator of P. gingivalis physiology and host-pathogen interactions.IMPORTANCECRISPR-Cas systems are established as adaptive immune elements, yet spacer arrays without known targets are frequently observed in bacteria and often lack a defined function. In P. gingivalis, a keystone periodontal pathogen, a non-coding CRISPR array has been shown to regulate biofilm formation, virulence in an invertebrate model, and the macrophage transcriptional response. This expands the recognized functions of CRISPR arrays to include the direct regulation of bacterial physiology and the modulation of host immune responses, identifying CRISPR spacers as potential targets for antimicrobial interventions. Furthermore, elucidating the role of CRISPR arrays in P. gingivalis may have broader clinical implications, given the established associations between periodontal health and systemic inflammatory diseases. Targeting spacer arrays to modulate bacterial virulence could influence the management of these conditions and enhance the translational relevance of such therapeutic strategies.},
}
@article {pmid41739553,
year = {2026},
author = {Valinsky, WC and Ray, RP and Schaefer, KS and Grimm, JB and Nicolini, C and Lavis, LD and Clapham, DE},
title = {Phenotypic CRISPR screens identify NLRX1 as an essential activator of the human mitochondrial permeability transition.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {9},
pages = {e2535298123},
pmid = {41739553},
issn = {1091-6490},
support = {NA//Janelia Research Campus (Janelia)/ ; },
mesh = {Humans ; Calcium/metabolism ; *Mitochondria/metabolism/genetics ; *Mitochondrial Proteins/metabolism/genetics ; Membrane Potential, Mitochondrial ; CRISPR-Cas Systems ; Mitochondrial Membrane Transport Proteins/metabolism/genetics ; Mitochondrial Membranes/metabolism ; Phenotype ; Clustered Regularly Interspaced Short Palindromic Repeats ; Permeability ; Calcium Channels/metabolism/genetics ; },
abstract = {The mitochondrial permeability transition (mPT) is an evolutionarily conserved destructive process that permeabilizes the inner mitochondrial membrane in response to calcium overload. The molecular mechanism underlying the mPT is not established. To unambiguously identify essential proteins, we designed two phenotypic assays for mitochondrial calcium overload and applied them to FACS-based CRISPR screening in human cells, ultimately evaluating 19,113 genes. The first screen studied mitochondrial membrane potential (MMP) collapse in response to calcium overload. Top-ranked genes were the essential proteins of the mitochondrial calcium uniporter complex, MCU and EMRE, reflecting that the calcium-induced MMP collapse results from mitochondrial calcium entry and not the mPT. The second screen measured the permeability of the inner mitochondrial membrane. Here, the fluorescent interaction of a membrane impermeant ~600 Da dye and a mitochondrial-targeted HaloTag protein was studied under mPT activating conditions; calcium overload and the thiol-reactive molecule phenylarsine oxide. With secondary validation, we identified four protein-encoding genes that delayed or prevented the mPT under knockout: NF2, REST, BPTF, and NRLX1. Knockout of the nonmitochondrial proteins BPTF, NF2, or REST increased mitochondrial calcium retention capacity (CRC). However, calcium release or sensitivity to cyclosporin A (CsA) persisted, indicative of mPT sensitizers. Only knockout of the mitochondrial matrix protein, NLRX1, increased CRC, abolished calcium release, and was CsA-insensitive. This top-ranked hit of the mitochondrial permeability screen meets the definition of an essential mPT activator. Integral membrane proteins, including all previously proposed mPT candidates, were not essential activators.},
}
@article {pmid41740027,
year = {2026},
author = {Duarte, DF and Lucena, LP and Gonçalves, MHO and Benko-Iseppon, AM and Aburjaile, F and Azevedo, V and Brenig, B and Gama, MAS and Souza, EB},
title = {Phylogenomic analysis of Paracidovorax citrulli strains reveals the presence of two lineages in Brazil.},
journal = {Genetics and molecular biology},
volume = {48},
number = {4},
pages = {e20250046},
pmid = {41740027},
issn = {1415-4757},
abstract = {Paracidovorax citrulli is the causative agent of bacterial fruit blotch in melons and watermelons. This study used comparative genomic approaches of 17 Brazilian P. citrulli strains obtained from melons and watermelons to classify them into groups I and II and try to understand their genomic differences. The genomes of P. citrulli presented general characteristics similar to those shown for the genomes of the type strain of P. citrulli and reference strains of groups I and II. A phylogenomic analysis revealed two distinct groups of P. citrulli, in which most Brazilian P. citrulli strains were grouped with the strain representing group I. CRISPR-Cas analysis revealed the presence of two proteins, Cas3 and Cas10, in all Brazilian P. citrulli genomes. In addition, we observed the presence of two plasmids (pAMC6 and pAC53) in three Brazilian P. citrulli strains, all closely related to group I. The prediction of effector proteins revealed the XopE/AvrPphe protein as a differential between the strains of groups I and II. The present study will contribute to a more detailed understanding of aspects of host-pathogen interactions and will help improve the detection of strains from these groups, thus elucidating the population dynamics of Brazilian strains of P. citrulli.},
}
@article {pmid41740122,
year = {2026},
author = {Yıldırım, K and Kavas, M},
title = {The role of CRISPR in modern plant breeding: overcoming breeding barriers and legislative challenges through transgene-free genome editing.},
journal = {Genome},
volume = {69},
number = {},
pages = {1-17},
doi = {10.1139/gen-2025-0063},
pmid = {41740122},
issn = {1480-3321},
mesh = {*Plant Breeding/methods/legislation &amp; jurisprudence ; *Gene Editing/methods/legislation &amp; jurisprudence ; *CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Crops, Agricultural/genetics ; *Genome, Plant ; Transgenes ; },
abstract = {Traditional plant breeding techniques-such as crossbreeding, mutation breeding, and marker-assisted selection-have significantly contributed to crop improvement over the past century. However, these methods are often limited by long breeding cycles, low precision, and the unintended transfer of undesirable traits. To address these challenges, transgenic breeding emerged as a powerful tool, enabling the introduction of specific foreign genes to confer desirable traits such as pest resistance or herbicide tolerance. While highly effective and precise, transgenic approaches face considerable regulatory and public acceptance barriers, particularly in regions with strict GMO legislation. The advent of CRISPR/Cas genome editing has revolutionized plant breeding by enabling precise, efficient, and targeted modification of native genes, significantly accelerating the development of improved crops. Among CRISPR-based methods, transgene-free genome editing has gained prominence for producing enhanced plant varieties without integrating foreign DNA, thus avoiding many regulatory constraints associated with GMOs. This review aims to provide a comprehensive overview of CRISPR-mediated genome editing technologies in crop innovation, emphasizing especially the use of transgene-free approaches in modern plant breeding and their transformative potential to overcome the limitations of conventional methods while providing a regulation-friendly pathway for crop improvement.},
}
@article {pmid41740934,
year = {2026},
author = {Sun, W and Zhu, S and Dong, Q and Tang, S and Liu, Q and Sha, Y and Chen, T and Wang, R and Chen, Y and Ying, H},
title = {Engineering Escherichia coli cell Factories for continuous 5'-cytidine monophosphate production via biofilm-anchored dual-enzyme cascade catalysis.},
journal = {Bioresource technology},
volume = {447},
number = {},
pages = {134267},
doi = {10.1016/j.biortech.2026.134267},
pmid = {41740934},
issn = {1873-2976},
mesh = {*Biofilms/growth &amp; development ; *Escherichia coli/metabolism/genetics/enzymology ; *Cytidine Monophosphate/biosynthesis ; Biocatalysis ; CRISPR-Cas Systems/genetics ; Uridine Kinase/metabolism ; },
abstract = {5'-cytidine monophosphate (5'-CMP) serves as a crucial intermediate for diverse nucleotide derivatives and finds extensive applications in the food and pharmaceutical industries. However, existing enzymatic production processes suffer from low catalytic efficiency and poor economic feasibility. In this study, we developed a continuous 5'-CMP production system based on a cell-enzyme co-immobilized biocatalytic platform. First, the pgaABCD gene cluster was integrated into ClearColi BL21(DE3) using CRISPR-Cas9, enhancing its biofilm-forming capacity by 168.93% and enabling robust cell immobilization on the carrier. Second, a dual-anchoring strategy utilizing ice-nucleation protein (INP) and autotransporter (AIDA-I) enabled surface display of uridine kinase (UDK) and acetate kinase (AckA) on the cell surface. This approach successfully addressed the instability and recovery issues of free enzymes by using biofilm engineering to co-immobilize cells and enzymes. The modified strain achieved a 5'-CMP productivity of 1.77 mmol/L/h, 5.98-fold higher than free intracellular enzyme catalysis, and was reused for ten consecutive cycles under the tested conditions while maintaining a cytidine conversion rate above 73.79%, and a 5'-CMP yield above 59.26%. This work demonstrates the first successful realization of continuous 5'-CMP biosynthesis and establishes an efficient route for its industrial production.},
}
@article {pmid41741438,
year = {2026},
author = {Liu, T and Lin, Y and Liu, Q and Liao, W and Lin, Y and Zhang, Y and Zhang, J and Cao, W and Yang, L and Hong, Z and Lu, Z},
title = {Target-stabilized base editors enable robust high-fidelity RNA editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41741438},
issn = {2041-1723},
support = {82327805//National Natural Science Foundation of China (National Science Foundation of China)/ ; KQTD20210811090117032//Shenzhen Science and Technology Innovation Commission/ ; },
mesh = {*RNA Editing/genetics ; *Adenosine Deaminase/genetics/metabolism ; Humans ; Animals ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; HEK293 Cells ; RNA-Binding Proteins/genetics/metabolism ; Mutation ; Gene Editing/methods ; Mice ; Amyotrophic Lateral Sclerosis/genetics ; },
abstract = {RNA base editing using engineered deaminases represents a powerful tool to correct mutations at the RNA level. However, widespread off-target effects, primarily arising from dissociated free deaminases, remain a significant challenge. Here, we devise the RECODE (RNA editing with conditionally stable and enhanced ADAR1 deaminase variants) system, which employs designer degron-tagged ADAR1 deaminase (ADAR1d) with guide RNA (gRNA)-regulated stability. By promoting degradation of gRNA-unbound ADAR1d, RECODE markedly reduces transcriptome-wide edits while maintaining high on-target efficacy. Engineering gRNA for target RNA-induced conformational switching confines ADAR1d stabilization to intended editing sites, further enhancing editing precision. With structure-guided rational engineering of ADAR1d, RECODE efficiently corrects an Amyotrophic Lateral Sclerosis-relevant FUS mutation and installs a therapeutic mutation to Angptl3 in vivo, which mitigate FUS mislocalization to neuronal axons and lower plasma lipids, respectively. These findings establish RECODE as a highly stringent and efficient RNA editing technology and underscore a general principle for enhancing the specificity of RNA-guided protein effectors.},
}
@article {pmid41741470,
year = {2026},
author = {Aird, EJ and Serrano-Benitez, A and Siegner, SM and Cannavo, E and Belotserkovskaya, R and Gueorguieva, N and Fielden, J and Cullot, G and Ammann, S and Bader, AS and Gupta, V and Andrieux, G and Raab, R and Del Rey González, M and Cathomen, T and Cejka, P and Corn, JE and Jackson, SP},
title = {ERCC6L2 ensures repair fidelity for staggered-end DNA double-strand breaks.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41741470},
issn = {2041-1723},
support = {855741-DDREAMM-ERC-2019-SyG//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 855741-DDREAMM-ERC-2019-SyG//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *DNA Helicases/metabolism/genetics ; *DNA Repair ; DNA-Binding Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; MRE11 Homologue Protein/metabolism/genetics ; CRISPR-Cas Systems ; Gene Editing ; Cell Cycle Proteins/metabolism/genetics ; DNA Repair Enzymes/metabolism/genetics ; Poly-ADP-Ribose Binding Proteins/metabolism ; HEK293 Cells ; Nuclear Proteins/metabolism ; Bacterial Proteins ; Acid Anhydride Hydrolases ; CRISPR-Associated Proteins ; },
abstract = {DNA double-strand breaks (DSBs) both pose threats to genome integrity and are commonly used for genome editing applications. Structural features of DSB ends play key roles in determining DNA repair pathway usage and outcomes during genome editing, but the cellular factors involved in these processes are only partially known. Through genome-wide CRISPRi screening, we identify ERCC6L2 as critical for repairing Cas12a-induced staggered DSBs but irrelevant for Cas9-induced blunt DSBs. We show that ERCC6L2 acts as a protection factor for staggered DSBs with either 5' or 3' polarity, preventing large deletions and translocations stemming from DNA damage induced by Cas12a, TALENs, or dual Cas9 nicks. Furthermore, ERCC6L2 loss hyper-sensitizes cells to multiple staggered DSBs induced by promiscuous Cas12a activity or etoposide-induced TOP2 trapping. By combining genetics and biochemical reconstitution, we find that ERCC6L2 counteracts MRE11-RAD50-NBS1 (MRN)-mediated resection by binding and melting staggered DNA ends, thereby promoting accurate end joining. Our data reveal a protective role of ERCC6L2 in staggered-end DSB repair, which suggests the molecular underpinnings of pathology in patients with ERCC6L2 mutations and cautions against using overhang-inducing genome editing tools for their treatment.},
}
@article {pmid41741636,
year = {2026},
author = {Mathiowetz, AJ and Meymand, ES and Parlakgül, G and van Hilten, N and Torres, EF and Artico, LL and Deol, KK and Lange, M and Pang, SP and Doubravsky, CE and Roberts, MA and Jorgens, DM and Zalpuri, R and Kang, M and Boone, C and Parks, BW and Zhang, Y and Morgens, DW and Newman, ET and Zhou, Y and Talukdar, S and Grabe, M and Ku, G and Levine, TP and Arruda, AP and Olzmann, JA},
title = {CLCC1 promotes hepatic neutral lipid flux and nuclear pore complex assembly.},
journal = {Nature},
volume = {652},
number = {8109},
pages = {462-470},
pmid = {41741636},
issn = {1476-4687},
support = {R01 HL147097/HL/NHLBI NIH HHS/United States ; P30 DK098722/DK/NIDDK NIH HHS/United States ; R01 DK128099/DK/NIDDK NIH HHS/United States ; R01 GM137109/GM/NIGMS NIH HHS/United States ; R01 GM112948/GM/NIGMS NIH HHS/United States ; R01 DK140367/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Nuclear Pore/metabolism ; *Liver/metabolism/cytology ; Hepatocytes/metabolism ; Lipid Droplets/metabolism ; Humans ; Molecular Dynamics Simulation ; Male ; Fatty Liver/metabolism/pathology/genetics ; Membrane Fusion ; *Lipid Metabolism ; CRISPR-Cas Systems ; Mice, Knockout ; Lipoproteins/metabolism ; Nuclear Envelope/metabolism ; Female ; },
abstract = {Imbalances in lipid storage and secretion lead to hepatic steatosis, the accumulation of lipid droplets in hepatocytes[1,2]. Our understanding of the mechanisms that govern the channelling of neutral lipids in hepatocytes towards cytosolic lipid droplets or secreted lipoproteins remains incomplete[3,4]. Here we performed a series of CRISPR-Cas9 screens under different metabolic states that led to the identification of CLCC1 as a critical regulator of neutral lipid storage and secretion in hepatocytes. Loss of CLCC1 resulted in the buildup of large lipid droplets in hepatoma cells and Clcc1 knockout in mice caused liver steatosis. Lipid droplets were present in the lumen of the endoplasmic reticulum of the Clcc1-knockout hepatocytes and exhibited properties of lipoproteins, indicating a profound shift in neutral lipid flux. The loss of CLCC1 also led to the accumulation of nuclear membrane herniations accompanied by a reduction in nuclear pores. Remote homology searches identified a domain in CLCC1 that is homologous to yeast Brl1 and Brr6, factors that promote nuclear envelope fusion during nuclear pore complex assembly. Molecular dynamics simulations and mutagenesis studies support a model in which CLCC1 mediates membrane bending and fusion. We propose that CLCC1 mediates membrane fusion to promote hepatic neutral lipid flux and nuclear pore complex assembly.},
}
@article {pmid41741641,
year = {2026},
author = {Yang, L and Renauer, PA and Tang, K and Saskin, J and Zhou, L and Zou, C and Lee, SH and Fox, M and Johnson-Noya, S and Weiss, B and Deng, S and Fang, P and Chen, B and Sferruzza, G and Fooladi, S and Zhao, K and Park, D and Zhang, F and Tu, J and Chen, J and Moliterno, J and Gunel, M and Peng, L and Chen, S},
title = {OR7A10 GPCR engineering boosts CAR-NK therapy against solid tumours.},
journal = {Nature},
volume = {652},
number = {8110},
pages = {740-751},
pmid = {41741641},
issn = {1476-4687},
mesh = {Humans ; Animals ; *Killer Cells, Natural/immunology/metabolism/cytology/transplantation ; Mice ; Female ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; *Neoplasms/therapy/immunology/pathology ; Tumor Microenvironment/immunology ; *Immunotherapy, Adoptive/methods ; Male ; Cell Line, Tumor ; *Receptors, G-Protein-Coupled/genetics/metabolism/immunology ; Xenograft Model Antitumor Assays ; Cell Proliferation ; CRISPR-Cas Systems/genetics ; },
abstract = {Chimeric antigen receptor (CAR)-natural killer (NK) cell therapies hold promise for solid tumours but remain limited because of poor tumour infiltration, persistence and resistance in the tumour microenvironment[1-4]. Here, to identify gain-of-function targets that enhance CAR-NK cell efficacy, we performed an unbiased in vivo CRISPR activation screen followed by a barcoded targeted in vivo open reading frame screen in primary human CAR-NK cells. We identified and comprehensively validated OR7A10, a G protein-coupled receptor (GPCR), as the top candidate. Engineering CAR-NK cells with OR7A10 cDNA (a CRISPR-independent method with a simple manufacturing strategy) enhanced their proliferation, activation, degranulation, cytokine production, death ligand expression, chemokine receptor expression, cytotoxicity, persistence, metabolic fitness and tumour microenvironment resistance. Moreover, exhaustion in primary human NK cells derived from multiple peripheral blood and cord blood donors was reduced. OR7A10 gain-of-function CAR-NK cells displayed strong in vivo efficacy across multiple solid tumour models. For example, 100% complete response with long-term tumour control and survival benefit in an orthotopic breast cancer mouse model were achieved. These findings establish OR7A10-engineered CAR-NK cells as a highly potent and scalable off-the-shelf therapeutic for solid tumours.},
}
@article {pmid41741653,
year = {2026},
author = {Saxton, DS and DeWeirdt, PC and Doering, CR and Roney, IJ and Laub, MT},
title = {A membrane-bound nuclease directly cleaves phage DNA during genome injection.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41741653},
issn = {1476-4687},
abstract = {From mammals to bacteria, the direct recognition and cleavage of viral nucleic acids is a potent defence strategy against viral infection, but it requires mechanisms for distinguishing self from non-self[1,2]. In bacteria, CRISPR-Cas and restriction-modification systems achieve this discrimination by recognizing specific DNA sequences or DNA modifications, respectively. Alternative mechanisms probably remain to be discovered. Here, we characterize SNIPE, an anti-bacteriophage defence system that constitutively localizes to the bacterial cell membrane in Escherichia coli to block phage λ infection. Using radiolabelled phage DNA and time-lapse microscopy to track phage genomes, we demonstrate that SNIPE directly cleaves phage DNA during genome injection. Based on proximity labelling, we find that SNIPE associates with host proteins essential for λ genome entry and with the λ tape measure protein, which facilitates λ genome injection across the inner membrane. SNIPE also defends against diverse siphoviruses, probably through direct interactions with their tape measure proteins. Our findings establish SNIPE as a widespread bacterial defence system that exploits the spatial organization of phage genome injection to specifically target viral DNA, representing a previously unknown strategy for distinguishing self from non-self in prokaryotic immune systems.},
}
@article {pmid41741655,
year = {2026},
author = {Lei, J and Yang, K and Cao, W and Qi, S and Du, X and Li, H and Wang, Y and Gan, J and Feng, Y and Li, Y and Zhang, W and Bai, B and Lin, X and Su, X and Zhang, Q and Liang, T and Yu, G},
title = {Pancreatic-targeted lipid nanoparticles based on organ capsule filtration.},
journal = {Nature},
volume = {652},
number = {8108},
pages = {220-229},
pmid = {41741655},
issn = {1476-4687},
support = {//Beijing Municipal Science &amp; Technology Commission/ ; //National Key R&amp;D Program of China/ ; },
mesh = {Animals ; *Nanoparticles/chemistry ; Mice ; *Pancreas/metabolism ; Humans ; *Lipids/chemistry ; Pancreatic Neoplasms/therapy/genetics ; RNA, Messenger/genetics/administration &amp; dosage/metabolism ; Female ; Gene Editing ; Male ; Endocytosis ; Cancer Vaccines/immunology/administration &amp; dosage ; Cytokines/genetics ; CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Capsules/chemistry ; Liposomes ; },
abstract = {Achieving pancreatic-targeted delivery marks a breakthrough in treating pancreatic diseases, yet precise delivery remains challenging[1]. Here we identify an explicit and universal principle for pancreatic-selective delivery and propose a pancreatic-targeted lipid nanoparticle (AH-LNP) for mRNA delivery. AH-LNP exhibits size enlargement after assembly with proteins, facilitating capsule-filter-mediated pancreas-selective accumulation and receptor-mediated endocytosis, thereby boosting the pancreatic-targeted ability. Benefiting from this, AH-LNP enables precise and efficient genome editing in the pancreas through the delivery of Cas9 mRNA and single guide RNA (sgRNA), exhibiting promising potential in the treatment of autoimmune pancreatic diseases. Furthermore, pancreatic-targeted delivery of mRNA encoding therapeutic cytokines through AH-LNP demonstrates superior antitumour efficacy when combined with a cancer vaccine or chimeric antigen receptor T cell therapy in multiple pancreatic cancer models. The safety and pancreatic mRNA delivery of AH-LNP were verified in multiple animal models, including non-human primates, demonstrating great promise for clinical translation. Our findings highlight the transformative potential of this pancreatic-targeted mechanism and the derived LNP platform, opening avenues for developing precision therapeutics against diverse pancreatic diseases.},
}
@article {pmid41741764,
year = {2026},
author = {Shivgotra, R and Soni, B and Bakrey, H and Kaur, P and Jain, SK},
title = {Bridging Gaps in Fungal Keratitis Management: Novel Diagnostics, Drug Delivery Systems, and Gene Therapies.},
journal = {AAPS PharmSciTech},
volume = {27},
number = {3},
pages = {},
pmid = {41741764},
issn = {1530-9932},
abstract = {Globally, fungal keratitis has become a major health concern, particularly in tropical and humid regions. The disease burden is exacerbated because of the underlying complexity of fungal pathogens, delayed or insufficient diagnosis, and limitations in existing therapeutic strategies, often leading to progressive corneal damage and impaired vision. Additional challenges include insufficient therapy optimization and variable efficacy of topical and systemic antifungal treatments, which may be influenced by factors such as rapid ocular drug clearance, ineffective ocular penetration, and the increasing prevalence of antifungal resistance. Conventional diagnostic techniques, such as microscopy and culture, continue to serve as reference standards but have limitations due to comparatively modest sensitivity and the prolonged turnaround times. In comparison, recent advances in diagnostic techniques, including CRISPR-based assays, PCR, MALDI-ToF MS, and in vivo confocal microscopy, as well as the novel drug-delivery nanocarriers, have been reported to show noticeable improvements in diagnostic accuracy and therapeutic outcomes. Furthermore, emerging DNA-based gene therapies and RNA-based therapeutics, along with advanced ocular drug carriers, have shown promising outcomes in preclinical research and early-phase clinical trials, suggesting potential advantages in enhanced tissue targeting and reduced therapeutic resistance. However, before widespread clinical acceptability, extensive clinical validation, long-term safety evaluations, and cost assessments are required, as existing findings are mainly limited to short-term and experimental research. This review focuses on the pathophysiology of fungal keratitis while underscoring the unmet diagnostic and therapeutic needs. It further explores the potential for developing translatable technologies aimed at predictive diagnosis and the effective management of this sight-threatening condition.},
}
@article {pmid41742301,
year = {2026},
author = {Lee, YI and Hart, CC and Henley-Beasley, CS and Herr, JS and Zerpa, E and Barton, ER and Hammers, DW and Sweeney, HL},
title = {Functional and structural pathologies in skeletal muscle of a rat model of Duchenne muscular dystrophy.},
journal = {Skeletal muscle},
volume = {16},
number = {1},
pages = {},
pmid = {41742301},
issn = {2044-5040},
support = {P50-AR052646/AR/NIAMS NIH HHS/United States ; P50-AR052646/AR/NIAMS NIH HHS/United States ; P50-AR052646/AR/NIAMS NIH HHS/United States ; P50-AR052646/AR/NIAMS NIH HHS/United States ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/pathology/physiopathology/genetics/metabolism ; *Muscle, Skeletal/pathology/physiopathology/metabolism ; Rats, Sprague-Dawley ; Disease Models, Animal ; Dystrophin/genetics/metabolism/deficiency ; Rats ; Male ; Muscle Contraction ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Duchenne muscular dystrophy (DMD) is a lethal pediatric degenerative muscle disease for which there is no cure. Robust preclinical models that recapitulate major clinical features of DMD are required to investigate efficacy of potential DMD therapeutics. Rat models of DMD have emerged as promising small animal models to accomplish this; however, there have been no comprehensive studies investigating the functional skeletal muscle decrements associated with the modeling of DMD in rats.<br><br>METHODS: CRISPR/Cas9 gene editing was used to generate a dystrophin-deficient Sprague-Dawley muscular dystrophy rat (MDR). Biochemical and immunofluorescent analyses were performed to confirm loss of dystrophin in striated muscles of this rat model. In situ and ex vivo muscle function was assessed in wild-type (WT) and MDR muscles at 3, 6, and 12 months of age, followed by histopathological analyses.<br><br>RESULTS: MDR muscle tissues exhibited loss of full-length dystrophin and reduced content of other dystrophin glycoprotein complex members. MDR extensor digitorum longus (EDL) muscles and diaphragms displayed pronounced and progressive muscle weakness beginning at 3 months of age, compared to WT littermates. EDLs also exhibit susceptibility to eccentric contraction-induced damage. Functional deficits in soleus muscles were less severe and were associated with a right shift in force-frequency relationship. MDR muscles display progressive histopathology including degenerative lesions, fibrosis, regenerative foci, and modest adipose deposition.<br><br>CONCLUSIONS: MDR is a preclinical model of DMD that exhibits many translational features of the human disease, including a large dynamic range of muscle decrements, that has high utility for the evaluation of potential therapeutics for DMD.},
}
@article {pmid41742365,
year = {2026},
author = {Yao, W and Li, Y and Du, Y and Gao, R and Tan, J and Fan, J and Liu, Y},
title = {Aptamer-Mediated Protein Corona In Situ Multiplex Microfluidic Detection for Cancer Diagnosis.},
journal = {Analytical chemistry},
volume = {98},
number = {9},
pages = {7123-7135},
doi = {10.1021/acs.analchem.6c00170},
pmid = {41742365},
issn = {1520-6882},
mesh = {Humans ; *Aptamers, Nucleotide/chemistry ; *Neoplasms/diagnosis/blood ; *Protein Corona/chemistry ; *Microfluidic Analytical Techniques ; *Biomarkers, Tumor/blood ; *Lab-On-A-Chip Devices ; CRISPR-Cas Systems ; Silicon Dioxide/chemistry ; },
abstract = {Liquid biopsy faces critical limitations in detecting low-abundance protein biomarkers due to serum complexity, single-target constraints, and insufficient sensitivity. Here, we present an integrated microfluidic protein aptamer corona-CRISPR (PACC) platform that synergizes nanoparticle protein corona (PC) enrichment, multiplexed aptamer recognition, and CRISPR-Cas12a-driven signal generation for facile and rapid point-of-care testing. Fe3O4@SiO2 nanoparticles capable of streamlining PC isolation via magnetic separation were employed to concentrate cancer-associated proteins from serum. A 6-plex aptamer panel, which selectively bound target proteins via forming protein-aptamer coronas (PACs), was used to initiate the in situ activation of CRISPR-Cas12a via complementary crRNAs for fluorescence signal generation. This entire workflow was miniaturized into a 3D-printed microfluidic chip and portable reader, achieving a fast sample-to-answer process. Clinical validation with 112 serum specimens demonstrated excellent diagnostic performance, achieving an area under the curve (AUC) of 0.95 and 88.24% accuracy. By integrating these modalities, the PACC platform overcomes serum matrix interference and biomarker scarcity, providing a scalable and low-cost avenue for detecting low-abundance protein biomarkers and advancing liquid biopsy toward precision oncology.},
}
@article {pmid41742419,
year = {2026},
author = {Zhu, M and Yuan, J and Meng, Q and Yu, J and Xu, X and Xu, M and Ren, X and Hu, Y and Wei, G and Jia, Z and Yuan, G and Zang, L and Liu, S and Yang, Y and Zheng, Y and Wang, J and Cong, T and Xie, W and Lan, X and Cong, L and Ma, T and Ding, S and Guo, W and Zhang, X and Li, Y},
title = {Minimizing far-extending chromatin perturbation in genome editing preserves stem cell identity.},
journal = {Cell stem cell},
volume = {33},
number = {3},
pages = {470-486.e14},
doi = {10.1016/j.stem.2026.01.015},
pmid = {41742419},
issn = {1875-9777},
mesh = {*Gene Editing/methods ; *Chromatin/metabolism/genetics ; Animals ; Mice ; CRISPR-Cas Systems/genetics ; Cell Differentiation/genetics ; *Neural Stem Cells/metabolism/cytology ; Mouse Embryonic Stem Cells/metabolism/cytology ; CCCTC-Binding Factor/metabolism ; },
abstract = {Although CRISPR-Cas9 holds therapeutic promise, broader application demands an understanding of complications in vast non-coding regions. We found that CRISPR-Cas9 can cause premature differentiation of neural stem cells in vivo and mouse embryonic stem cells in vitro, even when cleavage occurred at distant sites tens of kilobases away from the nearest regulatory elements. To investigate this, we employed an integrated assay for transposase-accessible chromatin (ATAC)/RNA sequencing (AR-seq) approach and identified editing-induced chromatin accessibility changes, with their scale varying by cell type. Cells with stemness are most affected, experiencing perturbations that extend over a hundred kilobases. Furthermore, even local DNA perturbations can disrupt CTCF- and condensate-associated chromatin architecture, causing distal transcriptional rewiring and, ultimately, loss of stemness identity. To minimize chromatin perturbations and preserve cell identity, we refined gene-editing strategies, including distance-aware sgRNA design, pharmacological attenuation of DNA resection, and alternative editing systems. This work paves the way for the safer and broader application of genome-editing technologies.},
}
@article {pmid41744735,
year = {2026},
author = {Hu, X and Su, J and Song, S},
title = {CRISPR/Cas System-Based Biosensors.},
journal = {Biosensors},
volume = {16},
number = {2},
pages = {},
pmid = {41744735},
issn = {2079-6374},
abstract = {Over the past decade, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins, originally identified as adaptive immune systems in bacteria and archaea that defend against invading nucleic acids, have revolutionized biological research [...].},
}
@article {pmid41747377,
year = {2026},
author = {Pan, MX and Lv, MM and Nie, YG and Su, M and Zha, CJ and Mei, RY and Ying, ZM},
title = {Ultrasensitive miRNA detection via magnetic bead-confined catalytic hairpin assembly enabling transcription-driven crRNA assembly and CRISPR/Cas12a activation.},
journal = {Biosensors &amp; bioelectronics},
volume = {302},
number = {},
pages = {118559},
doi = {10.1016/j.bios.2026.118559},
pmid = {41747377},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *CRISPR-Associated Proteins/genetics/chemistry ; DNA/chemistry/genetics ; *Endodeoxyribonucleases/chemistry/genetics ; Limit of Detection ; *Bacterial Proteins/chemistry/genetics ; Nucleic Acid Hybridization ; Transcription, Genetic ; },
abstract = {The integration of CRISPR/Cas12a with catalytic hairpin assembly (CHA), a strategy that predominantly relies on CHA to generate dsDNA activators for direct Cas12a activation, has emerged as a powerful tool in molecular diagnostics. However, two major challenges remain: the strict protospacer adjacent motif (PAM) dependence of the dsDNA and background leakage from hairpin hybridization. Herein, we report a bead-confined platform that transcription mediates crRNA reassembly and template activation of Cas12a for ultrasensitive miRNA detection. The target-triggered CHA assembly dynamically constructed a T7 transcription template from three initially locked hairpins (H1, H2, and H3), which not only transcribed scaffold RNA but also hybridized with its own product to form a DNA/RNA complex that activates Cas12a. The integration of the split T7 promoter with CHA effectively suppressed background suppression and enhanced detection sensitivity. Additionally, the magnetic beads increase local concentration and reaction kinetics, collectively contributing to a substantially enhanced detection sensitivity. Moreover, a crRNA assembly strategy designed for transcription-powered Cas12a not only circumvents the conventional PAM-dependent dsDNA activation pathway of Cas12a but also enables self-supplied crRNA without requiring additional activators. We demonstrated that the biosensor exhibits exceptional sensitivity for miRNA-21 detection, achieving a limit of 65.3 aM. Furthermore, the practicality of this method was preliminarily confirmed through accurately quantifying target levels in cell lines and human serum. Our method presents a viable solution with transformative potential, designed to address complex challenges in contemporary diagnostic applications.},
}
@article {pmid41747624,
year = {2026},
author = {Ming Wu, SC and Boheler, KR},
title = {Generation of an induced pluripotent stem cell and isogenic control line from a vascular Ehlers-Danlos Syndrome (vEDS) patient harboring a pathogenic c.755G&gt;T in the COL3A1 gene.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103945},
doi = {10.1016/j.scr.2026.103945},
pmid = {41747624},
issn = {1876-7753},
mesh = {Humans ; *Ehlers-Danlos Syndrome/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology/pathology ; *Collagen Type III/genetics/metabolism ; Cell Line ; Cell Differentiation ; Fibroblasts/metabolism ; CRISPR-Cas Systems ; Ehlers-Danlos Syndrome, Type IV ; },
abstract = {We report the generation and characterization of a collagen III-mutant human iPSC line (JHUi007-A) and an isogenic gene-edited control (JHUi007-A-1). Reprogramming of dermal fibroblasts, obtained from a patient with vascular Ehlers-Danlos syndrome (vEDS) carrying the COL3A1 c.755G>T variant, was performed using integration-free Sendai virus. Isogenic controls were produced by CRISPR/Cas9 gene editing. Both lines displayed typical morphologies, expressed stemness factors, formed derivatives of all three germ layers, and maintained a normal karyotype. These lines readily differentiated into vascular smooth muscle cells with cytoskeletal differences between vEDS and control cells, confirming the utility of this resource to study disease processes.},
}
@article {pmid41747626,
year = {2026},
author = {Yoo, DH and Bayarsaikhan, D and Lee, J and Im, YS and Bayarsaikhan, G and Kang, HA and Lee, B and Kim, YO},
title = {Generation and characterization of SOX17-specific EGFP expressing human induced pluripotent stem cell line, KSCBi017-A-4, using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103943},
doi = {10.1016/j.scr.2026.103943},
pmid = {41747626},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *SOXF Transcription Factors/metabolism/genetics ; *Green Fluorescent Proteins/metabolism/genetics ; Cell Line ; Cell Differentiation ; Endoderm/cytology/metabolism ; },
abstract = {We generated a human induced pluripotent stem cell (hiPSC) reporter line in which EGFP was inserted in-frame at the C-terminus of the endogenous SOX17 locus using CRISPR/Cas9-mediated homologous recombination. The targeted clone, KSCBi017-A-4, was isolated by puromycin selection and validated by PCR and Sanger sequencing. This SOX17-EGFP hiPSC line retains a normal karyotype and pluripotency and displays specific EGFP expression upon directed definitive endoderm differentiation. This reporter line provides a reliable tool for monitoring SOX17 expression during human endoderm specification.},
}
@article {pmid41747766,
year = {2026},
author = {Cai, Y and Zhuang, L and Wang, Z and He, L and Li, X and Liu, BF and Li, T and Zhang, G and Zhou, H and Huang, X and Li, Y},
title = {Gravity-Driven Formation of Water-in-Wax Spheres for Efficient One-Pot CRISPR Diagnostics.},
journal = {ACS nano},
volume = {20},
number = {9},
pages = {8055-8067},
doi = {10.1021/acsnano.6c01232},
pmid = {41747766},
issn = {1936-086X},
mesh = {*Gravitation ; *Water/chemistry ; Humans ; *Waxes/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microspheres ; CRISPR-Cas Systems ; Human papillomavirus 16/genetics/isolation &amp; purification ; },
abstract = {Rapid, decentralized molecular diagnostics are urgently needed for effective infectious disease control. Here, we present "Wax-Sphere CRISPR" (WS-CRISPR), a paradigm-shifting platform for CRISPR-based diagnostics centered on a gravity-driven, interfacial phase-change self-encapsulation mechanism. This system fundamentally decouples bioreagent engineering from specific reaction vessels, transforming conventional, labor-intensive manual encapsulation into a standardized, physics-driven assembly process that generates discrete wax microspheres. Guided by fluid mechanics and interfacial thermodynamics, aqueous CRISPR droplets spontaneously traverse air/molten wax/ethanol to self-encapsulate and solidify, enabling standardized, high-throughput fabrication without manual wax handling. Upon temperature modulation, the wax phase change triggers sequential recombinase polymerase amplification (RPA) and CRISPR detection within a sealed, one-pot vessel. As a clinically oriented demonstration, WS-CRISPR enables multiplexed detection and risk stratification of all 14 high-risk HPV genotypes (HPV16/18 vs others) with a detection limit of 1 × 10[-18] M in under 30 min. Integrated with widely accessible devices─including a thermocycler, hand-held fluorescence reader, and microfluidic platform─the system demonstrated 97.4% sensitivity and 100% specificity across 70 clinical samples. By solving the engineering bottlenecks of scalability and universality, WS-CRISPR offers a robust tool for decentralized, large-scale pathogen surveillance.},
}
@article {pmid41748831,
year = {2026},
author = {Moreno, DS and Carvalho, JP and Murray, E and Colombo, NSR and Lamas, A and Cobas, AC and Hill, C and Azeredo, J and Domingues, L},
title = {Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41748831},
issn = {1432-0614},
mesh = {*Listeria monocytogenes/drug effects ; *Probiotics ; *CRISPR-Cas Systems ; *Endopeptidases/genetics/pharmacology/metabolism ; *Saccharomyces boulardii/genetics/metabolism ; Humans ; Bacteriophages/enzymology ; *Anti-Bacterial Agents/pharmacology/metabolism ; Saccharomyces cerevisiae/genetics ; },
abstract = {Listeriosis is a foodborne infection caused by Listeria monocytogenes that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, Listeria is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of L. monocytogenes, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. Saccharomyces boulardii is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered Saccharomyces cerevisiae through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against L. monocytogenes, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast S. boulardii and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce L. monocytogenes levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for Listeria. Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic S. boulardii. KEY POINTS: CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes. Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI). Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome.},
}
@article {pmid41750315,
year = {2026},
author = {Schulze, A and Kainz, K and Bauer, MA and Carmona-Gutierrez, D},
title = {Editing Candida: Origins and Advances of CRISPR Tools.},
journal = {Biomolecules},
volume = {16},
number = {2},
pages = {},
pmid = {41750315},
issn = {2218-273X},
support = {10.55776/P37278//FWF Austrian Science Fund/ ; not applicable//University of Graz/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Candida/genetics/pathogenicity ; Humans ; Virulence/genetics ; Genome, Fungal ; Candidiasis/microbiology/genetics ; },
abstract = {Pathogens causing candidiasis encompass a diverse group of ascomycetous yeasts that have become essential models for studying fungal adaptability, pathogenicity, and host-pathogen interactions. Although many candidiasis-promoting species exist as commensals within host microbiota, several have acquired virulence traits that enable opportunistic infections, positioning them as a leading cause of invasive fungal disease in humans. Deciphering the molecular and genetic determinants that underpin the biology of organisms responsible for candidiasis has long been a central objective in medical and molecular mycology. However, research progress has been constrained by intrinsic biological challenges, including noncanonical codon usage and the absence of a complete sexual cycle in diploid species, which have complicated traditional genetic manipulation. CRISPR-Cas9 genome editing has overcome many of these limitations, providing a precise, efficient, and versatile framework for targeted genomic modification. This system has facilitated functional genomic studies ranging from single-gene deletions to high-throughput mutagenesis, yielding new insights into the mechanisms governing virulence, antifungal resistance, and stress adaptation. Since its initial application in Candida albicans, CRISPR-Cas9 technology has been refined and adapted for other clinically and industrially relevant species, including Nakaseomyces glabratus (formerly referred to as Candida glabrata), Candida parapsilosis, and Candida auris. The present work provides an overview of the evolution of genetic approaches employed in research directed against candidiasis-associated species, with a particular focus on the development and optimization of CRISPR-based systems. It highlights how recent advancements have improved the genetic tractability of these pathogens and outlines emerging opportunities for both fundamental and applied studies in fungal biology.},
}
@article {pmid41751014,
year = {2026},
author = {Wang, Q and Zheng, L and You, G and Dong, H and Chen, S and Wang, S and Chen, S},
title = {Navigating the Complexity: Advancing Diagnostic Strategies for Avian Reovirus in Chinese Poultry.},
journal = {Animals : an open access journal from MDPI},
volume = {16},
number = {4},
pages = {},
pmid = {41751014},
issn = {2076-2615},
support = {XTCXGC2021018, XTCXGC2021012//the '5511' Collaborative Innovation Project of Fujian Academy of Agricultural Sciences, China/ ; },
abstract = {Avian reovirus (ARV) infections pose a significant and evolving threat to China's poultry industry, the world's largest. Diverse farming systems-ranging from modern intensive operations to traditional waterfowl-poultry polyculture-foster a unique ecological niche for ARV, defined by complex serotypic and genotypic diversity, marked regional variations, potential interspecies transmission between chickens and waterfowl, and recurrent co-infections. Collectively, these factors undermine the efficacy of conventional diagnostic approaches. This review systematically outlines the current epidemic landscape of ARV in China, highlighting the molecular characteristics of prevailing strains (particularly those from waterfowl) and their roles in diagnostic evasion. We critically assess the performance and limitations of existing diagnostic techniques (virus isolation, ELISA, PCR/qPCR) within the Chinese epidemiological setting. Furthermore, we discuss innovative technologies-including multiplex qPCR, CRISPR-Cas systems, and next-generation sequencing (NGS)-that offer potential for developing next-generation diagnostics tailored to China's specific challenges. Finally, we propose future directions, with an emphasis on standardization, data sharing, and interdisciplinary collaboration to bridge the gap between cutting-edge innovation and on-farm application for precise ARV control.},
}
@article {pmid41751536,
year = {2026},
author = {Hawkins, V and Rudiger, SR and McLaughlan, CJ and Kelly, JM and Lehnert, K and Jacobsen, JC and Handley, RR and Henare, K and Verma, PJ and Snell, RG},
title = {Foundations of an Ovine Model of Fragile X Syndrome.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751536},
issn = {2073-4425},
support = {3914//Curekids/ ; 20/259//Health Research Council of New Zealand/ ; },
mesh = {Animals ; *Fragile X Syndrome/genetics/pathology ; *Disease Models, Animal ; *Fragile X Messenger Ribonucleoprotein 1/genetics ; Sheep/genetics ; Male ; Female ; CRISPR-Cas Systems ; Gene Editing ; Gene Knockout Techniques ; Humans ; },
abstract = {BACKGROUND: Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder characterised by intellectual disability, developmental delays, anxiety, and social and behavioural challenges. Currently, no effective treatments exist to address the root cause of FXS. Mouse models are the most widely used for studying molecular pathogenesis and conducting preclinical treatment testing. However, therapeutic interventions that show promise in rodent models have yet to succeed in clinical trials. After evaluating the current models, we have developed an ovine model to address this clinical translation gap. We expect this model to more accurately reflect the human condition in brain size, structure, and neurodevelopmental trajectory. We aim to establish this model as a valuable preclinical platform for testing therapies for FXS.<br><br>METHODS: To generate the sheep model, we used CRISPR-Cas9 dual-guide editing to knock out the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene in ovine embryos.<br><br>RESULTS: Two founder animals were created, one ram (male) and one ewe (female), both of which carried FMR1 gene knockouts. The ewe carries inactivating mutations on both alleles, with the edits in both animals resulting in no detectable Fragile X Messenger Ribonucleoprotein (FMRP) as expected. Both founders have undergone molecular characterisation and basic health checks, with the female founder showing increased joint flexibility, a characteristic of FXS. The ram has been used for breeding, with the successful transmission of the edited allele to his offspring. Importantly, specific lamb cohorts for postnatal treatment testing can be produced efficiently utilising accelerated breeding methods and preimplantation selection.},
}
@article {pmid41751548,
year = {2026},
author = {Machel Gica, NG and Gica, WT and La, H and Mi, Y and Zhou, Y},
title = {Precision Breeding for a Global Staple Food: A Systematic Review with a Strategic Framework for CRISPR-Cas Applications in Rice (Oryza sativa L.).},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751548},
issn = {2073-4425},
support = {2025GBJ002388//Chinese Government Scholarship Council/ ; },
mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Plant Breeding/methods ; Plants, Genetically Modified/genetics ; Genome, Plant ; Crops, Agricultural/genetics ; },
abstract = {Background: Rice is one of the world's main staple crops, and improving its productivity and resilience is important to achieving food security under varying climatic conditions. Objectives: This systematic review synthesizes the existing evidence on the application, technical limitations, and potential of the development of genome editing technologies (CRISPR-Cas) in rice (Oryza sativa L.), as well as presents a novel approach called the CRISPR Trait Prioritization and Readiness Framework (CTPRF). Methods: Peer-reviewed articles that reported applications of genome editing based on the CRISPR-Cas system in the genome of rice for trait improvement or functional genomics were identified through searches fromPubMed, Scopus, Web of Science, and Google Scholar with studies published between 2012 and 2025. Studies were screened on predefined inclusion criteria related to experimental validation, reporting of editing efficiency, and clear phenotypic results. Data on CRISPR systems, target genes, methods of delivery, traits modified, and phenotypic results were extracted and synthesized by comparative analysis. Results: A wide variety of different CRISPR systems have been used in rice, and our results indicate that NHEJ-mediated knockouts are effective in average genotypes with editing efficiencies in the range of 70-90%, but HDR and prime editing are still under 10%. The CTPRF is being introduced as a strategic decision support tool to evaluate traits from four dimensions: technical feasibility, phenotypic predictability, impact potential, and regulatory pathway. We use this framework for case studies in pioneering countries (USA, Japan, China) and show how it can be useful for guiding research investment and policy. Conclusions: CRISPR-Cas technologies have transformed rice breeding, but their introduction requires overcoming genotype-dependent barriers to transformation and negotiating patchwork regulatory environments. The CTPRF offers a roadmap for the acceleration of the development of climate-resilient and nutritious rice varieties for the action plan.},
}
@article {pmid41751560,
year = {2026},
author = {Lee, S and Park, S and Bang, H and Kim, SU and Park, YH and Wee, G and Chae, U and Kim, E},
title = {VPS35 Deficiency Markedly Reduces the Proliferation of HEK293 Cells.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751560},
issn = {2073-4425},
mesh = {Humans ; *Cell Proliferation/genetics ; *Vesicular Transport Proteins/genetics/deficiency/metabolism ; HEK293 Cells ; Apoptosis/genetics ; CRISPR-Cas Systems ; Mitochondrial Dynamics/genetics ; Mitochondria/metabolism/genetics ; Gene Knockout Techniques ; },
abstract = {Background/Objectives: The retromer protein complex is involved in various physiological processes, especially endosomal trafficking, and its dysregulation has been linked to Alzheimer's disease and Parkinson's disease, as well as VPS35 knockout (KO), causing early embryonic lethality. We aimed to investigate the cellular consequences of VPS35 deficiency. Methods: To investigate the effects of VPS35 loss, we used CRISPR/Cas9 to generate VPS35 KO human embryonic kidney 293 (HEK293) cells. We analyzed changes in retromer component expression, cell proliferation, apoptosis, and mitochondrial dynamics using Western blotting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and confocal microscopy. Results: VPS35 KO led to a significant reduction in cell proliferation and decreased expression of VPS29 and VPS26, both essential for retromer complex assembly. Consequently, retromer formation was impaired. Compared to control cells, KO cells exhibited elevated levels of cleaved caspase-3, poly(ADP-ribose) polymerase, cytochrome C, and p21, while the expression of Ki-67, CDK4, and cyclin D was reduced. Additionally, VPS35 deletion also promoted mitochondrial fragmentation, associated with increased expression of mitochondrial fission-related proteins. Finally, the rescue experiment using the human VPS35 gene confirmed that the recovery of VPS35 not only led to the recovery of the essential elements constituting the retromer but also the recovery of molecules related to the cell cycle, restoring cell death to a normal level. Conclusions: These findings suggest that VPS35 plays a critical role in cell growth and survival by modulating apoptosis, mitochondrial dynamics, and cell cycle progression.},
}
@article {pmid41751614,
year = {2026},
author = {Sun, Q and Guo, Y and Wang, L and Jia, L and Wei, P and Ma, S},
title = {CRISPR-Mediated Silkworm: The Oncoming Agricultural Revolutions and a Rising Model Organism.},
journal = {Genes},
volume = {17},
number = {2},
pages = {},
pmid = {41751614},
issn = {2073-4425},
support = {32570591//National Natural Science Foundation of China/ ; },
mesh = {*Bombyx/genetics/growth &amp; development ; Animals ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Agriculture/methods ; Silk/genetics ; Genome, Insect ; Genomics/methods ; },
abstract = {The silkworm (Bombyx mori) is essential to sericulture and is also becoming a key model organism in genomics and agriculture. For decades, genetic studies of the silkworm were limited by inefficient and inflexible genome tools. CRISPR genome editing allows precise and scalable alterations to genes regulating development, physiology, and industrial traits. This review summarizes silkworm genome-editing breakthroughs, highlighting CRISPR's evolution from simple gene knockouts to large-scale genome-wide screening. We highlight how these advancements contribute to disease resistance, higher yields, and the development of new silk-based materials, as well as how they influence the development and growth rate of the sericulture. The creation of high-quality reference genomes, pangenomes, and genome-wide screening systems has made the silkworm a major model for integrating multiple biological datasets and approaches, such as genomic, transcriptomic, and proteomic. By considering the unique biological characteristics of the silkworm, this provides new insights for research on silk biology, piRNA synthetic biology, and hormonal signaling regulation. Finally, we examine new areas at the intersection of CRISPR, pangenomics, and artificial intelligence (AI) and suggest future paths for molecular breeding, pest control, and synthetic biology. Moreover, AI-assisted prediction of CRISPR outcomes is utilized to inform the design of targeted trait modifications, representing an approach to enhancing biomanufacturing efficiency and eco-friendly silk production. Together, these advances have made the silkworm a flexible genetic platform and an important part of sustainable agriculture and biomanufacturing.},
}
@article {pmid41751799,
year = {2026},
author = {Cai, X and Liang, X and Zou, P and Xiao, R and Wang, Y},
title = {CRISPRi Screening Identifies Essential E. coli Virulence Factors for Placental Barrier Breach in a Maternal-Fetal Infection Model.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751799},
issn = {1422-0067},
support = {82574169//National Natural Science Foundation of China/ ; 7232009, 7244289//Natural Science Foundation of Beijing Municipality/ ; 20240484724//Cross-cooperation project of Beijing Science and Technology New Star Program/ ; Subject leaders-03-02//High Level Public Health Technical Personnel Construction Project/ ; //Research Foundation of Capital Institute of Pediatrics/ ; //Beijing Chaoyang District Postdoctoral Research Foundation 2024/ ; },
mesh = {Female ; Pregnancy ; Animals ; *Virulence Factors/genetics/metabolism ; *Escherichia coli Infections/microbiology/genetics ; *Escherichia coli/pathogenicity/genetics ; Rats ; *Placenta/microbiology/metabolism ; Humans ; Disease Models, Animal ; Escherichia coli Proteins/genetics/metabolism ; Host-Pathogen Interactions ; Trophoblasts/microbiology/metabolism ; CRISPR-Cas Systems ; *Pregnancy Complications, Infectious/microbiology ; Neonatal Sepsis/microbiology ; },
abstract = {Early-onset neonatal sepsis caused by Escherichia coli (E. coli) threatens neonates' lives due to the pathogen's high virulence and multidrug resistance. The mechanisms that enable its placental barrier breach are poorly understood. Using a clinically isolated ST95 ExPEC strain from a neonatal sepsis case, along with a pregnant rat model and an in vitro placental barrier model, we performed CRISPR interference screening. This screen targeted 264 virulence factor genes and identified virulence factors for motility, iron acquisition, hemolysin secretion, and adherence/invasion as critical. We demonstrated that hlyB is essential for uterine infection, and we elucidated a mechanism for ibeA that facilitates syncytial trophoblast cell layer penetration by interacting with the host receptor(s) PSF/VIM to enhance bacterial internalization. Host cells countered ibeA+ E. coli infection via a novel host defense pathway involving upregulation of ASPHD1. This study systematically mapped the virulence factors required for E. coli placental translocation and delineated key host-pathogen interactions.},
}
@article {pmid41751840,
year = {2026},
author = {Begum, SN and Hasan, SK},
title = {Prime Editing Driven Functional Genomics: Bridging Genotype to Phenotype in the Post-Genomic Era.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751840},
issn = {1422-0067},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Genomics/methods ; Phenotype ; Genotype ; Animals ; },
abstract = {The post-genomic era, defined by large-scale sequencing initiatives, has generated an unprecedented catalogue of human genetic variation. Yet, the vast majority of genetic variants remain classified as variants of uncertain significance or are located within poorly characterized non-coding regions, thereby hindering the effective translation of genomic data into meaningful biological understanding and clinical application. Bridging this genotype-to-phenotype gap requires precise, high-throughput functional genomics. Early CRISPR-Cas9 knockout and CRISPR interference/activation (CRISPRi/a) screens mapped gene-level functions but could not assess single nucleotide variants (SNVs). Bridging this genotype-to-phenotype gap demands precise, high-throughput functional genomics. Multiplexed assays of variant effect (MAVEs), like saturation genome editing, systematically test all possible mutations using CRISPR-Cas9 and donor libraries. Base editors allow targeted single-base changes without double-strand breaks but are limited in scope, while prime editing can introduce any small substitution, insertion, or deletion without double-strand breaks (DSBs) or donor templates. This review traces the evolution of functional screens from gene-level knockouts to saturation genomic editing (SGE), and highlights how prime editing is driving a new paradigm for the systematic functional characterization of thousands of variants across disease-relevant genes. We also detail the architecture, mechanism, and progressive optimization of PE systems and their delivery methods. Collectively, prime editing stands as a transformative platform poised to accelerate precision functional genomics and advance the diagnosis and treatment of genetic diseases.},
}
@article {pmid41751979,
year = {2026},
author = {Eskildsen, J and Dong, M and Hanak, T and Madsen, CK and Holme, I and Plaszkó, T and Vestergård, M and Nicolaisen, M and Thordal-Christensen, H and Brinch-Pedersen, H},
title = {Novel CRISPR/Cas9-Derived mlo Alleles in Barley: Resistance to Powdery Mildew and Microbiome Implications.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41751979},
issn = {1422-0067},
support = {NNF19OC0056580//Novo Nordisk Foundation/ ; BarleyMicroBreed, 101060057//EU Horizon research and innovation/ ; },
mesh = {*CRISPR-Cas Systems ; *Hordeum/genetics/microbiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Ascomycota/pathogenicity ; *Microbiota/genetics ; Alleles ; Plant Roots/microbiology/genetics ; *Plant Proteins/genetics ; Mutation ; },
abstract = {Barley grown in temperate regions is often challenged by powdery mildew disease. An effective solution is mildew resistance locus o (mlo)-based resistance, which is monogenic, durable, and broad-spectrum. While the pleiotropic effects of mlo mutations on above-ground tissues are well documented, their impact on the root-associated microbiome remains underexplored. We utilized CRISPR/Cas9 to generate novel mlo mutant lines and evaluated their resistance to causal fungus Blumeria hordei. We further examined if mlo knockout has any impact on the overall root microbiome diversity and composition under field-like conditions and applied DESeq2 to compare the abundance of microbial taxa between mutants and wild type. We created five novel resistant mlo lines, including the first mutants with amino acid alterations in the protein's extracellular region. Mutant lines showed significantly reduced B. hordei colony formation (0.5-5%). While microbial alpha and beta diversity were not significantly altered, a few microbial taxa displayed time-dependent shifts in abundance. Overall, our study demonstrates the effectiveness of CRISPR/Cas9 in generating mlo-based resistance. Moreover, the study revealed functionally important residues in the protein's extracellular region. Finally, we present the first evidence of limited mlo-associated effects on root microbiome diversity and relative abundance of microbial taxa.},
}
@article {pmid41752145,
year = {2026},
author = {Fayed, S and Amer, S and Badawy, M and Bou Malhab, L and Omran, N and Khoder, G and Ghemrawi, R and Haider, M and Hamoudi, R and Harati, R},
title = {The Role of CRISPR and Its Therapeutic Applications in Glioblastoma.},
journal = {International journal of molecular sciences},
volume = {27},
number = {4},
pages = {},
pmid = {41752145},
issn = {1422-0067},
support = {210111350//University of Sharjah/ ; 2201110368//University of Sharjah/ ; 23010902146//University of Sharjah/ ; VRI-20-10//ASPIRE Precision Medicine Research Institute Abu Dhabi/ ; },
mesh = {Humans ; *Glioblastoma/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Brain Neoplasms/therapy/genetics ; Animals ; *Genetic Therapy/methods ; },
abstract = {Glioblastoma multiforme (GBM) remains the most aggressive and treatment-refractory form of primary brain tumor in adults, characterized by rapid proliferation, intratumoral heterogeneity and resistance to current therapies. Despite therapeutic advancements in surgical resection, radiotherapy and chemotherapy, clinical outcomes remain poor, underscoring the need for innovative molecular strategies. This review examines the therapeutic potential of CRISPR/Cas9 genome-editing technologies in GBM, highlighting their ability to model, dissect and potentially correct the genetic alterations that drive GBM tumorigenesis. Key molecular targets, such as EGFR, PTEN, TP53, NF1 and PIK3CA, are discussed within the context of GBM's mutational and signaling landscape. We further outline emerging CRISPR applications in preclinical models, the current status of CRISPR-based clinical trials and the major barriers hindering translation, including off-target effects, immunogenicity and the challenge of delivering gene-editing systems across the blood-brain barrier. Particular emphasis is placed on delivery technologies, viral and non-viral vectors, including lipid nanoparticles, polymeric systems, inorganic nanocarriers and DNA nanostructures, which are rapidly evolving to improve precision, safety and CNS penetrance. Collectively, this review highlights CRISPR/Cas9 as a powerful tool whose integration with molecular neuro-oncology and precision medicine may ultimately shift GBM treatment toward more personalized and durable therapeutic interventions.},
}
@article {pmid41752921,
year = {2026},
author = {Panov, J and Elbert, A and Rosenthal, DS and Levi, M and Chumakov, K and Andino, R and Brodsky, L and Kaphzan, H},
title = {Spacio-Linear Screening for Ligand-Docking Cavities in Protein Structures: SLAM Algorithm.},
journal = {Life (Basel, Switzerland)},
volume = {16},
number = {2},
pages = {},
pmid = {41752921},
issn = {2075-1729},
support = {N/A//Tauber Foundation/ ; },
abstract = {Identifying structurally similar ligand-binding sites in unrelated proteins can facilitate drug repurposing, reveal off-target effects, and deepen our understanding of protein function. A number of tools were developed for structural screening, but many of them suffer from limited sensitivity and scalability. Using a data bank of crystallized protein structures, we aimed to discover novel protein targets for a ligand by leveraging a known ligand-binding query protein with a resolved structure. Here, we present SLAM (Spacio-Linear Alignment of Macromolecules), a novel alignment-based algorithm that detects local 3D similarities between ligand-binding cavities or protein-exposed surfaces of query and target proteins. SLAM encodes spatial substructure neighborhoods into short linear sequences of physicochemically annotated atoms, then applies pairwise sequence alignment combined with distance-correlation scoring to identify high-fidelity structural matches. Benchmarking using the Kahraman-36 dataset demonstrated that SLAM outperforms the state-of-the-art ProBiS algorithm in true-positive rate for predicting ligand-docking compatibility. Furthermore, SLAM identifies candidate ligands that may inhibit functionally critical domains of CRISPR-Cas proteins and predicts novel binding partners of toxic per- and polyfluoroalkyl Substance (PFAS) compounds (PFOA, PFOS) with plausible mechanistic links to toxicity. In conclusion, SLAM is a robust computationally efficient and flexible structural screening tool capable of detecting subtle physicochemical compatibilities between protein surfaces, promising to accelerate target discovery in pharmacology and elucidate protein-ligand interactions in environmental toxicology.},
}
@article {pmid41753780,
year = {2026},
author = {Palanisamy, V and Bosilevac, JM and Barkhouse, DA and Velez, SE and Dass, SC},
title = {Unraveling the Coevolutionary Dynamics of Phage and Bacterial Protein Warfare Occurring in the Drains of Beef-Processing Plants.},
journal = {Microorganisms},
volume = {14},
number = {2},
pages = {},
pmid = {41753780},
issn = {2076-2607},
support = {2020-67017-30776//USDA-NIFA/ ; },
abstract = {Phages, the most abundant entities on Earth, exhibit a complex interplay with bacteria, especially within environmental biofilms, resulting in an ecological arms race. This study investigates the interaction between phages and bacteria in the drains of beef-processing plants using high-throughput sequencing and metagenomic analysis. Metagenomic data collected from 75 drain samples from beef-processing plants were analyzed to investigate phage-bacterial interactions. First, assembled contigs were screened to identify viral sequences, which were then taxonomically annotated to determine the viral composition, including phages. Functional annotation of these viral sequences provided information about the viral genes and their roles in bacterial interactions specifically associated with attack and counterattack of bacteria. In parallel, bacterial contigs were examined to identify genes associated with antiphage defense systems, providing insights into the strategies adapted by bacteria to resist phage infection. Taxonomic annotation of viral sequences from the bulk metagenomic data revealed the presence of phages targeting Pseudomonas, Klebsiella, and Enterococcus. The higher abundance of Pseudomonas phages aligns with our previous study, where Pseudomonas was identified as the dominant bacterial genus, suggesting potential copersistence of phages and their hosts. Functional annotation of phage contigs revealed infective and lysis-related genes, highlighting their potential role in bacterial attack. Conversely, bacterial contigs encoded antiphage defense systems, including CRISPR-Cas, restriction-modification, and other defense-related genes. The study also uncovered the presence of anti-CRISPR proteins in phages, suggesting a counterattack on the bacterial defense. These findings provide evidence for phage attack, bacterial defense, and phage counterattack and may showcase the ongoing coevolutionary arms race between phages and bacteria. While this evidence looks promising, these results remain preliminary and further studies are needed to validate these findings. Still, this study provides a foundational understanding of bacteria-phage coexistence in beef-processing plant drains and paves the way for further explorations of these intricate interactions and their possible applications in controlling pathogenic microorganisms within biofilms.},
}
@article {pmid41754535,
year = {2026},
author = {Li, H and Wang, R and Li, J and Duan, W and Liang, Y and Sun, Q and Zhou, J and Zhang, Y},
title = {SHFL Post-Transcriptionally Restricts Coxsackievirus A16 In Vitro and In Vivo.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754535},
issn = {1999-4915},
support = {GJJKJ-2024-ZY//National Disease Control and Prevention Administration Public Health Talent Training Support Project/ ; ZDGWNLJS25-36//National Key Laboratory of Intelligent Tracking and Forecasting for Infectious Diseases (NITFID)/ ; 2021YFC2302003//National Key Research and Development Program of China/ ; },
mesh = {Animals ; Mice ; Virus Replication ; *Coxsackievirus Infections/virology ; *Enterovirus A, Human/genetics/physiology ; Humans ; *Enterovirus/genetics/physiology ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Hand, Foot and Mouth Disease/virology ; Cell Line, Tumor ; Viral Load ; Interferon-beta/pharmacology ; },
abstract = {Coxsackievirus A16 (CVA16), a major etiological agent of hand, foot, and mouth disease, is increasingly contributing to neurological complications, with no vaccines or virus-specific antivirals currently available. To identify CVA16-restricting host factors, we investigated the role of the interferon-stimulated gene shiftless (SHFL), previously implicated in the control of other RNA viruses. Using CRISPR-Cas 9, we generated SHFL knockout rhabdomyosarcoma cells and assessed viral replication, cytopathic effects, and replication stage dynamics. We evaluated disease progression and tissue injury in neonatal mice infected with a mouse-adapted CVA16 strain. SHFL expression was strongly induced during CVA16 infection and was inducible by exogenous interferon-β treatment, and its loss markedly increased infectious virus production, accelerated early replication, and exerted severe cytopathic effects. In vivo, SHFL deficiency led to rapid weight loss, pronounced neurological signs, increased viral burden across multiple tissues, and uniform mortality, together with high viral loads and extensive pathological damage in the central nervous system, lungs, and skeletal muscle. Transcriptomic analyses revealed SHFL-dependent modulation of adhesion- and mitogen-activated protein kinase-related pathways. Overall, our results suggest SHFL as a key determinant of host resistance to CVA16, acting mainly at the post-transcriptional stage to limit viral spread and tissue injury, and highlight SHFL-linked pathways as promising host-directed antiviral targets.},
}
@article {pmid41754561,
year = {2026},
author = {Wupori, K and Garnett, L and Bello, A and Strong, JE},
title = {CRISPR-Based Detection of Viral Hemorrhagic Fevers at the Point of Care.},
journal = {Viruses},
volume = {18},
number = {2},
pages = {},
pmid = {41754561},
issn = {1999-4915},
support = {8//Genomics Research and Development Initiative/ ; CSSP-2022-CP-2546//Canadian Safety and Security Program/ ; },
mesh = {Humans ; *Hemorrhagic Fevers, Viral/diagnosis/virology ; *Point-of-Care Systems ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Point-of-Care Testing ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Viral hemorrhagic fevers (VHFs) are highly lethal diseases that often present non-specific, influenza-like symptoms in their early stages, making clinical recognition and differentiation from other febrile illnesses difficult. This overlap underscores the critical need for diagnostic tests that are both sensitive and specific. Point-of-care (POC) diagnostic tests are an invaluable tool for detecting and controlling the spread of pathogens that threaten public health, such as VHFs, as these require fast, accurate diagnostics to ensure biosafety and appropriate mobilization of resources during outbreaks. Current molecular and serological diagnostic tests, while efficient and effective, lack the characteristics required of a POC test (POCT) to quickly and easily respond to a VHF outbreak while maintaining a low cost. Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tests have gained popularity as POCTs due to their inherent attractive qualities, including high sensitivity and specificity, adaptability, low cost, quick turnaround time, and ease of use. However, studies on the development of CRISPR-based POC diagnostic tests for VHFs are limited. This review summarizes the current CRISPR-based POCTs for VHFs, including Ebola virus (EBOV), Lassa virus (LASV), Dengue virus (DENV), and Crimean-Congo hemorrhagic fever virus (CCHF). The isothermal pre-amplification methods commonly paired with CRISPR-based tests, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), are also discussed.},
}
@article {pmid41755633,
year = {2026},
author = {Aguilar-González, A and Martos-Jamai, I and Ramos-Hernández, I and Molina-Estévez, FJ and Villao, NV and Puig-Serra, P and Rodríguez-Perales, S and Torres, R and Labun, K and Sánchez-Martín, RM and Díaz-Mochón, JJ and Martín, F},
title = {A novel Dual-guide CRISPR-Cas13 strategy improves specificity for single-nucleotide variant detection.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755633},
issn = {1362-4962},
support = {MCIN)/AEI/10.13039/501100011033//Spanish Ministry of Science and Innovation/ ; PID2022-141065OB-I00//European Union Next Generation/ ; CV20-77741//Consejer&#xed;a de Econom&#xed;a y Conocimiento/Project/ ; PI21/00298//Instituto de Salud Carlos III/ ; PI24/00888//Instituto de Salud Carlos III/ ; RD21/0017/0004//Instituto de Salud Carlos III/ ; RD24/0014/0005//Instituto de Salud Carlos III/ ; PI-0236-2024//Consejer&#xed;a de Salud y Familias/ ; PIP-0004-2025//Consejer&#xed;a de Salud y Familias/ ; GeneHumdi-CA21113//European Cooperation in Science and Technology/ ; FPU22/03455//Spanish Ministry of Science, Innovation and Universities/ ; RHJ-0053-2025//Consejer&#xed;a de Salud y Familias, Junta de Andaluc&#xed;a/ ; //European Social Fund/ ; //Universidad de Granada/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *SARS-CoV-2/genetics/isolation &amp; purification ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *COVID-19/diagnosis/virology/genetics ; RNA, Viral/genetics ; *Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins p21(ras)/genetics ; Sensitivity and Specificity ; Leishmania/genetics ; CRISPR-Associated Proteins/genetics ; },
abstract = {The emergence of CRISPR-Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13's heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first Dual-guide RNA system for Cas13 that improves mismatch discrimination and enhances target specificity. This system employs two distinct RNAs-dcrRNA and dtracrRNA-which cooperatively recognize the target and reduce off-target activity. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, distinguished KRAS G12D and G12C mutations, and differentiated mucocutaneous from cutaneous Leishmania sequences in analytical assays, with clinical validation confirming accurate detection of positive and negative samples. These results highlight the Dual-guide Cas13 platform's potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a substantial advance over conventional Cas13 systems, offering improved specificity while maintaining clinically relevant sensitivity, and provides a generalizable tool for next-generation molecular diagnostics and precision RNA targeting and regulation.},
}
@article {pmid41755634,
year = {2026},
author = {Hu, Z and Liu, Y and Han, Y and Li, M and Deng, K and Lu, X and Huang, Y and Liang, C and Wang, Y and Fu, Y and Xu, A},
title = {CRISPR/Cas9 screening with destabilized bicistronic fluorescent protein reporter revealed PABPN1 as a hub of regulators for alternative polyadenylation.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41755634},
issn = {1362-4962},
support = {2022YFA1103900//National Key Research and Development Program of China/ ; 32470586//National Natural Science Foundation of China/ ; 91942301//National Natural Science Foundation of China/ ; 81430099//National Natural Science Foundation of China/ ; 31930084//National Natural Science Foundation of China/ ; 32500472//National Natural Science Foundation of China/ ; 2023B1212060028//Guangdong Science and Technology Department/ ; 2022YFA1103900//National Key Research and Development Program of China/ ; },
mesh = {*Polyadenylation/genetics ; Humans ; *CRISPR-Cas Systems/genetics ; Genes, Reporter ; *Poly(A)-Binding Protein I/genetics/metabolism ; HEK293 Cells ; DEAD-box RNA Helicases/metabolism/genetics ; *Luminescent Proteins/genetics/metabolism ; Cell Proliferation/genetics ; Polypyrimidine Tract-Binding Protein/metabolism/genetics ; RNA-Binding Proteins/metabolism/genetics ; },
abstract = {Alternative polyadenylation (APA) is intricately intertwined with diverse biological processes. Efficient approaches for screening the regulatory factors of specific APA events are essential to elucidate their regulation mechanisms. Here, we first engineered a destabilized bicistronic fluorescent protein reporter (dBFPR) to enhance the sensitivity of APA detection. Then, we developed a robust high-throughput screening platform for APA regulators by integrating CRISPR/Cas9, dBFPR, and fluorescence-activated cell sorting. With this method, we successfully screened the library of RNA binding proteins and found that PTBP1, ELAVL1, and DDX3X play significant roles in regulating APA and promoting cell proliferation through interaction with PABPN1, suggesting that PABPN1 is an important hub for APA regulation.},
}
@article {pmid41755886,
year = {2026},
author = {Kaniganti, S and Saini, H and Chaitanya, AK and Hegde, N and Shah, P and Magar, ND and Rijal, R and Kaushik, JJ and Nanda, D and Sachan, S and Kumar, A and Bhoite, R and Jamedar, HR},
title = {CRISPR/Cas Genome Editing and Its Applications in Cereal Crop Improvement.},
journal = {Plant-environment interactions (Hoboken, N.J.)},
volume = {7},
number = {2},
pages = {e70133},
pmid = {41755886},
issn = {2575-6265},
abstract = {CRISPR/Cas-based genome editing has emerged as a transformative tool for precise genetic improvement of cereal crops. Recent advances in CRISPR technologies, including Cas9, Cas12, Cas13, base editing, and prime editing, have enabled targeted modification of genes and regulatory elements controlling yield, stress tolerance, and grain nutritional quality in major cereals such as rice, wheat, maize, and barley. This review summarizes current progress in CRISPR-mediated genome editing systems, delivery strategies, and representative applications in cereal crop improvement. Emphasis is placed on how genome editing reprograms enzymatic activities and biological pathways underlying complex agronomic traits rather than acting through single-gene effects. The review also discusses challenges related to trait complexity, regulatory considerations, and prospects for translating genome-edited cereal crops from laboratory research to field-level application. Collectively, this review highlights the potential of CRISPR/Cas genome editing as a powerful approach for developing high-yielding, resilient, and nutritionally improved cereal crops.},
}
@article {pmid41757335,
year = {2026},
author = {Shafiq, T and Khan, N and Kausar, T and Ahmed, W and Zhang, Z and Liang, Y and Duan, L},
title = {Red Blood Cell-Derived Extracellular Vesicles for Gene and RNA Therapeutics: Biological, Engineering, and Translational Challenges.},
journal = {International journal of nanomedicine},
volume = {21},
number = {},
pages = {579975},
pmid = {41757335},
issn = {1178-2013},
mesh = {Humans ; *Extracellular Vesicles/chemistry/metabolism ; *Genetic Therapy/methods ; *Erythrocytes/cytology/chemistry/metabolism ; CRISPR-Cas Systems ; Animals ; RNA, Messenger/genetics/administration &amp; dosage/therapeutic use ; *RNA/therapeutic use ; Gene Transfer Techniques ; },
abstract = {Gene therapy has great prospects of DNA/RNA manipulations and protein modulations. Its use in clinic is, however, stifled by risks of immunogenicity, low target specificity, and adverse effects. The red blood cell (RBC-EVs) extracellular vesicles can serve as a solution to this issue since they are biocompatible, long-term stable, and with low immunogenicity. RBC-EVs permit the accurate delivery of therapeutic cargo to space and time, thus minimizing systemic toxicity. This review presents the most recent developments on the expansion of the use of RBC-EVs to encapsulate the components of mRNA and CRISPR-Cas. Through the addition of the means to address these deficiencies, including stimulus-sensitive release mechanisms (eg, pH- or light-activated systems) and tissue-selective targeting approaches, RBC-EVs can be applied to enable the precise application in genetic diseases, inflammatory diseases, and cancer. Such innovations have the potential to overcome the clinical need and enable the biological complexity of mRNA- and CRISPR-Cas-based agents to provide a powerful delivery platform. Moreover, the review also demonstrates the unprecedented benefits of red blood cell EVs, which include immune evasion, scalability, and universal loading capacity, which can establish them as the next-generation delivery vehicles. Red blood cell EVs have the potential to increase the efficacy of precision medicine by increasing its feasibility. Lastly, we note the potential and translational issues in the provision of red blood cell EV-based mRNA and CRISPR-Cas therapeutic delivery of gene therapy.},
}
@article {pmid41757451,
year = {2026},
author = {Molina-Márquez, A and Kelterborn, S and Hegemann, P and Pérez-Rodríguez, M and Vigara, J and León, R},
title = {Characterization of Phytoene Desaturase Knockout Carotenoid-Deficient Microalgal Mutants Generated by Cas9-Ribonucleoprotein Complexes.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70811},
pmid = {41757451},
issn = {1399-3054},
support = {2019-110438RB-C22//Agencia Estatal de Investigaci&#xf3;n-MCIN/AEI/10.13039/501100011033/ ; PID2022-140995OB-C21//Agencia Estatal de Investigaci&#xf3;n-MCIN/AEI/10.13039/501100011033/ ; 426566805//German Research Foundation (DFG)/ ; //Hertie Foundation/ ; },
mesh = {*Carotenoids/metabolism ; *Oxidoreductases/genetics/metabolism ; *Ribonucleoproteins/metabolism/genetics ; Gene Knockout Techniques ; *Microalgae/genetics/metabolism ; *Chlamydomonas reinhardtii/genetics/metabolism ; Mutation/genetics ; Chloroplasts/metabolism/ultrastructure ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Phytoene desaturase (PDS; EC 1.3.5.5) is a key enzyme of the carotenoid biosynthetic pathway, catalyzing the desaturation of phytoene, precursor of all carotenoids. In this study, several PDS-knockout (PDS-KO) transformants of the chlorophyte microalga Chlamydomonas reinhardtii were generated using a reverse genetics strategy. Two single guide RNAs (sgRNA) were designed to target the first exon of the PDS gene, and pre-assembled Cas9 ribonucleoprotein (RNPs) complexes were delivered into microalgal nuclei by electroporation. Multiple white PDS-KO transformants were successfully obtained by this approach, and three independent transformant lines were subsequently characterized. By integrating ultrastructural, pigment and transcriptomic analyses of dark-grown cells of several PDS-KO carotenoid-deficient mutants in comparison with the parental strain, it was demonstrated that carotenoids are indispensable components of multiple cellular architectures. Chromatographic analysis confirmed that the only carotenoid accumulated in these transformants was phytoene, which lacks the critical structural and photoprotective functions of its colored derivatives. Transmission Electron Microscopy (TEM) observations revealed profound ultrastructure alterations, including poorly developed chloroplasts and effects on other cellular structures that were either absent or severely disorganized. Consistently, clustering differentially expressed genes into functional groups revealed downregulation of pathways associated with photosynthesis, chlorophyll and carotenoid biosynthesis, ribosome biogenesis, and vesicle and membrane trafficking in the PDS-KO lines. Conversely, upregulation of regulatory and retrotransposon-inducing genes was observed. These findings underscore the central metabolic role of colored carotenoids in plant cells, highlighting their essential contribution to cellular homeostasis and photosynthetic competence.},
}
@article {pmid41757824,
year = {2026},
author = {Chen, J and Davison, CW and Ellis, J and Blevins, B and Presley, W and Myers, MT and Kong, D and Hou, Z and Mian, SI and Prasov, L and Zhang, Y},
title = {CRISPR Base Editing Correction of TGFBI Mutations in Autosomal Dominant Corneal Dystrophies.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {67},
number = {2},
pages = {60},
pmid = {41757824},
issn = {1552-5783},
support = {P30 EY007003/EY/NEI NIH HHS/United States ; R35 GM137883/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Corneal Dystrophies, Hereditary/genetics/therapy/metabolism ; *Gene Editing/methods ; *Mutation ; *Transforming Growth Factor beta/genetics/metabolism ; *Genetic Therapy/methods ; Epithelium, Corneal/metabolism/pathology ; *Extracellular Matrix Proteins/genetics ; *CRISPR-Cas Systems ; Dependovirus/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {PURPOSE: Lattice and granular corneal dystrophy comprise two common TGFBI-associated autosomal dominant corneal disorders. Existing therapies are only temporizing and carry significant morbidity. Here, we develop a novel therapeutic approach using an adenine base editor (ABE) to correct common TGFBI mutations.<br><br>METHOD: We generated two human corneal epithelial (HCE) cell models harboring a copy of the most common disease-causing TGBFI mutations, R124C or R555W. These lines were electroporated with an ABE8e-NG-encoding mRNA and guide RNAs targeting the mutations. The resulting A&#x2022;T-to-G&#x2022;C editing efficiencies and off-target (OT) effects were assessed by amplicon sequencing. GFP-expressing adeno-associated viruses (AAVs) with different capsid types were transduced into HCE cells and healthy human corneal donor tissues, and GFP fluorescence was evaluated.<br><br>RESULTS: Using all-RNA delivery for ABE8e-NG, we achieved 91% and 62% correction of the pathogenic adenines in HCE TGFBIR124C/WT and TGFBIR555W/WT cells, without editing the wild-type allele. Indel formation was negligible (<0.2%), bystander adenine editing was minimal (<0.7%), and editing at top computationally predicted OT sites was modest (<1.2% at all but 1 of the 20 OT sites analyzed), suggesting minimal safety concerns. Correction of TGFBIR124C/WT in HCEs rescued the aberrant lysosomal localization of TGFBI. We further identified AAV1 as the most effective serotype for gene delivery into both human corneal donor tissue and HCE cells.<br><br>CONCLUSIONS: Our study demonstrates the feasibility and safety of CRISPR adenine base editing as a new therapeutic strategy for correcting common TGFBI mutations in corneal dystrophies, paving the way for further preclinical testing.},
}
@article {pmid41758321,
year = {2026},
author = {Pathak, A and Singh, J and Swati, and Dwibedi, V},
title = {Deciphering microbial biofilm: mechanism, infection, and advanced approaches for control.},
journal = {Folia microbiologica},
volume = {},
number = {},
pages = {},
pmid = {41758321},
issn = {1874-9356},
abstract = {Microbial biofilms are densely organised microbial communities that adhere to biotic and abiotic surfaces, encased within an extracellular polymeric substance (EPS). Microorganisms within these biofilm structures gain enhanced protection, versatility, and resistance to external stresses, antibiotics, and host immune systems. The biofilm formation follows a series of steps, including initial microbial adherence, microcolony establishment, EPS production, regulation by quorum sensing (QS), and dispersal. This flexibility enables biofilm survival in multiple environments, such as medical devices and natural systems, posing serious challenges in healthcare, agricultural, and industrial sectors. The review focuses on the mechanisms involved in biofilm formation and discusses the role of EPS in promoting biofilm stability and resistance to antimicrobials. It addresses biofilm-associated infections in medical environments, such as chronic wounds, cystic fibrosis, urinary tract infections (UTIs), and complications with implanted medical devices. The capacity of biofilm-forming microorganisms to evade immune responses and persist through extended antibiotic use highlights the urgent demand for novel therapeutic approaches. The discussion includes emerging strategies for biofilm control, including anti-biofilm agents, QS inhibitors, enzymatic treatments, and innovative combination therapies combining antibiotics with biofilm-disrupting agents. Emerging technologies, like antimicrobial peptides (AMPs), CRISPR-Cas systems, nanotechnology, and bioelectric therapies, present innovative biofilm disruption and removal approaches. This paper discusses the effectiveness of natural products, plant-derived compounds, and bacteriophage therapies for mitigating biofilm-associated infections linked to biofilms. The review examines the dynamic challenges posed by biofilms, particularly their role in chronic and device-related infections, which contribute to significant healthcare complications. The study highlights the significance of adopting new approaches to overcome biofilm-induced antimicrobial resistance (AMR) and improve therapeutic outcomes. Furthermore, this paper discusses the promising potential of emerging technologies, such as nanomaterials, QS interference, and biofilm-specific antimicrobial agents, in enhancing biofilm control and prevention measures across clinical, industrial, and environmental domains.},
}
@article {pmid41758452,
year = {2026},
author = {Bharti S, AK and Mukherjee, AG and Gopalakrishnan, AV and Gajendran, B and Vashishth, R and Prince, SE},
title = {From bench to bedside: stem cell therapy as a transformative approach against HIV.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {},
pmid = {41758452},
issn = {1573-4978},
mesh = {Humans ; *HIV Infections/therapy/virology/immunology ; *Hematopoietic Stem Cell Transplantation/methods ; Gene Editing/methods ; HIV-1 ; *Stem Cell Transplantation/methods ; Induced Pluripotent Stem Cells ; Translational Research, Biomedical ; Receptors, CCR5/genetics ; CRISPR-Cas Systems ; Virus Latency ; },
abstract = {Human immunodeficiency virus (HIV) remains a persistent global health burden, as combination antiretroviral therapy (ART) achieves sustained viral suppression but fails to eliminate long-lived latent reservoirs. Stem cell-based therapeutic strategies have emerged as transformative approaches with the potential to induce durable remission and, ultimately, a functional cure. Clinical proof-of-concept has been established through allogeneic hematopoietic stem cell transplantation (HSCT) using CCR5Δ32/Δ32 donor cells, demonstrating that durable resistance to viral entry can result in prolonged HIV remission. Building on these landmark observations, recent advances in autologous gene-edited hematopoietic stem and progenitor cells and induced pluripotent stem cell (iPSC)-derived immune effectors have accelerated the development of scalable, patient-specific interventions. The convergence of stem cell biology with precision genome-editing platforms, including CRISPR-Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs), has enabled targeted disruption of viral entry pathways and host dependency factors, while offering new strategies to address viral latency and immune reconstitution. Despite significant challenges related to treatment-associated toxicity, manufacturing complexity, long-term safety, and ethical considerations, rapid progress in cellular engineering and translational immunology continues to advance the field toward curative outcomes. This review critically synthesizes recent progress in stem cell-based HIV therapeutics, elucidates the underlying mechanistic frameworks, evaluates emerging clinical and preclinical evidence, and outlines future directions required to achieve a durable functional cure.},
}
@article {pmid41758644,
year = {2026},
author = {Adami, A and Garza, R and Dorazehi, F and Douse, CH and Jakobsson, J},
title = {Protocol for efficient CRISPRi-mediated silencing of retrotransposons in human pluripotent stem cells.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104398},
pmid = {41758644},
issn = {2666-1667},
mesh = {Humans ; *Gene Silencing ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retroelements/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism ; },
abstract = {Here, we present a workflow for transcriptional silencing of transposable elements (TEs) in human induced pluripotent stem cells (hiPSCs). We describe steps for designing guide RNAs (gRNAs) to target TE families or unique TE loci. We also detail procedures for validating the efficiency and specificity of large-scale CRISPRi-based silencing using a multiome approach combining bulk RNA sequencing, CUT&amp;RUN epigenetic profiling, and proteomics. This framework optimizes the performance and interpretation of in vitro functional studies based on transcriptional manipulation of TEs in hiPSC models. For complete details on the use and execution of this protocol, please refer to Adami et al.[1].},
}
@article {pmid41758946,
year = {2026},
author = {Saito, R and Umemura, Y and Makino, S and Fukaya, T},
title = {Decoding the molecular logic of rapidly evolving ZAD zinc finger proteins in Drosophila.},
journal = {Science advances},
volume = {12},
number = {9},
pages = {eady7568},
pmid = {41758946},
issn = {2375-2548},
mesh = {Animals ; *Drosophila Proteins/metabolism/genetics/chemistry ; *Zinc Fingers ; *Transcription Factors/metabolism/genetics ; *Evolution, Molecular ; Protein Binding ; *Drosophila/genetics/metabolism ; *Drosophila melanogaster/genetics/metabolism/embryology ; DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; CCCTC-Binding Factor ; Microtubule-Associated Proteins ; Nuclear Proteins ; },
abstract = {The zinc finger-associated domain (ZAD)-containing C2H2 zinc finger proteins (ZAD-ZnFs) represent the most abundant class of transcription factors that emerged during insect evolution, yet their molecular diversity and biological functions remain largely unclear. Here, we established a systematic CRISPR-based protein-tagging approach that enables direct, unambiguous comparison of nuclear localization and genome-wide binding profiles of endogenous ZAD-ZnFs in developing Drosophila embryos. Evidence is provided that a subset of ZAD-ZnFs forms nuclear condensates through the stacking of the N-terminal ZAD dimerization surface. Disruption of condensation activity leads to misregulation of genome-wide binding profiles and lethality, underscoring its functional and physiological significance in development. Integrative chromatin immunoprecipitation sequencing and Micro-C analyses reveal that many ZAD-ZnFs colocalize with core insulator proteins such as CCCTC-binding factor and Centrosomal protein 190 kD to control the formation of topological boundaries. We suggest that the diverse molecular functions of ZAD-ZnFs have evolutionarily arisen from their ancestral role as insulator-binding proteins.},
}
@article {pmid41759295,
year = {2026},
author = {Molina, CE and Knight, AL and Lisi, GP},
title = {Comparative thermodynamic and kinetic properties governing the nucleic acid interactions of CRISPR-Cas9 and Cas12a.},
journal = {Physical biology},
volume = {23},
number = {2},
pages = {},
doi = {10.1088/1478-3975/ae4b7f},
pmid = {41759295},
issn = {1478-3975},
mesh = {Thermodynamics ; Kinetics ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; *DNA/metabolism/chemistry ; *CRISPR-Associated Protein 9/metabolism/chemistry ; Gene Editing ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {Clustered regularly interspaced short palindromic repeat-associated proteins (CRISPR-Cas) biochemistry has been leveraged for genome editing applications in biochemical research and therapeutics. CRISPR-Cas9 and CRISPR-Cas12a are the two most widely used RNA-guided endonucleases and while Cas9 and Cas12a have a shared function, both have unique biophysical properties that alter their specificity and efficiency. The thermodynamic and kinetic properties governing their molecular interactions, recognition and binding of target DNA, and R-loop formation can differ. In some cases, these critical biophysical metrics have not been resolved. Distinctions between Cas9 and Cas12a enzymes are also prevalent in RNA:DNA hybrid binding affinities, DNA localization relative to the preferred PAM site and the DNA cleavage mechanism. In this review, we examine the thermodynamic and kinetic properties of both endonucleases, focused on the nucleic acid interactions that confer specificity and function. Complementing this biophysical overview, we discuss case studies in disparate model organisms that compare the genome editing and fidelity of Cas9 and Cas12a.},
}
@article {pmid41759376,
year = {2026},
author = {Xu, Y and Wu, Y and Zheng, H and Zhao, J and Chen, J and Liu, S and Han, M and Li, F and Zhou, F and Zhang, X and Cao, Y and Zhang, H and Zhang, C},
title = {CRISPR-based metabolic screening identifies PLCE1 as a pivotal regulator of oncolytic viral antitumor immunity via tumor immune microenvironment remodeling.},
journal = {Biochemical and biophysical research communications},
volume = {810},
number = {},
pages = {153505},
doi = {10.1016/j.bbrc.2026.153505},
pmid = {41759376},
issn = {1090-2104},
mesh = {*Tumor Microenvironment/immunology ; Animals ; *Oncolytic Virotherapy/methods ; *Oncolytic Viruses/immunology/genetics ; Mice ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Colorectal Neoplasms/therapy/immunology/genetics ; Humans ; *Phosphoinositide Phospholipase C/genetics/metabolism/antagonists &amp; inhibitors ; Female ; Mice, Inbred BALB C ; },
abstract = {As a promising cancer immunotherapeutic agent, oncolytic viruses (OV) can specifically kill tumor cells and elicit systemic antitumor immune responses. However, the intrinsic resistance of tumors to oncolytic virotherapy severely limits its therapeutic efficacy. This study identified phospholipase C epsilon 1 (PLCE1) as a key negative regulator of OV antitumor effects via CRISPR-Cas9 metabolic gene screening in MC38 colorectal cancer model. PLCE1 inhibitor U-73122 enhanced OV infection efficiency and immunogenic cell death in vitro. In vivo, U-73122 combined with OV synergistically reduced tumor volume and prolonged survival. The combination therapy has been shown to remodel the tumor immune microenvironment, leading to an increase in CD45[+] immune cells and CD8[+] T cells, including naïve subsets, and a decrease in FOXP3[+] Treg cells. This shift promotes T cell activation by modulating relevant genes and signaling pathways. This study provides a novel target for optimizing OV immunotherapy.},
}
@article {pmid41759529,
year = {2026},
author = {Cao, Z and Yu, S and Peng, J and Barrett, DR and Liu, Y and Sussman, JH and Chen, C and Thadi, A and Liu, L and Alikarami, F and Xu, J and Carroll, MP and Tan, K and Bernt, KM and Shi, J},
title = {CRISPR-based functional genomics for dissecting therapeutic dependency in primary acute myeloid leukemia samples.},
journal = {Molecular cell},
volume = {86},
number = {5},
pages = {968-985.e7},
pmid = {41759529},
issn = {1097-4164},
support = {R01 CA262260/CA/NCI NIH HHS/United States ; U01 CA243072/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/pathology/drug therapy/therapy ; *CRISPR-Cas Systems ; Animals ; *Genomics/methods ; Mice ; Single-Cell Analysis ; Mutation ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; Gene Editing ; Gene Expression Regulation, Leukemic ; Mice, Inbred NOD ; },
abstract = {Cancer functional genomics enables high-throughput target discovery and mechanistic investigation, yet its application has remained largely confined to mouse models and established human cancer cell lines. Direct functional interrogation of heterogeneous primary tumors offers a powerful opportunity to evaluate therapeutic targets and uncover cancer dependencies or resistance mechanisms. Here, we developed an optimized CRISPR-based platform for functional genomics in patient-derived xenograft and primary acute myeloid leukemia (AML) samples harboring diverse pathogenic mutations. Integrated in vitro and in vivo CRISPR-Cas9 knockout and CRISPR interference (CRISPRi) dropout screens validated known AML-biased targets and identified cis-regulatory elements essential for leukemic growth. Coupling pooled CRISPR perturbations with single-cell RNA sequencing (Perturb-seq) further resolved the perturbation-induced alterations in regulatory networks, cell cycle states, and cellular hierarchies in primary AML samples. Together, these studies establish a general and robust framework for leveraging CRISPR-based functional genomics to directly dissect cancer dependencies and cellular heterogeneity in primary AML patient samples.},
}
@article {pmid41759621,
year = {2026},
author = {Yan, X and Chen, M and Yang, S and Guo, Y and Dai, Y and Chen, Y and Zhong, H and Ma, T and Zha, D and He, Y and Li, B and Jia, X and Guo, L and Hu, J and Wei, Y and Chen, X},
title = {Mitochondrial genome editing tools: prospects in animal breeding.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.jgg.2026.02.018},
pmid = {41759621},
issn = {1673-8527},
abstract = {Mitochondria are vital organelles responsible for driving cellular energy metabolism and regulating key biological processes. Their circular mitochondrial DNA (mtDNA) encodes 13 subunits of the respiratory chain proteins but is susceptible to mutations due to high levels of reactive oxygen species and limited repair mechanisms. Mutant phenotypes manifest only when heteroplasmy surpasses a critical threshold. Understanding the consequences of mtDNA mutations has long been hampered by the lack of precise editing tools. Recently, CRISPR-free, protein-only mitochondrial base editors have enabled C·G-to-T·A and A·T-to-G·C transitions. These breakthroughs facilitate the creation of relevant disease models and offer unique opportunities for animal breeding, as specific mtDNA variants are known to influence economically important traits in livestock, including production, reproduction, and stress tolerance. This review summarizes recent advances in mitochondrial genome editing technologies, including CRISPR/Cas-based systems, restriction endonucleases, double-stranded DNA deaminase toxin A (DddA)-based cytosine and adenine base editors, and DddA-free base editors, along with their delivery strategies and optimization avenues. Furthermore, we outline the associations between mtDNA polymorphisms, copy number variation, and economic traits in livestock and poultry. Finally, we discuss the potential applications of mitochondrial genome editing in animal breeding and highlight the critical safety and ethical considerations that require careful attention.},
}
@article {pmid41759757,
year = {2026},
author = {Schmidt, GE and Weaver, EA and Kim, TH},
title = {CRISPR-based functional analysis of chicken IRF9 reveals distinct modulation of dsRNA stimulated innate immune pathways.},
journal = {Developmental and comparative immunology},
volume = {177},
number = {},
pages = {105577},
doi = {10.1016/j.dci.2026.105577},
pmid = {41759757},
issn = {1879-0089},
mesh = {Animals ; *Chickens/immunology/genetics ; Immunity, Innate/genetics ; *Interferon-Stimulated Gene Factor 3, gamma Subunit/genetics/metabolism ; *RNA, Double-Stranded/immunology ; *Avian Proteins/genetics/metabolism ; Interferon Type I/metabolism ; Signal Transduction ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation ; },
abstract = {The chicken immune system is distinct from mammalian models due to its reduced immune gene repertoire, yet it retains the ability to mount a highly effective immune response. In mammals, interferon regulatory factor 9 (IRF9) is a key transcriptional regulator of the type I interferon (IFN) pathway, stimulating the expression of hundreds of antiviral genes. Although IRF9 was previously thought to be absent in chickens, current chicken reference genome annotation (bGalGal1.mat.broiler.GRCg7b) lists a putative chicken IRF9. To investigate the function of this gene in chickens, we utilized a clustered regularly interspace short palindromic repeats (CRISPR) based transcriptional modulation platform to elucidate the role of the putative chicken IRF9 in the innate immune response. We analyzed the transcriptomes of IRF9 repressed cells stimulated with double stranded RNA at 0, 0.5, 1, and 6 h post-stimulation. Gene set enrichment analysis revealed that IRF9 repression resulted in the enrichment of pathways associated with regulating the type I IFN response, including the retinoic acid inducible gene I like (RIG-I like) receptor pathway and the Toll-like receptor pathway. Furthermore, concurrent transcriptional repression of type I IFN modulator IRF7 with transcriptional activation of IRF9 failed to rescue the expression of downstream IFN-stimulated genes. These results suggest chicken IRF9 plays a distinct regulatory role from canonical mammalian IRF9 in the type I IFN response and demonstrate a need for functional evidence-based classification of chicken IRFs.},
}
@article {pmid41761908,
year = {2026},
author = {Perry, TN and Mais, CN and Sanchez-Londono, M and Steinchen, W and Plitzko, PA and Randau, L and Pausch, P and Innis, CA and Bange, G},
title = {Structural basis of Cas8-independent Cas3 recruitment in Type I-F2 CRISPR-Cas.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41761908},
issn = {1362-4962},
support = {//Deutsche Forschungsgemeinschaft/ ; 260989694//DFG/ ; 324652314//DFG/ ; 405858511//DFG/ ; 3869//DFG/ ; //Inserm/ ; 5342-2023//Research Council of Lithuania/ ; //Marburg University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; DNA/chemistry/metabolism/genetics ; Models, Molecular ; DNA Helicases/chemistry/metabolism/genetics ; Protein Domains ; Protein Binding ; *Bacterial Proteins/chemistry/metabolism/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity in prokaryotes by targeting and degrading invasive genetic elements. Among them, the Type I-F2 system represents the most compact Type I CRISPR-Cas variant, distinguished by the complete absence of both large (Cas8) and small (Cas11) subunits. In other Type I systems, Cas8 is essential for protospacer adjacent motif (PAM) recognition and for triggering Cas3 recruitment, while Cas11 stabilizes the Cascade backbone and guides the nontarget DNA strand during R-loop formation. To elucidate how I-F2 executes interference in their absence, we determined the cryo-electron microscopy structure of the I-F2 Cascade bound to target DNA and Cas3. Our structure reveals that Cas5 alone mediates PAM sensing, while Cas7 subunits directly recruit Cas3, which adopts a helicase-loaded conformation compatible with DNA engagement. We show how the helicase and C-terminal domains of Cas3 capture the displaced nontarget strand to initiate directional unwinding and degradation. These findings uncover key mechanistic adaptations that enable efficient interference without canonical large and small subunits and emphasize the mechanistic diversity among closely related Type I systems, including I-E, I-F1, and I-F2. These insights provide a structural basis for engineering the hypercompact I-F2 system for genome editing and biotechnological applications.},
}
@article {pmid41762821,
year = {2026},
author = {Golla, DA and Sun, C and Haugh, L and Straub, N and Gao, X},
title = {Advances in multiplex precision genome editing in eukaryotic and prokaryotic systems.},
journal = {Current opinion in biotechnology},
volume = {99},
number = {},
pages = {103470},
doi = {10.1016/j.copbio.2026.103470},
pmid = {41762821},
issn = {1879-0429},
abstract = {Multiplex genome editing (MGE) enables coordinated modification of multiple genomic loci and is foundational for engineering complex biological traits. Traditional CRISPR-Cas nuclease-based strategies rely on DNA double-strand breaks (DSBs), which limit precision and pose scaling challenges for incorporating simultaneous edits across different loci. Recent advances in genome editing technologies that operate without generating DSBs have expanded the accuracy and feasibility of multiplexed genomic manipulation. This review focuses on emerging strategies for precise MGE, including base editing, prime editing, and related genome rewriting platforms. We highlight key engineering principles that impact the success of scalable multiplexing, including the choice of editing platform, edit size, and guide RNA architecture, and discuss applications across mammalian, plant, fungal, and bacterial systems. Together, these technologies establish MGE as a versatile framework for precise multigene control in biotechnology and agriculture.},
}
@article {pmid41762887,
year = {2026},
author = {Yu, Y and Sun, S and Song, X and Xiong, Z and Song, Z and Peng, C and Zhang, J and Ai, L},
title = {crRNA-engineered CRISPR/Cas12a system coupled with RPA for ultrasensitive detection of Lactiplantibacillus plantarum.},
journal = {Food chemistry},
volume = {509},
number = {},
pages = {148595},
doi = {10.1016/j.foodchem.2026.148595},
pmid = {41762887},
issn = {1873-7072},
mesh = {*CRISPR-Cas Systems ; *Lactiplantibacillus plantarum/genetics/isolation &amp; purification ; Probiotics/analysis ; *Bacterial Proteins/genetics/metabolism ; *RNA, Bacterial/genetics/metabolism ; Limit of Detection ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The growing probiotic industry requires rapid and precise strain detection methods. Here, a one-pot fluorescence platform integrating RPA with an enhanced CRISPR/Cas12a system (termed RPA-ECas12a) was developed for the detection of Lactiplantibacillus plantarum. Through rational 5'end DNA extension of the crRNA, an optimal variant (5'crRNA10) was identified, which increased the trans-cleavage catalytic efficiency of Cas12a by 33% (3.6 × 10[8] M[-1] s[-1]) compared to the wild-type crRNA. The resulting RPA-ECas12a platform detected L. plantarum with a limit of detection of 1.3 CFU/mL, a linear range from 10[1] to 10[7] CFU/mL, and excellent precision (CVs < 10%). The entire detection was completed within 45 min. The platform demonstrated high selectivity and robustness when applied to commercial probiotic powders, yogurts and other complex food matrices. This work not only provides a sensitive and rapid detection tool for probiotic authentication but also offers a generalizable crRNA-engineering strategy to enhance the performance of CRISPR/Cas12a in diagnostic.},
}
@article {pmid41762997,
year = {2026},
author = {Yu, D and Ren, H and He, P and Li, W and Tang, Q and Huang, L and Wei, J and Zhang, K and Liao, X},
title = {Stage-aware quantification of the SARS-CoV-2 3CL[pro] biomarker via CsPbBr3@COF-LZU1@AuNP electrochemiluminescence and Cas13a amplification.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129593},
doi = {10.1016/j.talanta.2026.129593},
pmid = {41762997},
issn = {1873-3573},
mesh = {*SARS-CoV-2/enzymology/isolation &amp; purification ; *Biosensing Techniques/methods ; Gold/chemistry ; *COVID-19/diagnosis/virology ; *Metal Nanoparticles/chemistry ; *Electrochemical Techniques/methods ; Humans ; *Luminescent Measurements/methods ; Metal-Organic Frameworks/chemistry ; CRISPR-Cas Systems ; Biomarkers/analysis ; Limit of Detection ; },
abstract = {Direct, activity-based quantification of the SARS-CoV-2 main protease (3CL[pro]) remains challenging in complex matrices. Here we report a water-compatible electrochemiluminescent (ECL) biosensor that integrates a CsPbBr3@COF-LZU1 emitter, a peptide-DNA conformational switch, and CRISPR/Cas13a-assisted amplification to convert protease activity into a robust optical "turn-on" signal. The covalent organic framework physically stabilizes perovskite nanocrystals in aqueous media and, together with a sparse Au nanoparticle layer, supports assembly of ferrocene-terminated reporters that impose an ultralow baseline via near-field/redox quenching. Target-specific cleavage unlocks an initiator that drives entropy-mediated T7 promoter formation and transcription, producing RNA activators that switch on Cas13a collateral cleavage; removal or distancing of ferrocene from the emitter restores photon output. Under optimized conditions the sensor exhibits a broad log-linear response from 10 to 10[8] aM with an ultralow detection limit of 4.31 aM, high analytical selectivity against common interferents, tight fabrication-to-fabrication precision (inter-electrode RSD ∼3%), and practical robustness (≈97% signal retention over 12 h, ≥90% after 7 days at 4 °C, and ∼92% after 120 ECL cycles). Stepwise ECL, cyclic voltammetry, and impedance analysis confirm layer-by-layer assembly and the intended mechanism of ferrocene-mediated quenching and Cas13a-driven recovery. Applied directly to minimally processed pharyngeal swab eluates, the platform resolves cohort-level differences across disease stages and captures the expected attenuation of 3CL[pro] activity in late-stage specimens, supporting stage-aware quantification in real clinical samples. The modular design-reprogrammable at the protease-cleavable motif, promoter template, and crRNA-points to a general route for sensitive, selective, and water-stable ECL assays of enzymatic activity with translational potential.},
}
@article {pmid41763219,
year = {2026},
author = {Aravind, KM and Del Vecchio, D},
title = {Resource competition shapes CRISPR-mediated gene activation.},
journal = {Cell systems},
volume = {17},
number = {3},
pages = {101511},
doi = {10.1016/j.cels.2025.101511},
pmid = {41763219},
issn = {2405-4720},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Humans ; Gene Editing/methods ; *Transcriptional Activation/genetics ; },
abstract = {CRISPR-mediated gene activation (CRISPRa) allows concurrent transcriptional control of many genes and is widely used in genome-wide screening, bioproduction, and therapeutics. Multi-gene control is possible due to the sequence specificity by which guide RNAs (gRNAs) recruit dCas9 and an activator protein to target genes. Still, the optimization of CRISPRa systems remains difficult. Here, we show that, despite sequence specificity, different gRNAs interfere with each other by competing for dCas9 and the activator protein. This competition breaks modularity and hinders CRISPRa. We also discover that gene activation is biphasic, wherein increased level of a gRNA leads to target repression instead of activation. We introduce a chemical reaction-network model that captures these effects and use it for improving the dynamic range of CRISPRa. Our results demonstrate that CRISPRa is not as modular or scalable as previously thought and establish a predictive modeling tool that enables systematic design and optimization of multi-gRNA CRISPRa systems. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid41763755,
year = {2026},
author = {Ma, C and French, N and Wu, X and Gupta, SK and Gupta, TB},
title = {Molecular detection of Clostridium and Bacillus species in foods: recent advances and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {229},
number = {},
pages = {118370},
doi = {10.1016/j.foodres.2026.118370},
pmid = {41763755},
issn = {1873-7145},
mesh = {*Clostridium/isolation &amp; purification/genetics ; *Bacillus/isolation &amp; purification/genetics ; *Food Microbiology/methods ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; CRISPR-Cas Systems ; In Situ Hybridization, Fluorescence ; Humans ; Food Contamination/analysis ; },
abstract = {Spore-forming bacteria, especially Clostridium spp. and Bacillus spp., are ubiquitous in food systems, and their ingestion can cause serious diseases in humans and animals. Their persistence in diverse food matrices and resistance to conventional treatments make rapid and accurate detection essential for effective monitoring and control. Traditional culture-based and biochemical assays remain the standard for identifying these bacteria but are often time-consuming, labor-intensive and limited in sensitivity. In contrast, nucleic acid-based methods provide rapid, specific and sensitive alternatives by directly targeting genetic markers of pathogenic or spoilage strains. This review summarizes how nucleic acid methods, including PCR, FISH, LAMP, RPA, WGS, and the emerging CRISPR/Cas systems, have been applied specifically to detect Clostridium spp. and Bacillus spp. in food systems. Each method offers unique advantages and limitations. PCR-based methods enable accurate quantification but require thermal cycling. FISH-based methods are simple but require microscopy and have limited validation in food. WGS-based methods provide strain-level characterization but depend on informatics and specialized equipment. Isothermal techniques such as LAMP- and RPA-based methods allow rapid field detection but involve complex primer design or poor discrimination of closely related genes. CRISPR/Cas-based platforms further enhance simplicity, specificity, sensitivity for on-site detection, though the validation for spore-forming bacteria remains limited. Overall, this review provides an overview of gene targets, methodological adaptations, and analytical performance of nucleic acid-based assays for detecting Clostridium spp. and Bacillus spp., highlighting current progress and future opportunities for improving food safety monitoring.},
}
@article {pmid41764730,
year = {2026},
author = {D'Souza, LJ and Young, JN and Coffman, H and Petrow, EP and Bhattacharya, D},
title = {A genome-wide CRISPR screen reveals novel determinants of long-lived plasma cell secretory capacity.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {2},
pages = {},
doi = {10.1093/jimmun/vkaf354},
pmid = {41764730},
issn = {1550-6606},
support = {R01AI129945//National Institutes of Health (NIH)/ ; P30CA023074//Research, Innovation &amp; Impact (RII) of the University of Arizona and National Cancer Institute/ ; S10 OD028466/GF/NIH HHS/United States ; },
mesh = {Animals ; *Plasma Cells/immunology/metabolism ; Mice ; Humans ; Myeloid Differentiation Factor 88/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Multiple Myeloma/immunology/genetics ; Mice, Inbred C57BL ; Mice, Knockout ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Plasma cell subsets vary in their lifespans and ability to sustain humoral immunity. We conducted a genome-wide CRISPR-Cas9 screen in myeloma cells for factors that promote surface expression of CD98, a marker of longevity in mouse plasma cells. A large fraction of genes found to promote CD98 expression in this screen are involved in secretory and other vesicles, including subunits of the V-type ATPase complex. Genetic ablation and chemical inhibition of V-type ATPases in myeloma cells and primary plasma cells, respectively, reduced antibody secretion. Mouse and human long-lived plasma cells had greater numbers of acidified vesicles than their short-lived counterparts, and this correlated with increased antibody secretory capacity. The screen also revealed a requirement for the signaling adapter MYD88 in CD98 expression. Plasma cell-specific deletion of Myd88 led to reduced survival and antibody secretion by antigen-specific cells in vivo and unresponsiveness to BAFF and APRIL ex vivo. These data reveal novel regulators that link plasma cell secretory capacity and lifespan.},
}
@article {pmid41766140,
year = {2026},
author = {Hartig, AM and Dai, W and Zhang, K and Rottinghaus, AG and Moon, TS and Parker, KM},
title = {Genetic Markers Remain Detectable in Genetically Engineered Microbes Biocontained with a CRISPR Kill Switch.},
journal = {Environmental science &amp; technology},
volume = {60},
number = {10},
pages = {7983-7994},
doi = {10.1021/acs.est.6c00321},
pmid = {41766140},
issn = {1520-5851},
mesh = {Escherichia coli/genetics ; Genetic Markers ; *CRISPR-Cas Systems ; Genetic Engineering ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Microorganisms, Genetically-Modified/genetics ; },
abstract = {Biocontainment strategies, such as kill switches, have been developed to avoid the unintended proliferation of genetically engineered microbes (GEMs) intended for open-release environmental applications. However, the presence of GEM DNA after successful biocontainment presents new environmental risks and challenges for monitoring. In this study, we investigated whether biocontainment using a CRISPR-Cas9 kill switch, which causes double-strand breaks in target genes essential for GEM growth, could resolve this challenge in a model Escherichia coli GEM. Surprisingly, the escape rates of the GEM as determined by CRISPR-targeted gene abundances were as high as 10[-1.6] to 10[-1.0] in LB media, despite the escape rates measured by colony forming units (cfu) being only 10[-6.2] under the same condition. This discrepancy suggested that the CRISPR-Cas9 kill switch prevents colony growth while still leaving a large fraction of target genes intact for detection by molecular methods. Within 1 h after biocontainment, these target genes remained predominantly inside an intact cell membrane and were resistant to degradation by DNase, though degradation was observed in river water over multiple days. Overall, a detailed understanding of the impact of the biocontainment mechanism on both the GEM and its DNA is needed to minimize unintended environmental risks.},
}
@article {pmid41766888,
year = {2026},
author = {Li, X and Zhao, Y and Guo, X and Bai, Y and Wang, J},
title = {Characterization and diversity of defense systems in Providencia pathogen.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1755933},
pmid = {41766888},
issn = {1664-3224},
mesh = {*Providencia/genetics/immunology/virology ; Phylogeny ; Genome, Bacterial ; *Enterobacteriaceae Infections/microbiology/immunology ; CRISPR-Cas Systems ; Bacteriophages ; Humans ; },
abstract = {INTRODUCTION: Providencia species are emerging opportunistic pathogens associated with multidrug-resistant infections, yet their molecular defense mechanisms against phage or mobile genetic elements remain poorly characterized.<br><br>METHODS: We present a comprehensive pan-genomic analysis of antiviral defense systems across 73 complete genomes (or chromosomes) of Providencia stuartii (n = 31) and Providencia rettgeri (n = 42), using DefenseFinder and CRISPRCasFinder. We further expanded analysis of contig/scaffold assemblies to confirm conservation of core defense profiles across assembly types. BacMGEnet was employed to derive spacer-MGE interaction networks. Phylogenetic reconstruction and gene gain and loss modeling were performed to assess evolutionary patterns. To validate functionality, we experimentally tested the anti-phage activity of Gabija and Septu in heterologous E. coli assays, including point mutation analysis of conserved residues.<br><br>RESULTS: We reveal a diverse and complex defense repertoire dominated by restriction-modification systems and CRISPR-Cas Class 1 Type I-F, with significant contributions from toxin-antitoxin, GAPS2, PsyrTA, and Mokosh systems. Notably, defense genes are non-randomly distributed, often clustering into genomic islands suggestive of horizontal acquisition. Expanded analysis confirms conservation of core defense profiles across assembly types, supporting the utility of lower-quality data when complete genomes are scarce. Comparative analysis uncovers species-specific differences, with P. rettgeri harboring a higher abundance of non-CRISPR systems. BacMGEnet-derived spacer-MGE interaction networks further highlight species-specific dynamics, dense, hub-driven networks in P. stuartii versus sparser networks in P. rettgeri. Correlation analysis indicates potential associations between specific defense systems and virulence or antibiotic resistance genes. Phylogenetic reconstruction and gene gain and loss modeling further highlight dynamic evolutionary patterns. Both Gabija and Septu systems conferred robust, phage-specific protection; point mutations in conserved residues (GajA E465K and PtuB H53K) abolished defense.<br><br>DISCUSSION: Our findings unveil a multi-layered, modular immune architecture in Providencia, providing crucial insights into its genome plasticity, phage resistance, and adaptation in clinical environments. This work establishes a foundation for understanding the role of defense systems in the evolution and pathogenicity of the Providencia genus.},
}
@article {pmid41769381,
year = {2026},
author = {Mandal, S and Baloch, AR and Yuan, X and Chen, J and Saribas, AS and Zhu, Y and Zhang, D and Jaijyan, D and Xu, J and Hossain, R and Sisto, I and Wang, H and Yang, X and Li, Q and Hu, W},
title = {Bipolar CD4-targeted dual-DARPin-55/57 lipid nanoparticle enables efficient CRISPR/Cas-mediated HIV-1 DNA excision and reactivation blockade in latent CD4 T cell lines.},
journal = {Materials today. Bio},
volume = {37},
number = {},
pages = {102939},
pmid = {41769381},
issn = {2590-0064},
abstract = {The persistence of HIV-1 latent reservoirs remains the principal barrier to a cure, as viral rebound occurs upon interruption of antiretroviral therapy. CRISPR/Cas genome editing offers a promising strategy to excise proviruses from host genome; however, the absence of a targeted and clinically viable delivery platform has hindered its translational application. Here, we report a chemistry-driven, CD4-targeted lipid nanoparticle (LNP) delivery platform employing a unique bipolar conjugation strategy to decorate dual CD4-targeted Designed Ankyrin Repeat Proteins (DARPins-55 and -57) on LNP (dual-DARPin-LNP). The N- and C-terminally modified DARPin-55/57 was thiolated stepwise, then bipolar maleimide-thiol coupling conjugated the thiolates to the maleimide-functionalized LNP surface. This coupling strategy ensured DARPin proper orientation on the LNP surface for efficient uptake by resting CD4 T cells. This dual-DARPin-LNP system was engineered for selective and efficient co-delivery of spCas9-GFP mRNA (Sp9m) and HIV-1-specific single-guide RNAs (sgRNAs) targeting LTR and Gag (LGsg) into HIV-1 latently infected CD4 T cells. In widely used HIV-1 latency models with defined proviral modifications (J-Lat 10.6 and 2D10 cell lines), dual-DARPin-LNP loaded with Sp9m/LGsg efficiently excised integrated HIV-1 proviral DNA, as confirmed by standard PCR genotyping, absolute digital PCR quantification, confocal microscopy, and flow cytometry. Importantly, proviral excision functionally blocked HIV-1 reactivation following stimulation with latency-reversing agents suberoylanilide hydroxamic acid (SAHA) and TNFα. Together, these findings establish a modular, non-viral, receptor-guided delivery platform for CD4 T cell targeting and provide proof-of-concept for precise HIV-1 DNA excision and reactivation blockade in established latency models. This new strategy represents a step toward next-generation curative interventions against persistent HIV-1 infection.},
}
@article {pmid41771837,
year = {2026},
author = {Schoger, E and Kim, R and Bleckwedel, F and Peralta, TM and Priesmeier, L and Fischer, JA and Stengel, L and Rocha, C and Santos, GL and Lutz, S and Boileau, E and Baumgarten, N and Schulz, MH and Dieterich, C and Müller, OJ and Cyganek, L and Cabrera-Orefice, A and Eberl, H and Maack, C and Streckfuss-Bömeke, K and Pavez-Giani, MG and Doroudgar, S and Sossalla, S and Zelarayán, LC},
title = {Enhancing KLF15 activity in cardiomyocytes: a novel approach to prevent pathological reprogramming and fibrosis via nuclease-deficient dCas9VPR.},
journal = {Signal transduction and targeted therapy},
volume = {11},
number = {1},
pages = {},
pmid = {41771837},
issn = {2059-3635},
mesh = {*Myocytes, Cardiac/metabolism/pathology ; *Kruppel-Like Transcription Factors/genetics/metabolism ; Animals ; CRISPR-Cas Systems/genetics ; *Fibrosis/genetics/pathology ; *Cellular Reprogramming/genetics ; Humans ; Mice ; Fibroblasts/metabolism/pathology ; Gene Expression Regulation/genetics ; *Transcription Factors/genetics ; },
abstract = {Transcriptional activity perturbation holds promise for selectively modulating harmful transcriptional networks, but its therapeutic potential remains largely unexplored. We employed a network-based analysis of single-cell heart transcriptomes to identify transcription factor activities linked to pathological cardiomyocytes in vivo. This analysis revealed that transcriptional activity of Krüppel-like factor 15 (KLF15) exhibited the most significant change in pathological cardiomyocytes, characterized by less effective repression of disease-associated genes in stressed hearts, which correlated with reduced KLF15 expression. To restore KLF15 activity, we utilized CRISPR/nuclease-dead (d)Cas9-based transcriptional enhancement (CRISPRa) in cardiomyocytes, which effectively abolished fetal reprogramming by simultaneously suppressing pathological gene expression and restoring metabolic homeostasis under sustained stress conditions. Furthermore, we identified a novel cell-nonautonomous anti-fibrotic effect mediated by cardiomyocyte-fibroblast crosstalk, and revealed the contribution of KLF15-dependent Alpha-2-glycoprotein 1, zinc-binding (AZGP1) regulation in this process. We also elucidated the upstream mechanisms of KLF15 regulation, highlighting its role as a cell-specific downstream target of the broad TGF-β canonical signaling pathway, along with its downstream-dependent mechanisms in human cardiomyocytes. Finally, to enhance the therapeutic potential of this approach, we engineered and validated an adeno-associated viral (AAV) vector with a small CRISPRa system for endogenous regulation in human cardiomyocytes suitable for clinical applications. Overall, we elucidated a regulatory circuit involving TGF-β, KLF15, and AZGP1, which coordinates critical pathological responses through cellular crosstalk between cardiomyocytes and fibroblasts. Importantly, we demonstrated the efficacy of CRISPRa as an epigenetic intervention restoring a critical transcriptional function disrupted in non-genetic heart failure. This approach provides a promising blueprint for future adaptation targeting additional non-hereditary pathologies.},
}
@article {pmid41771871,
year = {2026},
author = {Becerra, B and Wittibschlager, S and Patel, ZM and Kutschat, AP and Delano, J and Che, E and Tauber, A and Wu, T and Starrs, M and Horstmann, CS and Müller, S and Whittaker, MN and Sylvander, E and Lehner, M and Love, MI and Kleinstiver, BP and Jankowiak, M and Bauer, DE and Seruggia, D and Pinello, L},
title = {Nucleotide-resolution mapping of regulatory elements via allelic readout of tiled base editing.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41771871},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing/methods ; Alleles ; PAX5 Transcription Factor/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Regulatory Sequences, Nucleic Acid/genetics ; Binding Sites ; Nucleotides/genetics ; Mutation ; Enhancer Elements, Genetic ; },
abstract = {CRISPR tiling screens have enabled the characterization of regulatory sequences but are limited by low resolution arising from the indirect readout of editing via guide RNA sequencing and enrichment analysis. This study introduces an end-to-end experimental assay and computational pipeline, which leverages targeted sequencing of CRISPR-introduced alleles at the endogenous target locus following dense base-editing mutagenesis. As a proof of concept, we studied a putative CD19 enhancer, an immunotherapy target in leukemia, and identified alleles and single nucleotides crucial for CD19 regulation. Our visualization tools revealed transcription factor motifs corresponding to the top-ranked nucleotides. Validation experiments confirmed that mutations in MYB, PAX5, and EBF1 binding sites reduce CD19 expression. Critically, editing MYB and PAX5 motifs conferred resistance to CD19 CAR-T cell therapy, revealing how non-coding variants can drive immunotherapy escape. Taken together, this approach achieves nucleotide-resolution genotype-phenotype mapping at regulatory elements beyond conventional gRNA-based screens.},
}
@article {pmid41772759,
year = {2026},
author = {Donega, S and Gorospe, M and Harries, LW and Ferrucci, L},
title = {Loss of Splicing Homeostasis as a Hallmark of Aging.},
journal = {Molecular and cellular biology},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10985549.2026.2627235},
pmid = {41772759},
issn = {1098-5549},
abstract = {Alternative splicing is a fundamental mechanism that ensures accurate gene expression, supports cellular adaptability, and expands protein diversity beyond the limits of a fixed gene pool. With aging, splicing fidelity weakens, contributing to decline in RNA homeostasis and disrupting essential cellular functions, including mitochondrial oxidative phosphorylation, genome stability, and immune regulation, and in turn accelerating tissue and organ dysfunction. Evidence from senescent cells, aged tissues, and model organisms shows that altered levels of splicing factors and increased RNA polymerase II elongation rates impair co-transcriptional splicing and promote mis-spliced isoforms that reinforce senescence and drive pathology. Dysfunction of RNA-binding proteins further contributes to aberrant splicing, linking splicing defects to age-related diseases such as atherosclerosis, osteoarthritis, sarcopenia, and neurodegenerative disorders like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Therapeutic strategies to correct splicing defects, such as antisense oligonucleotides, RNA interference, CRISPR-Cas systems, ADAR-mediated editing, and RNA aptamers, can restore a homeostatic balance of mRNA isoforms. However, major challenges remain, including distinguishing adaptive physiological from pathological splicing 'noise' and achieving targeted delivery to tissues. Despite these obstacles, RNA splicing dysregulation represents a promising avenue to extend health span by reestablishing homeostatic RNA programs, and reinforces the idea that "transcriptomic instability" is a hallmark of aging.},
}
@article {pmid41773016,
year = {2026},
author = {Park, H and Yun, J and Lee, K and Kim, JH and Park, JH and Park, YJ and Park, JH and Lee, H and Kim, MG},
title = {Functional decoupling of crRNA enables customizable CRISPR diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773016},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; RS-2025-16063091//National Research Council of Science and Technology/ ; RS-2024-00411137//National Research Council of Science and Technology/ ; CRC22024-500//National Research Council of Science and Technology/ ; //National Research Foundation of Korea/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nucleic Acid Amplification Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {One-pot CRISPR-based diagnostics have transformed nucleic acid testing, yet their design customizability remains constrained. Because target programming and cis-cleavage activity are simultaneously determined during CRISPR RNA (crRNA) design, optimizing cleavage activity to match isothermal amplification inevitably requires altering the programmed crRNA sequence. This requirement fundamentally constrains the range of compatible target sequences, imposing limitations on the flexible design of diagnostic assays. Here, we establish a customizable one-pot system by decoupling the dual functions inherent in crRNA design to enable their independent control. In this strategy, target programming remains defined by the crRNA sequence, whereas cis-cleavage activity is regulated by the reaction energy barrier. We selectively modulate this energy barrier through the introduction of a crRNA-complementary RNA oligonucleotide, achieving cleavage regulation without altering the crRNA sequence. Consequently, this approach ensures that cis-cleavage activity matches isothermal amplification conditions independent of the programmed target sequence, thereby realizing a customizable CRISPR diagnostic system. We validated the clinical applicability of this system using 120 patient-derived samples, achieving sensitivity and specificity comparable to quantitative polymerase chain reaction. Collectively, this work resolves a fundamental constraint of CRISPR diagnostics and establishes a customizable and clinically deployable platform for next-generation nucleic acid testing.},
}
@article {pmid41773018,
year = {2026},
author = {Yang, T and Tang, M and Xu, L and Jiang, L and Jiang, L and Zou, Y and Wang, J and Liu, Z and Chen, F and Ban, Y and Ren, W and Cheng, W},
title = {A tailored phosphorothioate coordinator enables CRISPR/Cas in-situ amplification.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41773018},
issn = {1362-4962},
support = {U24A20751//National Natural Science Foundation of China/ ; 82372334//National Natural Science Foundation of China/ ; 82502827//National Natural Science Foundation of China/ ; CSTB2023NSCQ-LZX0022//Chongqing Education Commission/ ; CSTB2024NSCQ-QCXMX0006//New Chongqing Youth Innovative Talents Project/ ; //National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Phosphorothioate Oligonucleotides/chemistry/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics ; CRISPR-Associated Proteins/metabolism/genetics/chemistry ; RNA, Viral/genetics/metabolism ; RNA, Messenger/genetics/metabolism ; Uterine Cervical Neoplasms/virology/diagnosis ; Female ; HeLa Cells ; Endodeoxyribonucleases/metabolism/genetics ; },
abstract = {The CRISPR/Cas system is a powerful tool for molecular diagnostics, but its reliance on linear amplification constrains sensitivity, particularly for in situ imaging. Here, we discovered that phosphorothioate (PS)-modified activators can modulate Cas enzyme conformation via hydrophobic anchoring. By adjusting the PS modification sites, we achieved precise control over Cas activation and trans-cleavage resistance. Guided by this mechanism, we proposed a tailored design strategy featuring a "scattered" PS modification to engineer a linear "Coordinator" probe. This design effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, enabling the construction of a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). SACA achieves exponential amplification without external enzymes, enhancing Cas12a and Cas13a sensitivity by 50 000-fold and 10 000-fold, respectively. Furthermore, the superior biostability and structural simplicity of these linear probes endow SACA with excellent compatibility, facilitating precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells. This study not only advances the understanding of Cas enzyme regulation by chemically modified nucleic acids but also establishes a new paradigm for precise and efficient molecular diagnostics.},
}
@article {pmid41773916,
year = {2026},
author = {Sui, Z and Chen, B and Zhao, J and Deng, R and Xu, J},
title = {Pronounced Fluorescence Polarization Enhancement Driven by RPA-CRISPR/Cas12a Induced Nucleoprotein Assembly for Salmonella Analysis in Animal-Derived Food Matrices.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7822-7831},
doi = {10.1021/acs.analchem.5c08279},
pmid = {41773916},
issn = {1520-6882},
mesh = {*Fluorescence Polarization/methods ; *CRISPR-Cas Systems ; *Salmonella/isolation &amp; purification/genetics ; Animals ; *Nucleoproteins/metabolism/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Food Microbiology ; *CRISPR-Associated Proteins/metabolism ; *Recombinases/metabolism ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins/metabolism/genetics ; },
abstract = {Salmonella is one of the most hazardous foodborne pathogens, posing a serious threat to public health and food safety worldwide. Conventional recombinase polymerase amplification (RPA)-CRISPR/Cas12a detection assays predominantly rely on the trans-cleavage of fluorescent reporters; however, such signal-generation modes are inherently susceptible to photobleaching, signal drift, and fluctuation, thereby compromising quantitative accuracy and long-term signal stability in practical pathogen detections. To overcome these limitations, we developed a trans-cleavage-independent fluorescence polarization (FP) sensing platform for the rapid and quantitative detection of Salmonella. Unlike conventional reporter-cleavage-based readouts, the proposed system exploits target-induced nucleoprotein assembly to achieve direct, physical signal amplification. In this design, a FAM-labeled forward primer serves as an intrinsic molecular reporter, while exonuclease I (Exo I) selectively degrades unincorporated primers, effectively suppressing background interference. Upon recognition of Salmonella genomic DNA, RPA produces rigid double-stranded amplicons that restrict fluorophore rotational freedom, and subsequent crRNA-guided Cas12a binding further increases molecular size and hydrodynamic volume, resulting in a stepwise enhancement of FP signals. The assay exhibits excellent linearity over a concentration range of 3 × 10[1]-3 × 10[6] CFU mL[-1], with an ultralow detection limit of 5 CFU mL[-1]. In addition, it demonstrates outstanding photostability, reproducibility, and high specificity against nontarget bacteria. Importantly, reliable Salmonella detection was achieved in complex food matrices, including meat, eggs, and dairy products, with consistently high recoveries and strong tolerance to matrix interference, offering a promising alternative to conventional fluorescence-intensity-based CRISPR diagnostics in complex food systems.},
}
@article {pmid41774834,
year = {2026},
author = {Chen, Z and Lin, H and Yoon, C and Huang, H and Kim, Y and Meng, C and Jang, H and Xie, Z and Li, L and Liu, Y and Kim, JS and Zhang, H},
title = {Bismuthene-Based Nanoplatform for Synergistic Thermogenetic CRISPR and Photothermal Cancer Therapy.},
journal = {Nano letters},
volume = {26},
number = {10},
pages = {3407-3416},
doi = {10.1021/acs.nanolett.5c06006},
pmid = {41774834},
issn = {1530-6992},
mesh = {Humans ; *Photothermal Therapy/methods ; *CRISPR-Cas Systems ; Animals ; Female ; Mice ; Cell Line, Tumor ; *Triple Negative Breast Neoplasms/therapy/genetics/pathology ; *Nanostructures/chemistry ; *Hyperthermia, Induced/methods ; },
abstract = {Overcoming tumor thermotolerance within clinically safe temperature ranges remains a central limitation of photothermal therapy (PTT). Here we report a closed-loop therapeutic nanoplatform that integrates topologically enhanced photothermal conversion with thermally gated CRISPR/Cas9 regulation. Rationally engineered hexagonal bismuthene nanodiscs exhibit strong near-infrared responsiveness, enabling mild hyperthermia (∼45 °C) that activates a heat-sensitive CRISPR switch targeting CDK7. The resulting disruption of the CDK7-HSP70 stress axis lowers the thermal resistance threshold and reprograms tumor adaptation, thereby amplifying photothermal efficacy and promoting immunogenic cell death. In triple-negative breast cancer models, this gene-thermal feedback achieves >93% tumor inhibition with minimal systemic toxicity. This work establishes a genetically programmable, thermogenetic nanomaterial paradigm that links material design with gene logic for next-generation precision cancer therapy.},
}
@article {pmid41776280,
year = {2026},
author = {Shirai, Y and Kao, JA and Kumar, T and Matsuda, N and Nakagawa, R and Nureki, O and Extavour, CG and Daimon, T},
title = {HUH-tagged Cas9 as a platform for efficient ssODN-mediated knock-in via embryo and adult injection in insects.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41776280},
issn = {2399-3642},
support = {20H02999, 20K21311, 22K19179, 24H00511, 24K21869//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21J20658//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Overseas Research Fellow//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; Long-Term Fellow//Human Frontier Science Program (HFSP)/ ; JPJ008000//Cabinet Office, Government of Japan/ ; IOS-2220747//NSF | BIO | Division of Integrative Organismal Systems (IOS)/ ; },
mesh = {Animals ; *Gene Knock-In Techniques/methods ; *Tribolium/genetics/embryology ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism ; DNA End-Joining Repair ; *Oligodeoxyribonucleotides/genetics ; Recombinational DNA Repair ; },
abstract = {Recent advances in adult injection-based insect genome editing have enabled genetic manipulation of a wide range of insect species, including those previously considered difficult or even impervious to genetic modification. However, achieving efficient knock-in remains a significant challenge with this approach. Here, we demonstrate that fusing a HUH endonuclease tag to Cas9 significantly enhances both non-homologous end joining (NHEJ)-mediated knockout and homology-directed repair (HDR)-mediated knock-in via adult injection. This fusion increased knockout efficiency by up to fivefold in the beetle Tribolium castaneum through adult injection, likely due to its previously unrecognized nuclear localization activity. It also improved single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in efficiency, which we attribute to its characteristic ssDNA-tethering activity. To evaluate its versatility, we tested the HUH-tagged Cas9 in conventional embryo injection, which significantly enhanced HDR-mediated knock-in of an epitope tag in cricket and milkweed bug embryos. Our findings establish the HUH-tag as a versatile platform for improving both NHEJ- and HDR-based genome editing, providing a robust framework to advance genetic engineering across a broad spectrum of arthropods.},
}
@article {pmid41777069,
year = {2026},
author = {Zamperin, G and Palumbo, E and Castellan, M and Marciano, S and Fusaro, A and Monne, I},
title = {Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas-based rRNA depletion system.},
journal = {Veterinaria italiana},
volume = {62},
number = {2},
pages = {},
doi = {10.12834/VetIt.3908.38985.2},
pmid = {41777069},
issn = {1828-1427},
mesh = {*CRISPR-Cas Systems ; Animals ; *Metagenomics/methods ; *RNA, Ribosomal/genetics ; *Zoonoses/virology ; Genome, Viral ; },
abstract = {Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9-based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H-based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.},
}
@article {pmid41778754,
year = {2026},
author = {Hoyle, C and Pino, RD and Lai, SM and Green, JP and Adamson, A and Coutts, G and Lawrence, CB and Travis, M and Brough, D and Lopez-Castejon, G},
title = {Inflammatory responses following CRISPR modification of the nuclear localisation sequence in endogenous interleukin-1 alpha.},
journal = {Disease models &amp; mechanisms},
volume = {19},
number = {4},
pages = {},
doi = {10.1242/dmm.052705},
pmid = {41778754},
issn = {1754-8411},
support = {Fellowship//Dowager Countess Eleanor Peel Trust/ ; BB/Y004876/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; SRB1013//Dowager Countess Eleanor Peel Trust/ ; MR/T016515/1/MRC_/Medical Research Council/United Kingdom ; },
mesh = {Animals ; *Interleukin-1alpha/metabolism/genetics/chemistry ; *Nuclear Localization Signals/genetics/metabolism ; *Inflammation/genetics/pathology ; Lipopolysaccharides/pharmacology ; *Cell Nucleus/metabolism/drug effects ; Macrophages/metabolism/drug effects ; Mice ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *CRISPR-Cas Systems/genetics ; Peritonitis/genetics/pathology ; },
abstract = {Interleukin (IL)-1α is a pro-inflammatory member of the IL-1 cytokine superfamily and is important for inflammatory responses to infection and injury. Unlike pro-IL-1β, pro-IL-1α is mainly localised to the nucleus upon expression. This is mediated by a nuclear localisation sequence (NLS) responsible for its importin-dependent transport into the nucleus. This nuclear localisation and the presence of histone acetyl transferase (HAT)-binding domains within the pro-domain suggest a role of this cytokine in gene transcription regulation. In addition, nuclear trafficking of pro-IL-1α is proposed to regulate its secretion. To date, studies on the nuclear role of pro-IL-1α have used overexpression systems. Here, we generated a mouse strain in which the endogenous Il1a gene was edited using CRISPR to disrupt the NLS, yielding a mutated NLS (mNLS). Using an in vitro approach with murine macrophages we found that this NLS mutation did not affect pro-IL-1α RNA expression levels in response to lipopolysaccharide (LPS) but increased its protein expression levels. Moreover, we found that the transcriptional signature induced by LPS was not altered between WT and mNLS macrophages. Release of IL-1α in response to different stimuli such as ionomycin was not negatively impacted by disrupted nuclear localisation, although higher levels of IL-1α release were detected, potentially due to increased levels of pro-IL-1α. Inflammatory responses in an in vivo model of peritonitis and an influenza infection model were comparable between WT and mNLS mice. Thus, we have established a mouse model in which pro-IL-1α nuclear localisation is disrupted, although future research is required to reveal the importance of this nuclear localisation for IL-1α function.},
}
@article {pmid41779781,
year = {2026},
author = {Ju, X and Dong, L and Liu, T and Zhang, F and Sun, X and Schwoerer, MP and Ren, W and Gong, M and Ploss, A and Qin, W and Wu, X and Wang, L and Ding, Q},
title = {EIF4H and YBX1 are essential host factors for hepatitis E virus replication and pathogenesis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2529289123},
pmid = {41779781},
issn = {1091-6490},
support = {2023YFC2306900//National Key Research and Development Plan of China/ ; 82341084//MOST | National Natural Science Foundation of China (NSFC)/ ; 82272302//MOST | National Natural Science Foundation of China (NSFC)/ ; 82522053//MOST | National Natural Science Foundation of China (NSFC)/ ; 20251080029//Tsinghua University Dushi Program/ ; Not applicable//SXMU-Tsinghua Collaborative Innovation Center for Frontier Medicine/ ; Not applicable//High Meadows Environmental Institute/ ; T32 GM007388/GM/NIGMS NIH HHS/United States ; },
mesh = {*Hepatitis E virus/physiology/pathogenicity/genetics ; *Virus Replication ; Humans ; *Hepatitis E/virology/metabolism/pathology/genetics ; Animals ; Rats ; *Y-Box-Binding Protein 1/metabolism/genetics ; *Eukaryotic Initiation Factors/metabolism/genetics ; Host-Pathogen Interactions ; CRISPR-Cas Systems ; Hepatocytes/virology ; RNA-Binding Proteins ; },
abstract = {Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis worldwide, responsible for approximately 20 million infections annually. Despite the availability of a vaccine in China, no direct-acting antivirals are approved, and host factors required for HEV replication remain poorly defined. Here, using a genome-wide CRISPR/Cas9 knockout screen in a replicon system, we identified Eukaryotic Translation Initiation Factor 4H (EIF4H) and Y-Box Binding Protein 1 (YBX1) as essential host factors for HEV replication and pathogenesis. Knockout of either factor markedly impaired replication of HEV genotypes 1, 3, and 4, as well as HEV infection and production in hepatocellular carcinoma cells and human induced pluripotent stem cell-derived hepatocyte-like cells, while leaving SARS-CoV-2, hepatitis B virus, hepatitis C virus, and Zika virus unaffected, underscoring their HEV-specific roles. Mechanistically, EIF4H interacts with ORF1 via its methyltransferase-Y-papain-like protease region, and EIF4H deficiency alters the composition of the ORF1-associated replication complex. By contrast, YBX1 is dispensable for ORF1 translation and RNA binding but is specifically required for ORF1 proteolytic processing, a prerequisite for assembling a functional replication machinery. EIF4H knockout rats and liver-specific YBX1 knockout rats were largely resistant to rat HEV-C1 infection, showing profound reductions in viral shedding, suppressed hepatic and intestinal viral loads, and protection from liver pathology. Together, our findings establish EIF4H and YBX1 as essential host factors for HEV infection and pathogenesis and reveal potential targets for antiviral intervention.},
}
@article {pmid41779860,
year = {2026},
author = {Zhou, X and Pan, D and Zhou, J and Chen, W and Han, G and Zhang, R and Wang, C and Mao, Y and Du, Z and Zhang, F and Yue, H and Ma, J and Li, Z and Shen, RJ and Wang, B and Zhu, W and Peng, Y and Jin, K and Wu, DD and Wang, W and Zhou, B and Jin, ZB and Chen, L},
title = {Cis-regulatory evolution reveals sensory trade-offs as a genetic basis for temporal niche evolution in tapirs.},
journal = {Science advances},
volume = {12},
number = {10},
pages = {eadz4758},
pmid = {41779860},
issn = {2375-2548},
mesh = {Animals ; *Evolution, Molecular ; Mice ; *Regulatory Sequences, Nucleic Acid ; Biological Evolution ; CRISPR-Cas Systems ; Humans ; },
abstract = {Evolutionary shifts in diel activity patterns shape sensory remodeling across mammals, yet the genetic basis remains poorly understood. Tapirs represent a unique natural experiment, having reverted from a cathemeral ancestor to a nocturnal niche characterized by reduced vision but enhanced hearing and olfaction. Here, we investigate the genetic basis of this phenomenon by generating high-quality chromosome-level genomes for Tapirus terrestris and Tapirus indicus. Comparative analyses revealed extensive lineage-specific remodeling of genes and cis-regulatory elements linked to sensory pathways. Notably, functional validation via CRISPR-Cas9 editing of a tapir-specific conserved noncoding element (CNE74) upstream of the FLT1 gene in mice revealed coordinated sensory effects, including retinal degeneration and reduced visual acuity, yet enhanced auditory sensitivity. These findings suggest that regulatory element evolution may induce pleiotropic effects on competing sensory modalities, offering genetic insights into sensory evolution during temporal niche adaptation and potential relevance to human retinal vascular diseases.},
}
@article {pmid41781609,
year = {2026},
author = {Xiao, R and Hoffmann, FT and Xie, D and Wiegand, T and Palmieri, AI and Sternberg, SH and Chang, L},
title = {Structural basis of RNA-guided transcription by a dCas12f-σ[E]-RNAP complex.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41781609},
issn = {1476-4687},
abstract = {In both natural and engineered biological systems, RNA-guided proteins have emerged as critical transcriptional regulators by modulating RNA polymerase (RNAP) and its associated factors[1-3]. In bacteria, diverse clades of repurposed TnpB and CRISPR-associated proteins repress gene expression by blocking transcription initiation or elongation, enabling non-canonical modes of regulatory control and adaptive immunity[1,4,5]. A distinct class of nuclease-dead Cas12f homologues (dCas12f) instead activates gene expression through its association with unique extracytoplasmic function sigma factors (σ[E])[6], although the molecular basis has remained elusive. Here we reveal a new mode of RNA-guided transcription initiation by determining the cryo-electron microscopy structures of the dCas12f-σ[E] system from Flagellimonas taeanensis. We captured multiple conformational and compositional states, including the DNA-bound dCas12f-σ[E]-RNAP holoenzyme complex, revealing how RNA-guided DNA binding leads to σ[E]-RNAP recruitment and nascent mRNA synthesis at a precisely defined distance downstream of the R-loop. Rather than following the classical paradigm of σ[E]-dependent promoter recognition, these studies show that recognition of the -35 element is largely supplanted by CRISPR-Cas targeting, whereas the melted -10 element is stabilized through unusual stacking interactions rather than insertion into the typical recognition pocket. Collectively, this work provides high-resolution insights into an unexpected mechanism of RNA-guided transcription, expanding our understanding of bacterial gene regulation and opening new avenues for programmable transcriptional control.},
}
@article {pmid41781627,
year = {2026},
author = {Hoffmann, FT and Wiegand, T and Palmieri, AI and Glass-Klaiber, J and Xiao, R and Tang, S and Le, HC and Meers, C and Lampe, GD and Chang, L and Sternberg, SH},
title = {Exapted CRISPR-Cas12f homologues drive RNA-guided transcription.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41781627},
issn = {1476-4687},
abstract = {Bacterial transcription initiation is a tightly regulated process that canonically relies on sequence-specific promoter recognition by dedicated sigma (σ) factors, leading to functional DNA engagement by RNA polymerase (RNAP)[1]. Although the seven σ factors in Escherichia coli have been extensively characterized[2], Bacteroidetes species encode dozens of specialized, extracytoplasmic function σ factors (σ[E]) whose precise roles are unknown, pointing to additional layers of regulatory potential[3]. Here we uncover a mechanism of RNA-guided gene activation involving the coordinated action of σ[E] factor in complex with nuclease-dead Cas12f (dCas12f). We screened a large set of genetically linked dCas12f and σ[E] homologues in E. coli using RNA and chromatin immunoprecipitation experiments, revealing systems that exhibit robust guide RNA enrichment and DNA target binding with a minimal 5'-G target-adjacent motif. Recruitment of σ[E] was dependent on dCas12f and guide RNA, suggesting direct protein-protein interactions, and co-expression experiments demonstrated that the dCas12f-gRNA-σ[E] ternary complex was competent for programmable recruitment of the RNAP holoenzyme. Remarkably, dCas12f-RNA-σ[E] complexes drove potent gene expression in the absence of any requisite promoter motifs, with de novo transcription start sites defined exclusively by the relative distance from the dCas12f-mediated R-loop. Our findings highlight a new paradigm of RNA-guided transcription that embodies natural features reminiscent of CRISPR activation (CRISPRa) technology[4,5].},
}
@article {pmid41783940,
year = {2026},
author = {Berti, M and Ceriotti, S and Santi, L and Alberti, G and Beretta, S and Degl'Innocenti, S and Ruatti, C and Savoia, EO and Jofra-Hernandez, R and De Ponti, G and Bolamperti, S and Villa, I and Galeotti, F and Romano, A and Visigalli, I and Norata, R and Rocchi, M and Cristofori, P and Cossutta, M and Consiglieri, G and Tucci, F and Santorelli, L and Grumati, P and Ronfani, L and D'Adamo, P and Giustina, A and Angelozzi, M and Settembre, C and Mortellaro, A and Scala, S and Sanvito, F and Volpi, N and Aiuti, A and Bernardo, ME and Crippa, S},
title = {Development and characterization of a model of mucopolysaccharidosis type IVA for evaluating therapies targeting bone disease.},
journal = {Disease models &amp; mechanisms},
volume = {19},
number = {2},
pages = {},
pmid = {41783940},
issn = {1754-8411},
support = {20228H9T82//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; P20223MF7X_001//Ministero dell'Istruzione, dell'Universit&#xe0; e della Ricerca/ ; CN_00000041 - CUP G83C22000270001//NextGenerationEU/ ; TTAGTXEKFA//Else Kr&#xf6;ner-Fresenius-Zentrum f&#xfc;r Ern&#xe4;hrungsmedizin/ ; TELE-MB//Fondazione Telethon/ ; TELE-AA//Fondazione Telethon/ ; //Ospedale San Raffaele/ ; },
mesh = {Animals ; Disease Models, Animal ; *Mucopolysaccharidosis IV/therapy/pathology/blood/complications ; *Bone Diseases/therapy/pathology/complications ; Chondroitinsulfatases/metabolism/deficiency ; Glycosaminoglycans/metabolism ; Keratan Sulfate/metabolism/urine ; Chondroitin Sulfates/metabolism/urine ; Mice ; Humans ; Genetic Therapy ; Mice, Knockout ; Bone and Bones/pathology ; CRISPR-Cas Systems/genetics ; },
abstract = {Mucopolysaccharidosis type IVA (MPSIVA) is a lysosomal storage disease (LSD) caused by deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS), which causes the accumulation of keratan sulphate (KS) and chondroitin sulphate (CS). Patients with MPSIVA typically present with severe skeletal and joint disorders, which are not addressed by conventional therapies. Currently, no animal model accurately replicates the human disease, hindering the development of novel therapeutic interventions. To overcome this limitation, we established, by CRISPR-Cas9 technology, a Galns-/- mouse model that expresses a non-functional enzyme and accumulates CS and KS in the urine, plasma and distinct tissues, and glycosaminoglycans in the spleen. The mice exhibit shortened long bones, trabecular bone alterations and skeletal abnormalities in the growth plate. Additionally, we observed increased levels of inflammatory and oxidative markers in visceral organs and plasma. Our newly developed model of MPSIVA demonstrates clear and quantifiable signs of skeletal alterations, providing novel means of assessment of the safety and efficacy of innovative therapies, including hematopoietic stem and progenitor cell gene therapy, which has recently been shown to provide a beneficial effect on skeletal alterations in Hurler syndrome.},
}
@article {pmid41784267,
year = {2026},
author = {Wang, Z and Wu, Y and Wang, Z and Zhang, S and Liu, H and Nie, Y and Chen, K and Huang, Y and Zhou, Y and Cao, Y and Sun, L and Hao, R},
title = {crRNA scaffold remodeling controls CRISPR-Cas12a activity for enhanced performance.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41784267},
issn = {1362-4962},
support = {//Prevention and Control of Emerging/ ; 2025ZD01903403//National Science and Technology Major Project/ ; L234051//Beijing Municipal Natural Science Foundation/ ; L246011//Beijing Municipal Natural Science Foundation/ ; 2024-03-18//Training Plan for High-Level Public Health Technical Talents of Beijing Municipal Health Commission/ ; 2025ZD01903403//Prevention and Control of Emerging and Major Infectious Diseases-National Science/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Mycobacterium tuberculosis/genetics/isolation &amp; purification ; Klebsiella pneumoniae/genetics/isolation &amp; purification ; Nucleic Acid Conformation ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.},
}
@article {pmid41784340,
year = {2026},
author = {Zhou, C and Cheng, T and Zhou, J and Zhang, B and Liu, L and Jiang, G and Li, W and Wang, C},
title = {Synthetic Biofilms for Green Membranes: Engineering Low-Energy Filtration Systems.},
journal = {Environmental science &amp; technology},
volume = {60},
number = {12},
pages = {9357-9366},
doi = {10.1021/acs.est.5c15661},
pmid = {41784340},
issn = {1520-5851},
mesh = {*Biofilms ; Filtration ; *Membranes, Artificial ; Water Purification ; Biofouling ; Ultrafiltration ; },
abstract = {Membrane filtration is a key technology to modern water purification, yet its sustainability is compromised by biofouling, which increases energy consumption and ecological impacts. Conventional control strategies often struggle to balance efficacy and environmental footprint. In this study, an inducible, engineered quorum-quenching (QQ) bacterium was constructed via a genomic integration strategy, thereby achieving control over the biofilm structure in membrane filtration biofouling layers. By using the clustered regularly interspaced short palindromic repeat (CRISPR-Cas) targeted gene editing technology, the engineered bacteria that were constructed to express aiiO under l-ribose induction have achieved the regulation of biofilms. Validation using a gravity-driven membrane ultrafiltration system (UF-GDM) model showed that the engineered bacterium effectively reduced extracellular polymeric substances (EPS) components, increased the hydrophilic porosity of the residual biofilm, and decreased its stickiness. This approach reduced transmembrane pressure by 64.5%, increased total organic carbon (TOC) removal by 13.2%, and extended membrane lifespan by 16.1%. A technical-economic analysis indicates that the 100,000 m[3]/day treatment plant achieves an annual net profit increase of 31.52%, reaching 1.55 × 10[7] CNY, while reducing its net carbon footprint by 27.43%, with an annual net reduction of 2.96 × 10[5] kg CO2eq. This study provides a novel solution strategy for achieving biofouling resistance and sustainable, low-energy operation in membrane filtration processes, which contributes to the broader application and adoption of this technology.},
}
@article {pmid41784822,
year = {2026},
author = {Eweda, MA and Li, J and Hassan, U and Jalil, S and Jin, X},
title = {Peroxisomal acyl-CoA oxidase OsACX4 negatively regulates salt and drought stress tolerance by modulating cellular redox homeostasis in rice.},
journal = {Plant cell reports},
volume = {45},
number = {3},
pages = {},
pmid = {41784822},
issn = {1432-203X},
support = {517000-N12502ZJ//National Natural Science Foundation of China/ ; 2025ZDXT01-4//The Major Agricultural Technology Collaborative Extension Project/ ; LZ25C130006//Natural Science Foundation of Zhejiang Province/ ; 2021C02063-6//Science and Technology Department of Zhejiang Province/ ; },
mesh = {*Oryza/genetics/physiology/enzymology/metabolism ; Homeostasis ; Oxidation-Reduction ; *Plant Proteins/metabolism/genetics ; Droughts ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; Peroxisomes/metabolism/enzymology ; *Acyl-CoA Oxidase/metabolism/genetics ; Reactive Oxygen Species/metabolism ; Stress, Physiological/genetics ; *Salt Tolerance/genetics ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Oxidative Stress ; },
abstract = {OsACX4 knockout reduces peroxisomal oxidative stress, enhancing rice drought and salt tolerance through metabolic-redox rebalancing for climate-resilient breeding. Climate change is intensifying the frequency and severity of abiotic stress, such as salt and drought stresses, which severely limit rice productivity worldwide, necessitating the identification of molecular targets for crop improvement. This study provides the first comprehensive functional characterization of the peroxisomal acyl-CoA oxidase OsACX4 in rice (Oryza sativa L.) drought and salinity tolerance, revealing its unexpected role as a negative regulator of stress tolerance through modulation of cellular redox homeostasis. Through genome editing using CRISPR/Cas9-mediated knockout and overexpression approaches, we generated transgenic lines to investigate the function of OsACX4 under salt and drought stress. Knockout lines exhibited superior stress tolerance compared to the wild-type (WT) and overexpression lines, demonstrating significantly higher survival rates under severe stress conditions. Enhanced tolerance correlated with coordinated upregulation of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) activities. Knockout lines accumulated substantially higher proline (Pro) levels while maintaining markedly reduced reactive oxygen species (ROS) compared to overexpression lines. Transcriptional analysis revealed that OsACX4 disruption triggered upregulation of stress-responsive genes, including OsSOD1, OsDREB2A, OsDREB1B, and OsAPX1 under severe stress. Subcellular localization confirmed peroxisomal targeting of OsACX4, where its β-oxidation activity generates hydrogen peroxide (H2O2) as a metabolic by-product. Our results reveal a metabolic trade-off whereby stress-induced OsACX4 expression mobilizes energy reserves but compromises cellular redox homeostasis. The superior performance of knockout lines under both stresses demonstrates that strategic OsACX4 disruption enhances plant resilience, identifying this gene as a promising breeding target for developing climate-resilient rice varieties through precision genome editing.},
}
@article {pmid41785043,
year = {2026},
author = {Xu, X and Zhang, R and Hai, G and Wang, Y and Zheng, H and Cui, P and Kou, B and Jin, X and Peng, J},
title = {DNA Logic-Gated CRISPR/Cas13a and PNTs-Hemin Biomimetic Nanozyme for Ratiometric Detection of BRCA1 and circROBO1.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7580-7589},
doi = {10.1021/acs.analchem.5c07475},
pmid = {41785043},
issn = {1520-6882},
mesh = {*Hemin/chemistry ; Humans ; *BRCA1 Protein/analysis ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Biomimetic Materials/chemistry ; *DNA/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Logic ; Electrochemical Techniques ; },
abstract = {Although nanozyme-based biosensors show great promise for the early diagnosis of cancer, their application is often limited by poor catalytic activity at neutral pH and susceptibility to matrix interference. This study involved the construction of a biomimetic nanozyme through coordination-driven self-assembly on peptide nanotubes. This rigid framework enables the periodic arrangement of histidine residues to achieve precise axial coordination with the iron center of the hemin molecule, effectively mimicking the active site and catalytic microenvironment of natural horseradish peroxidase. Consequently, PNTs-hemin exhibits peroxidase activity 2.7 times that of free hemin under near-physiological conditions. To ensure detection specificity, an AND logic gate design was integrated, triggering CRISPR/Cas13a-mediated trans-cleavage only when both targets (BRCA1 and circROBO1) are present simultaneously. Furthermore, combining the nanozyme with graphdiyne-supported gold nanoparticles resulted in the formation of a cascade catalytic system that produced a ratiometric electrochemical reading (IFc/Ihemin). This effectively corrects for environmental fluctuations and false positive signals. This study presents an effective strategy that combines the specificity of molecular logic gates with biomimetic catalysis. This opens up new avenues for the precise diagnosis of multiple targets in complex biological samples.},
}
@article {pmid41785318,
year = {2026},
author = {Weber, LI and Timpen, LE and Egger-Hörschinger, AS and Schöpf, P and Ayhan, ND and Demmel, D and Hotze, M and Zhang, Y and Mehrabi, M and Puglisi, K and Stefan, E and Ghaffari-Tabrizi-Wizsy, N and Ramos-Pittol, JM and Kwiatkowski, M and Hartl, M},
title = {Reactivation of the silenced BASP1 gene suppresses oncogenic WNT signaling in human colorectal cancer cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2524159123},
pmid = {41785318},
issn = {1091-6490},
support = {P33662//Austrian Science Fund (FWF)/ ; },
mesh = {Humans ; *Wnt Signaling Pathway/genetics ; *Colorectal Neoplasms/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; *Nerve Tissue Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; beta Catenin/metabolism/genetics ; Promoter Regions, Genetic ; Gene Silencing ; Cell Movement ; },
abstract = {Starting from human colon cancer cells showing aberrant WNT/β-catenin/TCF signaling, hyperactivated MYC, and silenced BASP1, we generated stable cell lines overexpressing BASP1, either ectopically, or by reactivating the dormant BASP1 promoter using a lentiviral CRISPR-based system. BASP1 encodes a neuronal signaling protein and transcriptional corepressor, from which tumor-suppressive functions have been described in avian cell systems and in multiple human cancer cell types. Proteome and transcriptome analyses revealed activation of several tumor and metastasis suppressors in BASP1-expressing cells, which also show strong repression of the transformed phenotype in terms of contact inhibition, anchorage-independent growth, and tumor formation. Cells with reactivated BASP1 display a flat and differentiated morphology with enhanced migratory potential, accompanied by expression of multiple genes implicated in actin polymerization, focal adhesion, and neuronal migration. Furthermore, MYC protein expression is substantially repressed due to BASP1-mediated transcriptional MYC downregulation involving BASP1 interaction with β-catenin and binding to the MYC promoter. Upon BASP1 activation, multiple key proteins of the canonical WNT signaling pathway become suppressed. One of these BASP1 targets is the protein kinase TNIK catalyzing phosphorylation of TCF7L2, the latter required for transcriptional MYC activation. Results obtained with a preclinical TNIK inhibitor in human colorectal cancer cells show efficient abrogation of MYC expression and consequently impaired dimerization with its interaction partner MAX. The antagonistic BASP1 effect on MYC and the MYC dependency on TNIK could enhance the development of strategies to interfere with oncogenic functions of the cancer driver MYC.},
}
@article {pmid41785320,
year = {2026},
author = {Zhang, X and Richart, D and McFarlin, S and Cheng, F and Park, SY and Zhang-Chen, A and McFarlane, R and Xiao, C and Yan, Z and Qiu, J},
title = {Identification of CD164 as an essential entry receptor for divergent adeno-associated viruses.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {10},
pages = {e2525865123},
pmid = {41785320},
issn = {1091-6490},
support = {R21 AI166293/AI/NIAID NIH HHS/United States ; GM129525//HHS | National Institutes of Health (NIH)/ ; R21 AI180416/AI/NIAID NIH HHS/United States ; R01 HL174593/HL/NHLBI NIH HHS/United States ; AI166293//HHS | National Institutes of Health (NIH)/ ; AH-2126-20220331//Welch Foundation (The Welch Foundation)/ ; AI182645//HHS | National Institutes of Health (NIH)/ ; AI180416//HHS | National Institutes of Health (NIH)/ ; R21 AI182645/AI/NIAID NIH HHS/United States ; HL174593//HHS | National Institutes of Health (NIH)/ ; YAN23G0//Cystic Fibrosis Foundation (CFF)/ ; R01 AI150877/AI/NIAID NIH HHS/United States ; },
mesh = {*Dependovirus/genetics/physiology/metabolism ; Animals ; Mice ; Humans ; Mice, Knockout ; *Virus Internalization ; *Receptors, Cell Surface/metabolism/genetics ; HEK293 Cells ; *Antigens, CD/metabolism/genetics ; Genetic Vectors/genetics ; Transduction, Genetic ; CRISPR-Cas Systems ; *Receptors, Virus/metabolism/genetics ; Capsid/metabolism ; },
abstract = {Recombinant adeno-associated viruses (rAAVs) are widely used for in vivo gene delivery. While KIAA0319L, known as AAV receptor (AAVR), is essential for the transduction of multiserotype AAVs, it is dispensable for AAV4-related (Clade G) AAVs. We conducted a genome-wide CRISPR/Cas9 screen and identified CD164, a type I transmembrane sialomucin, as an essential entry receptor for Clade G AAVs. Ablation of CD164 expression substantially impaired both entry and transduction of Clade G AAVs. CD164-targeting antibodies and soluble CD164 ectodomain effectively blocked transduction. AAV4 capsids colocalized with CD164 at the plasma membrane and in endosomal compartments. In vitro, CD164 interacted with AAV4 or AAVrh32.33 capsids at high affinity. Importantly, systemic administration of rAAV4 or rAAVrh32.33 in CD164 knockout (KO) mice resulted in nearly complete loss of transgene expression. These findings establish CD164 as an essential entry receptor for Clade G AAV vectors and uncover a distinct AAVR-independent mechanism of AAV tropism.},
}
@article {pmid41785636,
year = {2026},
author = {Xue, S and Sun, H and Hou, X and Li, N and Xue, L and Dai, E and Wan, J},
title = {An off-target exonuclease activity in AsCpf1 undermines CRISPR diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {303},
number = {},
pages = {118578},
doi = {10.1016/j.bios.2026.118578},
pmid = {41785636},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics/analysis ; Humans ; DNA/genetics/chemistry ; *Exonucleases/chemistry ; Exodeoxyribonucleases/chemistry ; },
abstract = {The extensive utilization of CRISPR-Cas systems in molecular diagnostics stems from their crRNA-guided trans-cleavage capabilities. However, AsCpf1-based detection systems frequently exhibit unexplained sensitive variations. This research reveals that AsCpf1 maintains a crRNA-independent function, similar to exonuclease I, when utilized in standard buffers containing Mg[2+]. From a structural perspective, this exonuclease activity is independent of the RuvC domain-mediated canonical trans-cleavage activity. It is predicted by structural modeling to be potentially localized within the WED-PI domain. In the context of diagnostics, the effective target concentration is diminished by AsCpf1-mediated degradation of the free 3' ends of target DNA, which impairs detection sensitivity. To mitigate this interference, we demonstrate that 3' end capping effectively restores detection performance. This approach was validated in a CRISPR-EXPAR-based microRNA biosensor, which exhibited approximately 10-fold improvement in sensitivity following 3' end capping. Overall, this investigation characterizes a previously unidentified exonuclease activity within the AsCpf1 system and establishes practical design criteria to improve the robustness and accuracy of CRISPR-based diagnostic tools.},
}
@article {pmid41785880,
year = {2026},
author = {Petersen, A&#xd8; and Damholt, B and Grove, M and Hink, J and Marotte-Hurbon, T and Söderqvist, J and Troy, A and Zdravkovic, M and Bayer, L and Brunner, K and Bryde, T and Clube, J and Gencay, YE and Gram, A and Haaber, JK and Hallström, B and Jasinskytė, D and Pascal, R and Petersen, M and Semsey, S and Torio, AS and Turcu, IC and Smrekar, F and Taur, Y and Satlin, MJ and Sommer, MOA and van der Helm, E and Grøndahl, C},
title = {Safety, recovery, and pharmacodynamics of CRISPR-Cas therapeutic SNIPR001: a phase 1, randomised, double-blind, first-in-human, dose-escalation study.},
journal = {The Lancet. Microbe},
volume = {7},
number = {4},
pages = {101257},
doi = {10.1016/j.lanmic.2025.101257},
pmid = {41785880},
issn = {2666-5247},
mesh = {Humans ; Double-Blind Method ; Adult ; Middle Aged ; Male ; Female ; Young Adult ; Aged ; Escherichia coli/virology ; Adolescent ; *CRISPR-Cas Systems ; Feces/microbiology ; Gastrointestinal Microbiome ; Healthy Volunteers ; *Bacteriophages/genetics ; *Escherichia coli Infections/therapy ; },
abstract = {BACKGROUND: Patients with haematological cancer who receive stem-cell transplantation are at risk of bloodstream infections, often caused by multidrug resistant gut pathogens such as Escherichia coli. SNIPR001 is a cocktail of four CRISPR-Cas-armed bacteriophages that reduce colonisation of E coli in the gastrointestinal tract in animal models and is designed to not affect other members of the commensal microbiota. We aimed to investigate the safety and tolerability of SNIPR001 in healthy participants.<br><br>METHODS: In this randomised, placebo-controlled, double-blind, first-in-human, dose-escalation trial conducted at a single centre (Medpace Clinical Pharmacology Unit; Cincinnati, OH, USA), we sequentially enrolled healthy participants (aged 18-65 years) with more than 10[7]E coli colony-forming units per gram of stool into cohorts 1, 2, and 3, pending a safety review of the previous enrolment group where applicable. Participants in each cohort were randomly assigned to treatment or placebo using a unique three-digit participant identification number. Participants were orally administered 10[8] plaque-forming units (PFU) per dose (cohort 1), 10[10] PFU per dose (cohort 2), and 10[12] PFU per dose (cohort 3) of SNIPR001 or placebo (phosphate-buffered saline buffer), twice daily for 7 days. All personnel, except for a pharmacy staff member who prepared both SNIPR001 and placebo vials, were masked to the administered dose and assignment; masking was ensured by fully covering the surface of each vial. Participants were followed up to day 187. The primary outcome was the incidence and severity of adverse events and medically attended adverse events from the first administration of the study drug until 4 weeks after the last dose administration on day 35 of the study. Recovery and biodistribution of SNIPR001 in faeces, blood, and urine; pharmacodynamics, including the ability of SNIPR001 to reduce E coli levels in stool (assessed using a linear mixed-effects model); and microbiome composition (using Bray-Curtis dissimilarity) were secondary outcomes. Primary safety analyses were assessed per-protocol (ie, all enrolled participants who received at least one administration of the study drug). This trial was conducted under an Investigational New Drug application from the US Food and Drug Administration, is registered with ClinicalTrials.gov (NCT05277350), and is closed to new participants.<br><br>FINDINGS: The trial was carried out between March 24, 2022, and Nov 30, 2022. 36 eligible participants were randomly assigned to receive SNIPR001 or placebo in cohorts 1 (six assigned to 10[8] PFU per dose and two assigned to placebo), 2 (six to 10[10] PFU per dose and two to placebo), and 3 (12 to 10[12] PFU per dose and eight to placebo). The mean age of participants was 42&#xb7;1 years (SD 13&#xb7;8), with 14 (39%) female participants and 22 (61%) male participants. During the trial and 4-week follow-up period, only mild and moderate adverse events were observed, with most adverse events occurring in the placebo group (13, six, one, and nine for participants receiving either placebo or SNIPR001 at 10[8], 10[10], and 10[12] PFU twice a day, respectively). The number of participants who had adverse events was not significantly higher in treatment groups than in the placebo group (p=0&#xb7;94, one-sided Fisher's exact test). The most frequently reported adverse events were headaches and diarrhoea. No grade 3-4 adverse events were reported and no serious adverse events were reported in the SNIPR001 dose groups. During and after the dosing period, the gut microbiota composition did not significantly differ between the treatment and placebo groups (p>0&#xb7;05, two-sided Mann-Whitney U test of Bray-Curtis distances, false discovery rate [FDR]-corrected). Functional SNIPR001 was recovered from stool samples in concentrations proportional to the administered dose but was not meaningfully detected in plasma (only one sample) or urine (only one sample). SNIPR001 was undetected in all samples 6 months after the last dosing, which is a favourable pharmacokinetic property and meets regulatory expectations. We observed the largest reduction in E coli levels compared with placebo 2 weeks after treatment initiation at day 14 (78%; -0&#xb7;65 log10 [SE 0&#xb7;64] for 10[12] PFU SNIPR001 twice-daily group), according to a linear mixed-effects model for the highest dose population; however, this change was not statistically significant (p=0&#xb7;811, linear mixed-effects model, FDR-corrected).<br><br>INTERPRETATION: This first-in-human study of SNIPR001 supported its safety, tolerability, and restriction to the gastrointestinal tract, while not systemically disrupting the gut microbiome. These results justify further clinical development of SNIPR001 in an ongoing phase 1b/2a trial.<br><br>FUNDING: Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) and SNIPR Biome.},
}
@article {pmid41787916,
year = {2026},
author = {Saboor, M and Jasem Alblooshi, M and Adel Alkaabi, A and Ramazan Soozaei, F and Hamad Alketbi, M},
title = {CRISPR in Thalassemia: Global Research Trend Analysis.},
journal = {Hemoglobin},
volume = {50},
number = {2},
pages = {141-155},
doi = {10.1080/03630269.2026.2634815},
pmid = {41787916},
issn = {1532-432X},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *beta-Thalassemia/genetics/therapy ; *Thalassemia/genetics/therapy ; Genetic Therapy/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bibliometrics ; Pattern Analysis, Machine ; },
abstract = {β-Thalassemia is a prevalent inherited disorder of β-globin chains. The clustered regularly interspaced short palindromic repeats (CRISPR) genome editing system has emerged as a potential curative strategy. We conducted a bibliometric analysis to map global research trends in CRISPR-based thalassemia research. Original and review research articles were retrieved from the Scopus database using the search terms [TITLE-ABS-KEY ('βeta thalassemia' OR 'β thalassemia' OR thalassemia*) AND TITLE-ABS-KEY ('gene edit*' OR crispr* OR 'clustered regularly interspaced short palindromic repeats')] AND [LIMIT-TO (DOCTYPE, 're') OR LIMIT-TO (DOCTYPE, 'ar')] for analysis. Bibliometric mapping and network visualization were performed using VOSviewer to analyze publication trends, authorship networks, international collaborations, keyword clusters, and citation metrics. Major CRISPR-based therapeutic strategies for thalassemia were reviewed to place experimental and clinical developments within a translational framework. The analysis demonstrates a clear transition from foundational genomic studies to translational applications, with leading contributions from the United States and China. Two dominant therapeutic strategies have emerged: direct correction of the HBB gene in hematopoietic stem cells and fetal hemoglobin reactivation via BCL11A repression. The latter strategy culminated in regulatory approval of exagamglogene autotemcel (Casgevy). Advances in base editing, prime editing, and strategies to improve engraftment are expected to enhance the precision and long-term efficacy of next-generation approaches. Clustered regularly interspaced short palindromic repeats-based research on thalassemia continues to expand, supported by extensive international collaboration and growing clinical translation. Future large-scale implementation will require advances in bioprocess engineering, cost reduction for ex vivo manufacturing, and adaptable treatment models for diverse healthcare systems.},
}
@article {pmid41787951,
year = {2026},
author = {Chatla, K and Roper, B and Ayalew, L and Ko, P and Lippold, S and Doma, M and Camperi, J},
title = {Assessing mRNA and sgRNA Quality for Cell and Gene Therapy Applications Using Nanopore Direct RNA Sequencing.},
journal = {Analytical chemistry},
volume = {98},
number = {10},
pages = {7452-7461},
doi = {10.1021/acs.analchem.5c06819},
pmid = {41787951},
issn = {1520-6882},
mesh = {*RNA, Messenger/genetics/analysis ; Humans ; *Sequence Analysis, RNA/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics/analysis ; *Genetic Therapy ; *Nanopore Sequencing/methods ; *Nanopores ; },
abstract = {Recent advances in RNA technology have enabled the development of diverse therapeutics spanning vaccines, immunotherapies, and genome-editing platforms. Ensuring clinical efficacy and safety requires precise characterization and control of RNA critical quality attributes (CQAs). Nanopore direct RNA sequencing (NDRS) has emerged as a powerful single-molecule analytical approach capable of simultaneously resolving sequence and structural features consistent with regulatory expectations. In this study, we establish NDRS as a comprehensive, multiattribute analytical platform by integrating novel strategies to assess key CQAs in a single assay. Following workflow optimization, NDRS accurately determined full-length mRNA sequences and evaluated transcript integrity. Notably, we developed the first sequencing-based method for quantifying 5' capping efficiency directly from native RNA molecules. Additionally, we demonstrated, for the first time, full-length sequencing of 100-nucleotide single-guide RNA (sgRNA) molecules by incorporating a 5' RNA oligo adapter, enabling complete identity verification. Quantitative results for poly(A) tail length, integrity, and capping efficiency were consistent with established orthogonal techniques, including chromatography and mass spectrometry. Moreover, functional correlation studies with Cas9 mRNA and sgRNA used in CRISPR-Cas9 editing revealed that increased mRNA degradation led to decreased knockout efficiency. Together, these findings position NDRS as a versatile and unified analytical platform for comprehensive characterization of mRNA and sgRNA, supporting quality assurance, comparability, and control in the development and manufacturing of next-generation RNA therapeutics.},
}
@article {pmid41789142,
year = {2026},
author = {Xu, C and Zeng, C and Wang, M and Wei, X and Song, M and Liu, X and Wang, W and Chen, Q and Ji, X and Luo, P and Ma, L and Sun, Y and Gou, H and Zhu, Z and Li, X and Lv, YX and Liu, P and Zhu, JK},
title = {mRNA-engineered CRISPR-Cas epigenetic editors enable durable and efficient gene silencing in vivo.},
journal = {Innovation (Cambridge (Mass.))},
volume = {7},
number = {3},
pages = {101151},
pmid = {41789142},
issn = {2666-6758},
abstract = {Programmable epigenetic editors (EEs) that achieve long-term gene expression modulation without altering the DNA sequence hold immense therapeutic potential. However, the clinical translation of current CRISPR-based epigenome editors is impeded by substantial challenges, particularly their large molecular size, which limits efficient in vivo delivery. Here, we report the rational design and engineering of compact, mRNA-delivered EEs (CRISPR OFF-EE) using Streptococcus pyogenes Cas9 (SpCas9), intein-split-SpCas9, or the smaller Cas-SF01 (a Cas12i3 variant). Combined with optimized mRNA architecture and lipid nanoparticle (LNP) delivery, a single intravenous LNP administration of the optimized OFF-EE V2 mRNA, along with selected guide RNAs (gRNAs) targeting Pcsk9 in mice, resulted in an ∼83.2% reduction in circulating PCSK9 levels and a corresponding ∼51.4% reduction in low-density lipoprotein cholesterol (LDL-C) levels, persisting for at least 180 days. SF01-based EEs showed higher specificity with fewer off-target methylation events than SpCas9-based counterparts. Our optimized LNP formulation also demonstrated a favorable safety profile with predominantly liver-tropic activity. These findings establish a robust and versatile platform for advancing in vivo therapeutics based on precise and durable epigenetic silencing using transiently delivered, engineered mRNA editors.},
}
@article {pmid41790502,
year = {2026},
author = {Guo, S and Liang, Z and Liao, S and Chen, H and Xue, C and Yue, Z and Jiang, Y and Fang, X},
title = {CRISPR-Enhanced β-Carotene Biosynthesis: Droplet Microfluidics-Driven Pathway Optimization and High-Yield Terpenoid Strain Screening.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {10},
pages = {8497-8507},
doi = {10.1021/acs.jafc.5c10513},
pmid = {41790502},
issn = {1520-5118},
mesh = {*beta Carotene/biosynthesis ; *Terpenes/metabolism ; Metabolic Engineering/methods ; *Microfluidics/methods/instrumentation ; *Saccharomyces cerevisiae/metabolism/genetics ; Biosynthetic Pathways ; CRISPR-Cas Systems ; Mevalonic Acid/metabolism ; },
abstract = {Terpenoids are highly abundant and valuable natural products, yet high-throughput screening platforms for microbial producers remain scarce. To address this, we developed a microfluidic screening platform leveraging the intrinsic fluorescence of β-carotene. Using an engineered yeast chassis (YsL4) with a reinforced mevalonate pathway, we optimized cultivation conditions and achieved perfect (100%) phenotypic specificity in a 1:1 coencapsulation assay between producer (YsL4) and nonproducer (Ys011) strains. Screening a genome-wide CRISPRa/i library via multiround fluorescence-activated droplet sorting identified 15 target genes. Validation confirmed that 10 targets (5 for overexpression, 10 for knockout) significantly enhanced β-carotene production. A key mutant, YL19 (ΔHMO1), achieved a titer of 33.71 mg/L in shake-flasks─a 149.15% increase over the parent strain─with a concurrent lycopene reduction indicating redirected carbon flux toward β-carotene biosynthesis. This integrated platform enhances screening efficiency and provides a new paradigm for identifying critical terpenoid biosynthetic targets.},
}
@article {pmid41791263,
year = {2026},
author = {Cho, ES and Hu, JC and Kim, JW},
title = {Generation and characterization of a murine amelogenesis imperfecta model.},
journal = {Archives of oral biology},
volume = {186},
number = {},
pages = {106563},
doi = {10.1016/j.archoralbio.2026.106563},
pmid = {41791263},
issn = {1879-1506},
mesh = {Animals ; *Amelogenesis Imperfecta/genetics/pathology/diagnostic imaging ; Mice ; *Disease Models, Animal ; *Amelogenin/genetics ; X-Ray Microtomography ; Gene Knock-In Techniques ; Exons/genetics ; Mutation ; Dental Enamel/pathology ; Immunohistochemistry ; Alternative Splicing ; Genotype ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {OBJECTIVE: Amelogenesis imperfecta (AI) refers to a group of rare yet complex genetic disorders that affect the quantity and/or quality of tooth enamel. Recently, in AI patients, we identified mutations that disrupt a conserved alternative splicing pattern of the AMELX gene, which encodes amelogenin, the most abundant enamel matrix protein. These mutations led to the retention of exon 4, which is normally skipped during the pre-mRNA splicing process, resulting in the characteristic pitted, hypoplastic, and hypomineralized enamel defects. To observe the impact of retention of exon 4 within AMELX, a gene edited knock-in mouse model was generated.<br><br>DESIGN: A single-nucleotide knock-in mouse model was generated using CRISPR/Cas9 technology to introduce a silent mutation (NM_001415990.1: c.120&#x202f;T>C, p.(Ala40=)) that abrogated alternative splicing of exon 4. Following genomic sequence validation, the successfully-targeted mice were propagated, and their offspring genotyped for characterization. Micro-computed tomography analysis and immunohistochemistry analysis were performed on the hemi-mandibles of the wild-type and the knock-in mice.<br><br>RESULTS: The enamel of the knock-in mice was chalky white and lacked translucency, due to faulty mineralization. This defective enamel broke down soon after tooth eruption. During the maturation stage, the ameloblast layer lost its cellular polarity and homogeneity, and intermingled with adjacent cell types to form disorganized clusters.<br><br>CONCLUSIONS: The validated and characterized Amelx c.120&#x202f;T>C mouse model provides a useful platform for investigating the molecular pathophysiology associated with retention of the exon 4 sequence. Following systemic characterization, this mouse model will serve as an important tool for assessing therapeutic strategies aimed at ameliorating the disease phenotype.},
}
@article {pmid41791393,
year = {2026},
author = {Rapp, J and Verhülsdonk, A and Garcke, A and Stadelmann, A and Farke, N and Troßmann, F and Kronenberger, T and Alvarado, A and Petras, D and Link, H},
title = {The metabolome of an E. coli CRISPRi library identifies benefits of minimal metabolite levels and targets for engineering.},
journal = {Cell systems},
volume = {17},
number = {4},
pages = {101518},
doi = {10.1016/j.cels.2025.101518},
pmid = {41791393},
issn = {2405-4720},
mesh = {*Escherichia coli/metabolism/genetics ; *Metabolome/genetics ; *Metabolic Engineering/methods ; *CRISPR-Cas Systems/genetics ; Metabolic Networks and Pathways/genetics ; Gene Library ; Metabolomics/methods ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Tandem Mass Spectrometry ; },
abstract = {Metabolite concentration changes can have broad consequences on the function and robustness of metabolic networks. Here, we measured the metabolome response of 1,515 CRISPR interference (CRISPRi) E. coli strains targeting all genes in the iML1515 metabolic model. Metabolites that are hardly measurable in wild-type E. coli accumulated in specific CRISPRi strains, indicating that they are normally maintained at low levels. We confirmed metabolite accumulation using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and generated putative reference spectra for 102 metabolites for which no MS[2] data had previously been available. We show that minimal metabolite levels are beneficial because they (1) enable substrate level regulation of enzyme activity, (2) prevent competitive inhibition, and (3) suppress side reactions. However, minimal metabolite pools also limit flux through engineered pathways. For example, low levels of farnesyl diphosphate (frdp) constrained a synthetic carotenoid pathway, and we show that the knockdown of octaprenyl diphosphate synthase (IspB) increased frdp levels and carotenoid production. A record of this paper's transparent peer review process is included in the supplemental information.},
}
@article {pmid41791397,
year = {2026},
author = {Low, SJ and O'Neill, MT and Fernando, JA and Kerry, WJ and Prestedge, J and Wild, N and Chahal, S and Pollock, GL and Papadakis, G and Krysiak, M and Williams, E and Azzato, F and Tran, T and Fairley, C and Bradshaw, C and Chen, MY and Lim, CK and Williamson, DA and Pasricha, S},
title = {CRISPR-Cas-based diagnostics for point-of-care detection of sexually transmitted infections: a laboratory development and evaluation study.},
journal = {The Lancet. Microbe},
volume = {7},
number = {4},
pages = {101289},
doi = {10.1016/j.lanmic.2025.101289},
pmid = {41791397},
issn = {2666-5247},
mesh = {Humans ; *CRISPR-Cas Systems ; *Sexually Transmitted Diseases/diagnosis/microbiology ; Neisseria gonorrhoeae/genetics/isolation &amp; purification ; Sensitivity and Specificity ; Chlamydia trachomatis/genetics/isolation &amp; purification ; *Point-of-Care Systems ; *Point-of-Care Testing ; Treponema pallidum/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; Gonorrhea/diagnosis ; Female ; Nucleic Acid Amplification Techniques/methods ; Simplexvirus/genetics/isolation &amp; purification ; Male ; Chlamydia Infections/diagnosis ; },
abstract = {BACKGROUND: Timely, point-of-care diagnosis of sexually transmitted infections (STIs) is crucial for enabling prompt treatment and reducing transmission. We aimed to develop a portable, multiplexed, CRISPR-based assay panel for the detection of Neisseria gonorrhoeae (including the ciprofloxacin resistance marker gyrA S91F), Chlamydia trachomatis, Treponema pallidum, and herpes simplex virus (HSV).<br><br>METHODS: In this laboratory development and evaluation study, we developed and optimised four multiplexed, CRISPR-based, diagnostic STI assays for point-of-care use. The complete assay panel comprised a CRISPR TP-HSV (cTP-HSV) panel for the detection of T pallidum and pan-HSV, with reflex testing to distinguish HSV-1 from HSV-2, and a CRISPR NG-CT (cNG-CT) panel for the detection of N gonorrhoeae and C trachomatis, with reflex testing to detect N gonorrhoeae using two additional genome regions and to identify the gyrA S91F mutation. Each pathogen was targeted at two independent genomic regions by isothermal amplification and CRISPR-Cas reaction using Cas12a and Cas13a, each with distinct fluorescent reporters. Analytical specificity and limits of detection (LODs) were determined, and a retrospective, masked concordance study was conducted on genomic DNA from 900 clinical samples (400 for cTP-HSV and reflex testing and 500 for cNG-CT and reflex testing), using quantitative PCR as the reference standard. The diagnostic accuracy of the test was assessed by analysis of receiver operating characteristic curves.<br><br>FINDINGS: The overall sensitivity of the TP-HSV CRISPR assay was 82&#xb7;5% (95% CI 74&#xb7;0-88&#xb7;7) for T pallidum and 94&#xb7;4% (90&#xb7;2-97&#xb7;0) for pan-HSV; LODs were 6&#xb7;2 copies per &#x3bc;L for T pallidum and 7&#xb7;8 copies per &#x3bc;L for HSV. Reflex testing gave sensitivities of 97&#xb7;0% (91&#xb7;1-99&#xb7;3) for HSV-1 and 96&#xb7;0% (89&#xb7;7-98&#xb7;7) for HSV-2. The NG-CT CRISPR assay had an overall sensitivity of 80&#xb7;0% (74&#xb7;0-84&#xb7;9) for N gonorrhoeae and 73&#xb7;0% (65&#xb7;5-79&#xb7;3) for C trachomatis, with a LOD of 3&#xb7;9 copies per &#x3bc;L for both pathogens. Reflex testing for the detection of the gyrA S91F mutation in N gonorrhoeae showed an overall sensitivity of 63&#xb7;1% (55&#xb7;1-70&#xb7;4); however, this was dependent on sample type, with a sensitivity of 85&#xb7;7% (46&#xb7;7-99&#xb7;5) in genital samples and 61&#xb7;2% (52&#xb7;8-68&#xb7;9) in extragenital samples. For all pathogens, assay sensitivity was positively correlated with pathogen load. Area under the curve (AUC) values were 0&#xb7;90 for T pallidum and 0&#xb7;99 for pan-HSV in the TP-HSV assay, with values of 0&#xb7;99 for HSV-1 and 0&#xb7;97 for HSV-2 obtained in the reflex HSV-1-HSV-2 assay. For the cNG-CT assay, AUC values were 0&#xb7;90 for N gonorrhoeae and 0&#xb7;85 for C trachomatis, with a value of 0&#xb7;72 obtained for gyrA S91F in the reflex cNG-gyrA assay.<br><br>INTERPRETATION: Our multiplexed, CRISPR-based, point-of-care platform achieved performance consistent with WHO target product profiles for N gonorrhoeae and T pallidum. Proof-of-concept detection of the gyrA S91F resistance marker highlights its potential for resistance-guided therapy. Although optimisation is required before large-scale deployment, this suite offers a promising approach for rapid, decentralised, and resistance-informed STI diagnosis, particularly in resource-limited settings.<br><br>FUNDING: Victorian Government Department of Health, Australian Government Department of Health, Disability and Ageing and Aged Care, and Australian Research Council.},
}
@article {pmid41791700,
year = {2026},
author = {Xiang, Z and Guo, K and Xi, J and Xu, S and Duan, J and Wen, S and Liu, Z and Wang, X and Zhao, P and Zhang, X},
title = {CRISPR/Cas9-mediated knockout of SPI51 reveals an essential role of protease inhibitors in silk fiber formation.},
journal = {Journal of insect physiology},
volume = {170},
number = {},
pages = {104962},
doi = {10.1016/j.jinsphys.2026.104962},
pmid = {41791700},
issn = {1879-1611},
mesh = {Animals ; *Silk/metabolism/chemistry ; CRISPR-Cas Systems ; *Bombyx/genetics/metabolism/growth &amp; development ; *Protease Inhibitors/metabolism ; *Insect Proteins/genetics/metabolism ; Fibroins/metabolism ; Gene Knockout Techniques ; },
abstract = {Silkworm silk is a natural protein fiber composed mainly of fibroin and sericin, with protease inhibitors representing an additional abundant constituent. However, the impact of protease inhibitors on the structure and properties of silk fibers has not yet been studied. In this study, we focused on the Kunitz-type protease inhibitor SPI51, the most abundant protease inhibitors of cocoon. CRISPR/Cas9 editing was used to generate a homozygous mutant of SPI51 (SPI51[KO]), resulting in premature translation termination at the 33rd amino acid. After knocking out SPI51, the mechanical properties of silk were significantly reduced compared with those of the wild type. Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) results revealed that this deterioration was associated with significantly reduced β-sheet content and lower crystallinity. Morphological observations showed that the fibroin area of SPI51[KO] silk was significantly smaller than that in the wild type. Further Western blot analysis showed that fibroin heavy chain (Fib-H), fibroin light chain (Fib-L), and fibrohexamerin (P25) were decreased after knocking out SPI51, which resulted in a reduction of silk fibroin layer and affected structure and mechanical properties. Our results provide valuable insights into the balance between proteases and protease inhibitors in the silk gland and reveal for the first time the roles of the protease inhibitor in silk protein synthesis and the structural and mechanical properties of silk fibers.},
}
@article {pmid41793913,
year = {2026},
author = {Li, X and Liu, L and Luo, C and Chen, Z and Shu, B},
title = {Efficient CRISPR/Cas9 system established via co-cultivation of plantlets and Agrobacterium tumefaciens for positive transgenic calluses generation and regeneration in cultivated strawberry (Fragaria × ananassa).},
journal = {Plant physiology and biochemistry : PPB},
volume = {232},
number = {},
pages = {111195},
doi = {10.1016/j.plaphy.2026.111195},
pmid = {41793913},
issn = {1873-2690},
mesh = {*Fragaria/genetics/growth &amp; development ; *Agrobacterium tumefaciens/genetics ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics ; Gene Editing/methods ; Coculture Techniques ; Regeneration/genetics ; },
abstract = {Recently, an Agrobacterium-mediated CRISPR/Cas9 editing system was successfully applied in a gene function analysis, highlighting its great value for improving strawberry genetics. However, the resulting low transformation rates and long regeneration cycles have limited its extensive application. Based on the biological characteristics of crown branching, an Agrobacterium tumefaciens-mediated CRISPR/Cas9 gene editing system was developed to increase the transformation rate and decrease the regeneration time of cultivated strawberry. Two single guide (sg)RNAs were designed for the strawberry anthracnose-related transcription factor, WRKY (FxaC_17g55530), and its alleles. These sgRNAs were inserted into pKSE401G using pCBC-DT1T2; sgRNAs for subtilisin-like protease (FxaC_22g21540) were designed and cloned in a similar manner. After 10 days of co-cultivating plantlets (without media supply of carbon) and GV3101, 65 (61.9%) and 72 (68.6%) GFP-positive calluses for the two genes were respectively obtained from the crown of 105 plantlets. The positive calluses were removed from the crown and placed on Murashige and Skoog media containing 3 mg/L thidiazuron and 0.2 mg/L indole-3-butyric acid. After 50-80 days, 3-5 positive shoots were obtained from different positive calluses for each gene. The three T0 lines for FxaC_17g55530 and FxaC_22g21540 were found to be successfully edited at the target sites of both sgRNA1 and sgRNA2 or either sgRNA1 or sgRNA2. Overall, a quick and effective CRISPR-Cas 9 gene editing system was developed for cultivated strawberry, highlighting the applicability of gene editing in breeding and gene function analysis.},
}
@article {pmid41794473,
year = {2026},
author = {Safenkova, IV and Kamionskaya, MV and Serchenya, TS and Sviridov, OV and Dzantiev, BB and Zherdev, AV},
title = {CRISPR/Cas12a and fork-shaped probe enhance LAMP-LFT integration for equipment-free detection of Listeria monocytogenes.},
journal = {Food research international (Ottawa, Ont.)},
volume = {230},
number = {},
pages = {118592},
doi = {10.1016/j.foodres.2026.118592},
pmid = {41794473},
issn = {1873-7145},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Listeria monocytogenes/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Food Microbiology/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; DNA, Bacterial/genetics ; Food Contamination/analysis ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Listeria monocytogenes is an important foodborne pathogen associated with high mortality rates, especially among vulnerable populations, and therefore requires diagnostic methods that are not only highly sensitive and rapid but also suitable for use in resource-limited settings. In this study, we developed an isothermal amplification assay integrated with a lateral flow test (LFT) for reliable detection of L.monocytogenes. Two assay formats were designed and compared: (1) loop-mediated isothermal amplification (LAMP) with LFT detection of fluorescein- and biotin-labeled amplicons, and (2) LAMP combined with CRISPR/Cas12a, using LFT to detect a cleaved fork-shaped enhanced probe labeled with three fluoresceins. Both LFT formats utilized a common conjugate of gold nanoparticles and anti-fluorescein antibodies (anti-FAM), but differed in the test zone immobilization strategy: streptavidin for LAMP, and anti-FAM for LAMP-CRISPR/Cas12a. Among 12 tested (primer - label) combinations, the most effective was identified, but the sensitivity of the LAMP-LFT format was limited by high signal variability. In contrast, the LAMP-CRISPR/Cas12a assay, targeting LAMP amplicons with guide RNA, achieved a detection limit of 0.9 copies/reaction-representing > 20,000-fold improvement in detectable DNA concentration compared with LAMP-LFT-and comparable to fluorescence-based detection techniques. The LAMP-CRISPR/Cas12a-LFT assay was first reported to detect L.monocytogenes cells following thermal lysis (10 min at 95 °C), with a single-cell detection limit (0.2 cells/reaction in buffer, 1 cells/reaction in spiked milk) and an analysis time of 80 min. These results demonstrate the potential of the approach for sensitive, equipment-free detection of foodborne pathogens in complex food matrices.},
}
@article {pmid41795439,
year = {2026},
author = {Zhu, L and Yang, C and Bernards, R and Wang, C},
title = {CLIM-TIME links genetic cancer drivers to immune landscapes.},
journal = {Cell},
volume = {189},
number = {5},
pages = {1263-1265},
doi = {10.1016/j.cell.2026.01.014},
pmid = {41795439},
issn = {1097-4172},
mesh = {Humans ; *Neoplasms/genetics/immunology/therapy ; Immunotherapy ; Tumor Microenvironment/immunology/genetics ; Animals ; CRISPR-Cas Systems ; Genes, Tumor Suppressor ; },
abstract = {Immunotherapy resistance is associated with immune-privileged microenvironments, yet the interacting role of tumor-intrinsic genetics remains unclear. In this issue of Cell, Wang et al. introduce CLIM-TIME, a spatially resolved in vivo CRISPR screening platform linking loss of tumor suppressor genes to distinct metastatic immune architectures and divergent responses to immunotherapy.},
}
@article {pmid41796459,
year = {2026},
author = {Matsuoka, T and Oda, K and Iwashita, K and Watanabe, J},
title = {Identification of DeuA, an Aspergillus oryzae-derived deuterolysin-like metalloprotease, as the predominant thermostable protease in soy sauce.},
journal = {Journal of bioscience and bioengineering},
volume = {141},
number = {6},
pages = {445-449},
doi = {10.1016/j.jbiosc.2026.02.010},
pmid = {41796459},
issn = {1347-4421},
mesh = {*Aspergillus oryzae/enzymology/genetics ; *Soy Foods/microbiology/analysis ; *Metalloproteases/genetics/metabolism/chemistry ; Fermentation ; Enzyme Stability ; *Fungal Proteins/genetics/metabolism/chemistry ; *Peptide Hydrolases/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; },
abstract = {The thermostable proteases present in soy sauce can degrade proteins in processed foods, such as boiled eggs and fish cakes, leading to undesirable textural changes in the product. In this study, we identified DeuA, a deuterolysin-like metalloprotease from Aspergillus oryzae, as the major contributor to thermostable protease activity during soy sauce fermentation. Using CRISPR/Cas9-based genome co-editing, we generated deuA-knockout mutants and mutants of the related gene deuB, which encodes a deuterolysin-like metalloprotease, and evaluated their enzymatic activity levels under solid-state culture conditions mimicking soy sauce koji fermentation. The ΔpyrGΔdeuA strain exhibited a marked reduction in thermostable protease activity, with residual activity barely detectable in both the koji extracts and the final soy sauce, whereas knockout of deuB had no significant effect. These results indicate that DeuA is the predominant contributor to thermostable protease activity in soy sauce. The knockout of deuA did not affect other key brewing parameters such as the nitrogen or sugar contents, indicating the potential of this gene as a target for strain improvement. Our findings establish DeuA as an essential thermostable protease in soy sauce and provide a foundation for the development of brewing strains with improved industrial applicability that will not affect the textural stability of processed foods.},
}
@article {pmid41796733,
year = {2026},
author = {Lin, J and Wang, Y and Zeng, B and Chen, Z and Lin, X and Zeng, T},
title = {CRISPR-Cas12a/Cas13a in cancer molecular diagnosis.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {587},
number = {},
pages = {120934},
doi = {10.1016/j.cca.2026.120934},
pmid = {41796733},
issn = {1873-3492},
abstract = {Cancer remains a leading cause of global mortality, with early diagnosis being pivotal for improving treatment outcomes. Traditional tissue biopsy is limited by its invasiveness, inability to capture tumor heterogeneity, and failure to support dynamic monitoring. Liquid biopsy has emerged as a non-invasive alternative, enabling the analysis of circulating tumor biomarkers (e.g., ctDNA, miRNAs, exosomes) in bodily fluids. However, current liquid biopsy technologies (e.g., NGS, ddPCR) suffer from high costs, complex workflows, poor standardization, and insufficient sensitivity for low-abundance biomarkers. The CRISPR-Cas systems, particularly Cas12a and Cas13a, have revolutionized molecular diagnostics due to their programmable sequence recognition, robust signal amplification via trans-cleavage/collateral cleavage activity, and compatibility with point-of-care testing (POCT). Cas12a targets DNA molecules, enabling sensitive detection of gene mutations and DNA methylation, while Cas13a specifically recognizes RNA, facilitating direct analysis of miRNAs and viral RNAs. Additionally, these systems have been extended to non-nucleic acid biomarkers (e.g., proteins, exosomes) through signal conversion strategies. This review summarizes the latest advances in CRISPR-Cas12a/Cas13a-based biosensors for cancer molecular diagnosis, including the detection of gene mutations, epigenetic modifications, miRNAs, tumor-associated viruses, and non-nucleic acid biomarkers. We critically analyze current challenges (e.g., PAM dependence, matrix interference, multiplexing limitations, clinical validation gaps) and discuss future perspectives, such as engineering PAM-less Cas variants, integrating nanotechnology, microfluidics, and artificial intelligence/artificial intelligence (AI), and advancing clinical standardization. This review aims to provide a comprehensive reference for the development and clinical translation of CRISPR-based cancer diagnostic technologies.},
}
@article {pmid41797538,
year = {2026},
author = {Fang, M and Yap, J and Fei, M and Gong, M and Li, N and Lu, Y and Yu, M and Xu, Y and Wu, F and Gao, H and Sun, D},
title = {LysR-type regulator LrhA promotes CRISPR-Cas immunity in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41797538},
issn = {1362-4962},
support = {32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; 31930003//National Natural Science Foundation of China/ ; 2020C02031//Key Research and Development Program of Zhejiang Province/ ; LHDMY23H160003//Natural Science Foundation of China/ ; YS2022005//Natural Science Foundation of China/ ; 2026C02A1080//Zhejiang Lingyan Research and Development Program/ ; LMRY26H200010//Joint Funds of the Zhejiang Provincial Natural Science Foundation of China/ ; },
mesh = {*Escherichia coli/genetics/immunology/virology ; *CRISPR-Cas Systems/genetics ; *Escherichia coli Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Promoter Regions, Genetic ; *Transcription Factors/genetics/metabolism ; Operon ; Plasmids/genetics ; Trans-Activators/genetics ; },
abstract = {The CRISPR-Cas defense system safeguards prokaryotes against foreign genetic elements. Its activity is determined by the combined effects of adaptation and interference. However, the dynamic regulation of these two processes remains not fully understood. In this study, we identify the LysR-type transcriptional regulator LrhA, which is differentially expressed in various Escherichia coli strains, as a novel CRISPR-Cas activator that plays a critical role in modulating host defense levels. In a representative strain expressing a high level of LrhA, the regulator enhances CRISPR-Cas-mediated adaptive immunity against bacteriophage infection by promoting cas gene transcription through direct interaction with the promoter of the cas operon. Moderate activation of cas genes by weakly expressed LrhA in another representative strain efficiently accelerates the clearance of horizontally transferred CRISPR-targeted plasmids by enhancing spacer acquisition via interference-driven adaptation. This divergence, likely a result of genome evolution, suggests that adaptive immunity is optimized with intermediate transcription levels of cas genes by triggering positive feedback between adaptation and interference. Collectively, our findings highlight the crucial role of LrhA in fine-tuning host defense responses.},
}
@article {pmid41799190,
year = {2026},
author = {Zhao, P and Li, H and Cai, Z and Zhang, X and Wen, X and Liu, Z and Jiang, S and Jiang, X and Wang, J and Dang, Z and Liu, M and Xie, F and Ma, X},
title = {Molecular hydrogen triggers TRPC4-TRPC4AP-dependent reversible calcium transients via extracellular influx.},
journal = {Theranostics},
volume = {16},
number = {9},
pages = {4843-4864},
pmid = {41799190},
issn = {1838-7640},
mesh = {Animals ; Mice ; *TRPC Cation Channels/metabolism/genetics ; *Calcium/metabolism ; Mice, Inbred C57BL ; *Calcium Signaling/drug effects ; *Hydrogen/metabolism/pharmacology ; Humans ; Cell Movement/drug effects ; Molecular Dynamics Simulation ; Molecular Docking Simulation ; Brain/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {RATIONALE: Hydrogen gas (H2) produces pleiotropic therapeutic actions, but the exact molecular targets and ion-channel-based signaling cascades that underlie these benefits remain elusive. H2 may regulate calcium ion (Ca[2+])-dependent processes, but the direct involvement of H2 in Ca[2+] signaling and its underlying molecular mechanisms are unknown. We propose that H2 functions as a gaseous messenger that selectively opens a plasma-membrane Ca[2+] channel to evoke Ca[2+] transients ([Ca[2+] i]t) while avoiding cytotoxic overload, thereby offering a mechanism for its diverse biological effects.<br><br>METHODS: This study employed real-time calcium imaging and CRISPR-Cas9 gene editing, with live-cell imaging to monitor real-time calcium signal intensity in living cells. Two-photon in vivo imaging was applied to detect real-time Ca[2+] signals in the brain and dorsal skin of C57BL/6 mice carrying adeno-associated virus-delivered calcium sensors. Live-cell F-actin staining and a wound healing (scratch) assay were used to assess the effects of H2 on cell motility. Protein-protein docking and molecular dynamics simulations were performed to analyze the interaction interface and binding forces between TRPC4 and TRPC4AP in three-dimensional space. Additionally, RNA sequencing was performed to validate downstream biological effects and transcriptional regulation triggered by H2.<br><br>RESULTS: H2 elicited rapid and reversible [Ca[2+] i]t across multiple cell types in a Ca[2+]- and concentration-dependent manner, an effect that was absent in TRPC4&#x207b;/&#x207b; or TRPC4AP&#x207b;/&#x207b; cells. In vivo imaging in mice expressing a genetically encoded Ca&#xb2;&#x207a; sensor showed that H2 inhalation elevated Ca[2+] signals in the motor cortex (M1 region) and dorsal skin. Functionally, live-cell imaging and wound-healing assays confirmed that H2-induced Ca[2+] transients enhanced cell motility. Mechanistically, protein&#x202f;docking revealed a dual-arginine cluster within the CIRB domain of TRPC4; its interaction with TRPC4AP was essential for H2-evoked Ca[2+] influx. Mutating these arginines to alanine residues completely abolishing the response. H2 triggered proton efflux and increased intracellular pH. Molecular dynamics simulations indicated that altered pH modulates the binding force between TRPC4 Arg730/Arg731 and TRPC4AP. Transcriptomic analysis further demonstrated that H2 activates calcium-related channels and promotes cytoskeletal remodeling and cell migration.<br><br>CONCLUSIONS: This study identifies H2 as a novel gaseous signaling molecule that can regulate Ca[2+] channels via the TRPC4-TRPC4AP axis. The 730Arg-731Arg motif in TRPC4 serves as a critical H2-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic framework for developing gas-controlled H2 regenerative therapeutics.},
}
@article {pmid41799755,
year = {2026},
author = {Vadrot, N and Moulin, M and Ferreiro, A and Richard, P and Buendia, B},
title = {LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2.},
journal = {International journal of medical sciences},
volume = {23},
number = {3},
pages = {741-757},
pmid = {41799755},
issn = {1449-1907},
mesh = {Animals ; Cell Differentiation/genetics ; *Myocytes, Cardiac/metabolism ; Cell Proliferation/genetics ; CRISPR-Cas Systems/genetics ; Mice ; *Membrane Proteins/genetics/metabolism ; MEF2 Transcription Factors/genetics/metabolism ; Protein Isoforms/genetics/metabolism ; Rats ; *Aging/genetics ; Cell Line ; Gene Knockdown Techniques ; Myocardium/metabolism ; Humans ; Heart/growth &amp; development/physiology ; DNA-Binding Proteins ; },
abstract = {Haploinsufficiency of Lap2 alpha (LAP2a), a nuclear partner of Lamins A/C, has been associated with cardiac disease in rare cases, but LAP2a function remains largely unknown. To investigate the functional role of LAP2a in cardiomyocytes, we generated clones of embryonic myocardium-derived H9C2 cells in which LAP2a expression was specifically reduced through gene editing of the LAP2a gene Tmpo by CRISPR-Cas9. Downregulation (+/-) and absence (-/-) of LAP2a expression led to a decreased proliferation capacity of cardiomyocytes in vitro. Upon differentiation, the expression of myocardial markers (alpha cardiac Actin 1/Actc1, cardiac Troponin T2/Tnnt2, Myosin-2/Myh2 and Myosin-7/Myh7) was higher in LAP2a -/- cells compared to LAP2a +/- or LAP2a +/+ cells, with consistently higher expression of their upstream regulator Mef2c in LAP2a-devoid cells. These results suggest that LAP2a promotes cardiomyocyte proliferation and negatively modulates cardiomyocyte differentiation, through mechanisms including Mef2c regulation. Accordingly, normal protein expression of LAP2a was downregulated upon cardiomyocyte differentiation, contrary to LAP2b and a LAP2b-related shorter isoform. The latter tended to increase upon differentiation in all cells, most significantly in the LAP2a -/- clone. In postnatal mouse hearts, LAP2a levels were higher in the right than in the left ventricle, and lowest in the septum. The LAP2a:LAP2b ratio was much lower in murine hearts than in H9C2 cells, and decreased significantly upon ageing, specifically in the left ventricle. Finally, our data show that expression of the nuclear envelope proteins LEMD2 and Lamin A might be influenced by LAP2a upon cardiac differentiation. Our results show that LAP2 expression is finely regulated upon cardiac differentiation in vitro and is dependent on age and heart compartment in vivo. They contribute to clarifying the potential impact of genetic LAP2a defects and their connection with heart disease, possibly including reduced cardiomyoblast proliferation, increased cardiomyocyte differentiation and altered nuclear envelope remodelling.},
}
@article {pmid41800529,
year = {2026},
author = {Yan, J and Wang, S and Xiong, S and Luo, X and Li, Y and Deng, X},
title = {Cas12a Trans-Cleavage of Hairpins Triggers a CHA Cascade for an Ultrasensitive SERS Aptasensor.},
journal = {ACS sensors},
volume = {11},
number = {3},
pages = {2828-2839},
doi = {10.1021/acssensors.6c00041},
pmid = {41800529},
issn = {2379-3694},
mesh = {*Spectrum Analysis, Raman/methods ; *Aptamers, Nucleotide/chemistry/metabolism ; *Biosensing Techniques/methods ; Limit of Detection ; DNA/chemistry ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/chemistry ; CRISPR-Cas Systems ; Humans ; Gold/chemistry ; Bacterial Proteins ; },
abstract = {Ultrasensitive detection of non-nucleic acid biomarkers using CRISPR/Cas12a remains a major challenge due to the lack of intrinsic signal amplification. Moreover, linear DNA reporters fail to maintain efficient downstream signal amplification after trans-cleavage, while pre-amplification procedures often lead to nonspecific signals, thereby compromising assay accuracy, particularly in complex biological matrices. Here, a highly SERS aptasensor is developed by harnessing CRISPR/Cas12a-driven trans-cleavage of hairpin substrates to trigger catalytic hairpin assembly (Cas12a-CHA), achieving robust cascade signal amplification. Target recognition is converted into customizable DNA triggers that precisely activate Cas12a, while a thymine-rich DNA/RNA reporter with dT5 motifs facilitates high enhances trans-cleavage efficiency, sustaining continuous CHA cycles. The integration of AuNF@4-MBA@Ag@H2 SERS nanotags that generate abundant plasmonic hotspots, the system provides significantly enhanced Raman readout. Benefiting from synergistic molecular amplification and nanostructure engineering, the aptasensor achieves an ultralow detection limit of 1.97 × 10[-17] g/mL, nearly 20,000-fold higher sensitivity than traditional sandwich assays, along with a broad dynamic range and high specificity. Furthermore, it exhibits excellent uniformity, reproducibility, stability, and recovery in spiked serum samples, using FGF2 used as a representative biomarker to validate its performance, highlighting great potential for clinical diagnostics and real-world applications.},
}
@article {pmid41802204,
year = {2026},
author = {de Paula, JA and de Araújo, MRB and Sousa, EG and Prates, FD and Castro, DLC and Fonseca, PAS and Brenig, B and Felice, AG and Pacheco, LGC and Viana, MVC and Azevedo, VAC and de Castro Soares, S},
title = {Clonal clusters of multidrug-resistant Brazilian Corynebacterium striatum strains reveal putative virulence traits.},
journal = {Journal of applied microbiology},
volume = {137},
number = {4},
pages = {},
doi = {10.1093/jambio/lxag070},
pmid = {41802204},
issn = {1365-2672},
support = {88887.950984/2024-00//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; 32001010068P4//Federal University of Minas Gerais/ ; 311249/2023-0//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; APQ-01323-15//Funda&#xe7;&#xe3;o de Amparo &#xe0; Pesquisa do Estado de Minas Gerais/ ; },
mesh = {Brazil ; *Drug Resistance, Multiple, Bacterial/genetics ; Virulence/genetics ; Phylogeny ; *Corynebacterium/genetics/pathogenicity/drug effects ; Genome, Bacterial ; Microbial Sensitivity Tests ; Humans ; Virulence Factors/genetics ; Anti-Bacterial Agents/pharmacology ; *Corynebacterium Infections/microbiology ; Fimbriae, Bacterial/genetics ; },
abstract = {OBJECTIVES: This study presents the comparative analysis of 26 multidrug-resistant (MDR) C. striatum strains isolated in Brazil.<br><br>METHODS: Additional genomes from international sources were incorporated. The analyses encompassed in vitro antimicrobial susceptibility testing and an in silico workflow for genomic similarity comparison, phylogenetic reconstruction, genomic clustering, pangenome analysis, mobilome content, virulence prediction, and functional annotation of unique proteins and putative virulence clusters.<br><br>RESULTS: Strong in silico evidence of clonality among several Brazilian isolates was obtained at the same time that some strains consistently indicated a divergent genomic profile. There are 196 unique coding sequences (CDSs) across the Brazilian IHPs. Of particular interest, strain IHP2030 carried an exclusive fimbria, sharing less than 50% similarity with other fimbriae in the dataset. Yet, structural predictions suggested conservation of key structural domains typically associated with fimbrial proteins. Mobilome content analysis revealed that IHPs strains were overall similar, differing primarily in the number of insertion sequences and in the presence or absence of CRISPR-Cas defense systems. Regarding virulence, an exclusive cluster in IHP2050 and IHP2060 suggests adaptive advantages associated with their respective environments of isolation.<br><br>CONCLUSION: This study reveals a complex genomic landscape among Brazilian MDR C. striatum strains, marked by clonal dissemination alongside strain-level genetic variation in accessory genomes, mobilome composition, and virulence-associated gene repertoires, providing genomic evidence of diversification within hospital-associated lineages.},
}
@article {pmid41802999,
year = {2026},
author = {Hao, M and Zhou, M and Pan, F and Liu, T and Li, Y and Su, N and Ashfaq, A and Song, M and Wang, H and Wang, W and Liu, J and Li, C and Fu, L and He, P and Hu, Q and Mei, D and Cheng, H},
title = {Efficient CRISPR/Cas-SF01 genome editing tools with high editing efficiency in allotetraploid oilseed rape.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70221},
pmid = {41802999},
issn = {1744-7909},
support = {2025BEA003//the Major Program (JD) of Hubei Province/ ; CAAS-CSNCB-202303//Innovation Program of Chinese Academy of Agricultural Sciences/ ; 2025AFB468//Hubei Provincial Natural Science Foundation of China/ ; CARS-12//Earmarked Fund for China Agriculture Research System/ ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas9 has been widely utilized for plant genome editing, but the protospacer adjacent motif (PAM) requirement limits its editing scope. CRISPR/Cas12i3 belongs to the type-VI Cas system that has gained extensive attention due to its smaller size and less restricted canonical TTN PAM sequence. In this study, we explored the newly developed Cas-SF01 system (Cas12i3 variant) for genome editing in oilseed rape. We established an efficient protoplast transformation system in oilseed rape to compare editing efficiency between Cas-SF01 and Cas9. Cas-SF01 shows cleavage activities at the tested 5'-TTN-3' PAM sites with editing outcomes sharing considerable similarities with the CRISPR-Cas9 system in protoplast. Cas-SF01 also induces high efficiency mutagenesis for multiple target sites in stable transformed oilseed rape lines, generating mutants with multilocular silique and male sterile phenotypes. Furthermore, Cas-SF01-derived cytosine base editors (CBEs) were developed to produce targeted C-to-T base edits. Compared to SpCas9, Cas-SF01 has an expanded PAM range and effectively recognizes TTN PAMs, which has substantially broadened the scope of editable sites within the rapeseed genome. No mutations were identified at the putative off-target sites among the edited plants. This study developed a robust, first-of-its-kind Cas12 system in the allotetraploid Brassica napus, expanding the scope of editing and enriching genome-editing toolkits for biological research and genetic improvement.},
}
@article {pmid41803127,
year = {2026},
author = {Zhou, R and Liu, Y and Zhang, Q and Yin, Z and Tong, J and Zhang, C and Zhang, L and Li, X and Zhao, Y and Zhang, S and Liu, Z and Chen, W and Ji, N and Zhang, H and Li, Z and Yin, H and Zuo, S and Wei, Y},
title = {Structural and mechanistic insights into the dual-nuclease defense protein Upx as an anti-phage system.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-70435-x},
pmid = {41803127},
issn = {2041-1723},
support = {2022YFC3400400//National Science Foundation of China | Key Programme/ ; 23ZYCGSY00750//Tianjin Science and Technology Committee (Tianjin Municipal Science and Technology Commission)/ ; },
abstract = {Nucleic acid degradation is a common strategy for prokaryotic anti-phage systems, as exemplified by the CRISPR-Cas system. The PD-(D/E)-XK nucleases constitute a widely distributed family in these defenses. Notably, most members exhibit a single nuclease domain, while variants containing dual nuclease domains within a single polypeptide remain underexplored, and their molecular mechanisms largely obscure. Here, we biochemically and functionally study a single-protein system containing an uncharacterized PD-(D/E)-XK defense protein (Upx). As revealed by single-particle electron cryo-microscopy (cryo-EM) structure, the C-terminal domain (CTD) harboring the conserved PD-(D/E)XK catalytic core is buttressed by the N-terminal domain (NTD) and the middle domain (MD). Functional assays demonstrate that the nucleic acid binding capability of the CTD is enhanced by the MD. The NTD also displays a noncanonical, basal exonuclease activity that is auto-inhibited by MD. IP-MS experiments identify Upx-interacting phage proteins, and substrate profiling defines its physiological preferences, collectively pointing to its potential physiological targets. Notably, the phage protein gp16 was found to relieve MD-mediated inhibition of the NTD, suggesting a virus-triggered mechanism for activating Upx's dual nuclease activity. Together, these findings establish Upx as a single-protein dual-nuclease anti-phage system, expanding our understanding of bacterial immunity and informing antiviral strategy development.},
}
@article {pmid41803497,
year = {2026},
author = {Perez Taboada, V and Wu, Y and Cassidy, R and Medvedev, KE and Loeff, L and Nemudraia, A and Nemudryi, A},
title = {Bacterial Schlafen proteins mediate phage defence.},
journal = {Nature microbiology},
volume = {11},
number = {4},
pages = {1037-1048},
pmid = {41803497},
issn = {2058-5276},
support = {R00AI171893//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; 1T32GM156737-01//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Escherichia coli/virology/genetics/metabolism ; RNA, Transfer/metabolism ; *Coliphages/physiology ; *Bacterial Proteins/metabolism/genetics ; *Ribonucleases/metabolism/genetics ; Humans ; Virus Replication ; },
abstract = {Human Schlafen proteins restrict viral replication by cleaving tRNA, thereby suppressing protein synthesis. Although the ribonuclease domain of Schlafen proteins is conserved across all domains of life, its function in prokaryotes has remained unclear. Here we demonstrate that prokaryotic Schlafen nucleases are widespread antiviral effectors that protect bacteria from bacteriophages and are fused to a diverse array of phage-sensing domains. We expressed seven Enterobacterales Schlafen systems in Escherichia coli, identifying two that confer defence against coliphages. We focused on a system where Schlafen nuclease is fused to a previously unknown immunoglobulin-like sensor domain and demonstrated that it recognizes tail assembly chaperones of T5-like phages. Upon activation, the Schlafen nuclease cleaves both E. coli and phage-encoded tRNAs and restricts T5 phage by reducing its burst size. Our findings redefine Schlafens as an ancient, mechanistically conserved family of immune effectors, revealing the deep evolutionary origin of tRNA-targeting antiviral immunity in humans.},
}
@article {pmid41804827,
year = {2026},
author = {Parada, F and Cabedo-Díaz, P and Cerda, A and Osorio-Navarro, C and Toledo, JA and Villalobos-González, L and Handford, M and Pimentel, P},
title = {CRISPR/dCas9-Mediated BRL3 Activation Enhances Growth and Metabolic Resilience Under Osmotic Stress in Nicotiana tabacum.},
journal = {Physiologia plantarum},
volume = {178},
number = {2},
pages = {e70816},
doi = {10.1111/ppl.70816},
pmid = {41804827},
issn = {1399-3054},
support = {3240290//ANID-FONDECYT Postdoctoral Project/ ; 3210631//ANID-FONDECYT Postdoctoral Project/ ; 1231417//ANID-FONDECYT Regular Project/ ; RF23F0002//ANID Fortalecimiento de Centros Regionales Project/ ; NCN2024_047//ANID-Millennium Science Initiative Program/ ; },
mesh = {*Nicotiana/drug effects/growth &amp; development/metabolism ; *Osmotic Pressure ; CRISPR-Cas Systems ; *Brassinosteroids/metabolism ; *Genetic Vectors/pharmacology ; Transcriptional Activation ; Plant Leaves/anatomy &amp; histology/drug effects ; Plants, Genetically Modified/growth &amp; development/metabolism ; },
abstract = {Brassinosteroids (BRs) are crucial plant hormones that influence growth and stress adaptation. However, the specific function of the BR receptor BRL3 under osmotic stress remains largely unexplored outside Arabidopsis thaliana. In this study, we used a CRISPR/dCas9-based transcriptional activation (CRISPRa) system to upregulate the Nicotiana tabacum BRASSINOSTEROID INSENSITIVE-LIKE 3 receptor (NtBRL3) and assessed its impact on osmotic stress tolerance. Synthetic activation vectors were constructed using Loop Assembly, featuring dCas9-6TAL-VP128 modules driven by either a constitutive (CaMV35S) or ABA-inducible (SlAREB) promoter, paired with dual sgRNAs targeting the NtBRL3 promoter. Transient Agrobacterium-mediated transformation followed by PEG treatment was used to impose osmotic stress. RT-qPCR confirmed a 3- to 4-fold activation of NtBRL3 transcripts in CRISPRa-infiltrated leaves. The stress-inducible SlAREB promoter produced the strongest improvements, yielding nearly four-fold higher leaf biomass and a five-fold increase in root biomass relative to PEG-stressed controls. Both constructs reduced malondialdehyde (MDA) accumulation, indicating diminished oxidative damage, and modulated osmoprotectant balance, including reduced root proline and increased total soluble solids, particularly under SlAREB-driven activation. Histological segmentation revealed promoter-dependent anatomical remodeling, with NtBRL3-activated plants exhibiting a higher frequency of enlarged leaf cells and expanded tissue domains, consistent with brassinosteroid-mediated structural plasticity. Collectively, these findings demonstrate that CRISPR/dCas9-mediated transcriptional activation of NtBRL3 enhances osmotic stress resilience in tobacco through coordinated biomass recovery, oxidative stress mitigation, osmolyte homeostasis, and tissue remodeling. This transient, non-integrative CRISPRa approach provides a robust synthetic biology framework for dissecting BR signaling and engineering stress-tolerant crops.},
}
@article {pmid41805130,
year = {2026},
author = {Yu, W and Yuan, L and Zhou, W and He, L and Huang, X and Yu, J and Deng, J and Zhang, T and Hu, Y and Zhang, Y and Chen, S},
title = {Orn-mediated c-di-GMP regulates the CRISPR-Cas system to confer stress response in Mycobacterium tuberculosis.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41805130},
issn = {1362-4962},
support = {2021YFA1300901//National Key R&amp;D Program of China/ ; 2022YFA1303500//National Key R&amp;D Program of China/ ; GZNL2024A01024//Guangzhou National Laboratory/ ; //National Key Research and Development Program of China/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/metabolism/drug effects ; *CRISPR-Cas Systems/genetics ; *Cyclic GMP/analogs &amp; derivatives/metabolism ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Oxidative Stress/genetics ; *Stress, Physiological/genetics ; Promoter Regions, Genetic ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {Mycobacterium tuberculosis (Mtb) possesses a type III-A CRISPR-Cas system and has anti-plasmid immune activity. However, whether this system exerts other additional functions remains to be characterized. Here, we investigated the in vivo roles of the Mtb CRISPR-Cas system. We show that this system is transcriptionally dependent and exhibits limited ability to counteract exogenous nucleic acids, primarily through the Csm6 protein rather than the Cas10 HD domain. We further demonstrate that this system plays a role in mitigating oxidative stress and antibiotic treatment, a function mainly mediated by the Cas10 HD domain. Importantly, through transposon library screening, we identified oligoribonuclease (Orn) as a regulatory protein of the Mtb CRISPR-Cas system. Deletion of the orn gene resulted in elevated c-di-GMP levels. A subsequent biotin-labeled c-di-GMP pull-down assay identified the transcriptional regulator Rv3058. Knockdown of rv3058 significantly increased cas6 promoter activity, and its transcriptional repressor function was directly modulated by c-di-GMP. This regulatory pathway enhances stress defense by activating multiple protective pathways, including DNA repair, cell envelope maintenance, and iron homeostasis regulation. Together, we conclude that the regulation of the CRISPR-Cas system by Orn-mediated c-di-GMP contributes to oxidative and antibiotic stress responses in Mtb.},
}
@article {pmid41805294,
year = {2026},
author = {Anfang, M and Yahya, RH and Caldararu, O and Ben Yaakov, S and Landau, U and Berman, A and Hu, Y and Belew, ZM and Crocoll, C and Xu, D and Nour-Eldin, HH and Mayrose, I and Shani, E},
title = {Targeting redundant gene families: A multiplexed, tissue-specific CRISPR toolbox for Arabidopsis genetic screens.},
journal = {Cell reports},
volume = {45},
number = {3},
pages = {117055},
pmid = {41805294},
issn = {2211-1247},
mesh = {*Arabidopsis/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genetic Testing/methods ; *Multigene Family ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Organ Specificity/genetics ; Genome, Plant ; },
abstract = {Genome-scale targeted CRISPR libraries for forward genetic screens in plants are powerful tools for functional analysis, but they suffer from limited spatial control, single sgRNA design, and poor handling of genetic redundancy. We develop multiplexed CRISPR libraries in which each construct contains two sgRNAs that simultaneously target multiple members of a gene family. The libraries can also function at the cell-type-specific and tissue levels. A double-barcoding strategy enables efficient tracking and identification of sgRNA combinations at the plant level without individually sequencing each line. Using this platform, we generate over 1,000 Arabidopsis lines that express sgRNAs targeting 707 transporter genes across 114 gene families involved in nutrient uptake. The multiplexed design increases gene coverage and editing efficiency, underscoring its improved targeting capability to reveal hidden phenotypes. This toolbox provides a scalable resource for multi-targeted genome editing and spatially precise forward genetic screens in plants.},
}
@article {pmid41805349,
year = {2026},
author = {Han, F and Xu, Y and Wang, W and Li, Z and Zhang, Z and Du, R and Xu, Q},
title = {Methylobacterium as a Dual-Function Platform: Advances in C1-Based Biomanufacturing and Plant-Associated Applications.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {11},
pages = {8936-8947},
doi = {10.1021/acs.jafc.5c08026},
pmid = {41805349},
issn = {1520-5118},
mesh = {*Methylobacterium/metabolism/genetics ; Metabolic Engineering ; *Plants/microbiology/metabolism ; *Carbon/metabolism ; },
abstract = {One-carbon (C1) substrates are promising feedstocks for microbial bioproduction. Methylobacterium, known for its exceptional C1 utilization capacity, has emerged as a model microbial chassis for sustainable biomanufacturing. In this review, we first outline the C1 assimilation pathways in Methylobacterium and underscore its potential for producing valuable native metabolites. Furthermore, we then survey the genetic tools available for engineering this genus, including plasmid-based methods, transposon mutagenesis, homologous recombination, and CRISPR/Cas systems. Notably, recent advances in metabolic engineering have significantly expanded its biosynthetic scope, enabling the biosynthesis of diverse non-native compounds. Beyond its biomanufacturing potential, Methylobacterium also serves as a versatile plant growth-promoting bacterium, enhancing plant health and productivity through hormone synthesis, nutrient mobilization, stress mitigation, and induced systemic resistance. Collectively, this work highlights the dual potential of Methylobacterium as a sustainable microbial cell factory for biomanufacturing and a beneficial bioinoculant for agriculture.},
}
@article {pmid41805859,
year = {2026},
author = {Pang, C and Yang, Q and Zhong, Y and Ye, J and Lv, Y and Xie, S and Tang, Y and Ye, X and Zhang, F and Li, C and Zhang, J and Sun, L and Ai, S and Gao, X},
title = {HAND1 controls the lineage bifurcation of trophoblast and amnion from human pluripotent stem cells.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {83},
number = {1},
pages = {},
pmid = {41805859},
issn = {1420-9071},
support = {32270836//National Natural Science Foundation of China/ ; 32570950//National Natural Science Foundation of China/ ; 82270307//National Natural Science Foundation of China/ ; 32200660//National Natural Science Foundation of China/ ; 2022A1515012664//Basic and Applied Basic Research Foundation of Guangdong Province/ ; 2023A1515010309//Basic and Applied Basic Research Foundation of Guangdong Province/ ; },
mesh = {Humans ; *Trophoblasts/cytology/metabolism ; *Cell Lineage ; Cell Differentiation ; *Amnion/cytology/metabolism ; *Pluripotent Stem Cells/cytology/metabolism ; *Basic Helix-Loop-Helix Proteins/metabolism/genetics ; Wnt Signaling Pathway ; beta Catenin/metabolism ; CRISPR-Cas Systems ; Gene Regulatory Networks ; Female ; },
abstract = {Trophoblast and amniotic lineages, representing key extra-embryonic tissues, can be differentiated from human pluripotent stem cells (hPSCs) under chemically defined conditions. However, the regulatory mechanisms coordinating the fate decision between these lineages during PSC differentiation remain incompletely understood. Leveraging CRISPR/Cas9-mediated loss-of-function screening in lineage-reporter PSCs, we identified the transcription factor HAND1 as a critical determinant controlling the bifurcation of trophoblast and amniotic lineages. Genetic ablation of HAND1 effectively abrogated the amniotic differentiation capacity of PSCs while concomitantly enhancing their trophoblast differentiation potential. Conversely, ectopic HAND1 overexpression impaired trophoblast differentiation. Notably, forced HAND1 expression in human trophoblast stem cells (TSCs) induced transcriptional reprogramming toward an amniotic fate, indicating its lineage-instructive capability. Mechanistic analyses demonstrated that HAND1 interacts with the TCFs and Wnt signaling effectors β-catenin to form a transcriptional complex that antagonistically modulates the balance between trophoblast- and amnion-associated gene regulatory networks. Collectively, our findings establish HAND1 as a master regulator orchestrating the amniotic versus trophoblast lineage choice during human PSC differentiation, thereby illuminating fundamental regulatory mechanism underlying extra-embryonic lineage specification.},
}
@article {pmid41805869,
year = {2026},
author = {Moghe, AS and Nandi, SS and Bhonde, RR and Kamyab, SS and Sawant, SA and Karandikar, MN},
title = {Engineering of cell line assembled enteric organoid for enterovirus infection.},
journal = {Archives of virology},
volume = {171},
number = {4},
pages = {},
pmid = {41805869},
issn = {1432-8798},
support = {5/3/8/57/2020-ITR Date:11.03.2021//Indian Council of Medical Research/ ; },
mesh = {Humans ; *Organoids/virology ; Virus Replication ; Cell Line ; *Enterovirus Infections/virology ; *Enterovirus A, Human/physiology ; Epithelial Cells/virology ; *Enterovirus/physiology ; CRISPR-Cas Systems ; Coculture Techniques ; },
abstract = {The non-polio-enteroviruses are ubiquitous pathogens infecting over a billion people in the world. An alarming number of enterovirus-associated acute flaccid paralysis, encephalitis, hand, foot, and mouth disease, conjunctivitis and diarrhoea cases are reported worldwide. Despite their clinical significance, vaccine development has been hindered due to lack of suitable in vitro models for preclinical investigations. The present study was undertaken to develop a cell line assembled organotypic model of human intestine for replication of enteroviruses. An enterovirus specific PSGL1 receptor was introduced in intestinal epithelial HCT-8 cell line employing CRISPR/cas9 gene editing. It was co-cultured with human colon (CCD-18) and endothelial (HUVEC) cell lines with peripheral blood mononuclear cells in hanging drops and rotating wall vessel bioreactor to yield three-dimensional organoids. Histological analysis of the organoids showed presence of columnar epithelium cells with prominent intracytoplasmic mucin, hyperchromatic nuclei and presence of CK, CK20, MUC 2 and Villin markers characteristic of epithelial cells. Infection with Enterovirus A71 (EV-A71) demonstrated significantly higher viral titre in organoids compared to individual cell lines. Collectively, these findings determine, for the first time, a cell line-derived enteric organoid model that supports robust enterovirus replication, offering a cost-effective and physiologically relevant system for virology research and preclinical applications.},
}
@article {pmid41806413,
year = {2026},
author = {Jeong, Y and Lee, J and Choi, S and Shin, D and Jang, S and Son, SU and Kang, T and Jung, J and Hwang, J and Lim, EK},
title = {On-site detection of airborne foodborne pathogens using a field-deployable recombinase polymerase amplification and CRISPR/Cas12a cleavage activity assay.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118571},
doi = {10.1016/j.bios.2026.118571},
pmid = {41806413},
issn = {1873-4235},
mesh = {CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/instrumentation ; *Biosensing Techniques/instrumentation ; *Food Microbiology ; *Air Microbiology ; *Foodborne Diseases/microbiology ; Humans ; Staphylococcus aureus/isolation &amp; purification/genetics ; Listeria monocytogenes/isolation &amp; purification/genetics ; *Bacteria/isolation &amp; purification/genetics ; Limit of Detection ; Food Contamination/analysis ; Recombinases/chemistry ; Bacillus cereus/isolation &amp; purification/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {With the global increase in single-person households, the demand for meal kits is increasing, leading to the development of large-scale food production systems and complex supply chains. However, under the influence of global warming, these systems can be susceptible to food contamination, particularly by airborne foodborne bacteria. Conventional methods for detecting airborne bacteria involve complex, time-consuming, and labor-intensive processes, which limit their applicability for field use and rapid food hygiene surveillance. In the present study, we developed a field-deployable diagnostic platform by combining recombinase polymerase amplification with CRISPR/Cas12a cleaVage Activity (RCCVA assay) for the rapid and sensitive identification of airborne foodborne bacteria. Airborne bacteria were collected using a self-developed electrostatic air sampler and analyzed using a portable isothermal amplification device. The RCCVA assay was designed to detect four major foodborne pathogens: Staphylococcus aureus, Salmonella enteritidis, Listeria monocytogenes, and Bacillus cereus. The limit of detection was measured as 274.9, 4.5, 9.5, and 28.5 culture-forming units (CFU)/mL, respectively, within 45 min. This platform enables early on-site detection of airborne pathogens within approximately 1 h (for the analytical phase) and shows potential for real-time monitoring in food processing environments, thereby contributing to improved public health and food safety.},
}
@article {pmid41806414,
year = {2026},
author = {Kim, H and Kim, D and Han, H and Lee, C and Roh, YH and Han, TS and Lim, EK and Park, J and Ahn, JK and Kang, T and Jung, J and Lee, CY},
title = {On-site microRNA detection with 'off-the-shelf' glucose meter empowered by chimeric probe connecting CRISPR/Cas13a activation to kinases-driven glucose phosphorylation.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118568},
doi = {10.1016/j.bios.2026.118568},
pmid = {41806414},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation &amp; purification/blood/analysis ; Humans ; *Biosensing Techniques ; Phosphorylation ; CRISPR-Cas Systems/genetics ; *Glucose ; Limit of Detection ; *Stomach Neoplasms/blood/diagnosis/genetics ; Biomarkers, Tumor/genetics/blood ; *Blood Glucose Self-Monitoring/instrumentation ; },
abstract = {MicroRNAs (miRNAs) are promising biomarkers for cancer diagnosis due to their stability in body fluids and disease-specific expression profiles. However, current detection methods suffer from limitations including cumbersome workflows, heavy instrumentation for signal readout, or vulnerability in minimizing instrumentation. To address these challenges, we describe a novel point-of-care miRNA detection platform executable with "off-the-shelf", personal glucose meter (PGM), termed 'KEY-FACT (Kinases Ensemble-driven glucose phosphorYlation upon Fuel-Aided CRISPR acTivation)'. Upon recognition of target miRNA, a fuel-assisted toehold-mediated strand displacement reactions liberate guide RNAs (gRNAs) to activate Cas13a to cleave a chimeric reporter probe, producing 2',3'-cyclic adenosine monophosphates (cAMP). Subsequent dephosphorylation and kinases ensemble-mediated phosphorylation/dephosphorylation cycles lead cAMP to consume a large amount of glucose. A user can immediately measure resulting glucose level change with PGM on the spot. This strategy allows sensitive, prompt detection of miR-135b, a gastric cancer (GC) biomarker, with a limit of detection (LOD) of 1.4 pM within 2 h. KEY-FACT is specific to the target miRNA and is applicable to body fluids such as human serum with dilution (95.2% < recovery rates <104.3%, coefficients of variation ≤13%). Owing to its simple probe design, KEY-FACT was readily expanded to detect another GC biomarker, miR-21, with comparable sensitivity (LOD = 1.5 pM). The proposed platform fulfills minimal instrumentation and thus enables cost-effective, field-deployable analysis, paving the way for practical, on-demand miRNA diagnostics.},
}
@article {pmid41806830,
year = {2026},
author = {Escobar, M and Malik, SA and Srinivasa, MA and Mendez-Sosa, MA and Miller, JM and Lydon, SL and Luong, SN and Mathew, PR and Abouleisa, RRE and Chakravarty, S and Pathan, S and Mohamed, TMA and Ghanta, RK and Hilton, IB},
title = {CRISPR-Cas-based activation of PPARGC1A boosts endogenous mitochondria and enhances cardiac function after myocardial infarction.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {},
number = {},
pages = {},
pmid = {41806830},
issn = {1525-0024},
support = {R01 HL166280/HL/NHLBI NIH HHS/United States ; R01 HL163258/HL/NHLBI NIH HHS/United States ; R01 HL147921/HL/NHLBI NIH HHS/United States ; R35 GM143532/GM/NIGMS NIH HHS/United States ; R15 HL168688/HL/NHLBI NIH HHS/United States ; 25TPA1463933/AHA/American Heart Association-American Stroke Association/United States ; 917025/AHA/American Heart Association-American Stroke Association/United States ; R01 HL174616/HL/NHLBI NIH HHS/United States ; },
abstract = {Insufficient energy supply due to impaired mitochondria has emerged as a key pathological factor in the development of heart failure (HF) after myocardial infarction (MI). Unfortunately, no current therapeutic strategies directly augment myocardial energy production. While mitochondrial biogenesis is orchestrated by the activity of multiple genes, activation of PPARGC1A, a key regulator, can increase cellular mitochondria; however, supraphysiological levels of PPARGC1A result in adverse tissue remodeling and heart dysfunction. CRISPR activation (CRISPRa) technologies present a unique opportunity to address these shortcomings, as they enable tunable control over endogenous target gene expression. Here, we demonstrate that transcriptional activation of PPARGC1A using CRISPRa increases cellular mitochondria in human cell types. This effect is mediated through the activation of transcriptional programs driving mitochondrial biogenesis, mitochondrial function, and cellular bioenergetics. These activated transcriptional programs synergize to increase ATP production and reserve capacity in human cardiomyocytes. CRISPRa targeting of PPARGC1A in vivo increases cardiac mitochondria to recover heart ejection fraction in an acute MI model. Furthermore, CRISPRa acts on the adult human heart to increase PPARGC1A protein and cellular mitochondria, elevating mitochondrial function in both normal and HF-diagnosed hearts. These results provide the first proof of concept that endogenous gene activation via CRISPRa can improve heart function after MI.},
}
@article {pmid41807051,
year = {2026},
author = {Gur Dedeoglu, B and Noyan, S and İlhan, KNK},
title = {Non-coding RNAs regulation in breast cancer pathogenesis.},
journal = {Epigenomics},
volume = {},
number = {},
pages = {1-20},
doi = {10.1080/17501911.2026.2642583},
pmid = {41807051},
issn = {1750-192X},
abstract = {Breast cancer represents a molecularly heterogeneous disease shaped by complex genetic, epigenetic, and transcriptional dysregulation. Non-coding RNAs (ncRNAs) including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) as well as small nucleolar RNAs (snoRNAs), piwi-interacting RNAs (piRNAs), and small nuclear RNAs (snRNAs), have emerged as key epigenetic regulators that integrate multiple layers of gene control. Through interactions with chromatin-modifying enzymes, RNA-binding proteins, and signaling effectors, ncRNAs modulate transcriptional activity, chromatin accessibility, and post-transcriptional stability of target genes. miRNAs predominantly act as post-transcriptional repressors, whereas lncRNAs and circRNAs exert transcriptional and epigenetic control via scaffolding, miRNA sponging, and chromatin remodeling; some circRNAs even encode functional peptides. Aberrant ncRNA expression contributes to proliferation, metastasis, metabolic reprogramming, immune evasion, and therapeutic resistance, with distinct expression signatures associated with triple-negative, HER2-positive, and hormone receptor - positive breast cancers. Owing to their stability and detectability in plasma and exosomes, ncRNAs hold promise as minimally invasive biomarkers for early detection and disease monitoring. Moreover, therapeutic strategies targeting ncRNAs, such as antisense oligonucleotides, RNA interference, CRISPR/Cas-based editing, and ncRNA-derived vaccines, are advancing toward clinical translation. Collectively, ncRNAs redefine the epigenetic landscape of breast cancer, offering a framework for integrated diagnostic and therapeutic approaches in precision oncology.},
}
@article {pmid41807728,
year = {2026},
author = {Godsil, M and Wei, N and Meeske, AJ},
title = {Conditional activation of Cas13 enforces lysogeny in a native type VI-A CRISPR host.},
journal = {Nature microbiology},
volume = {11},
number = {4},
pages = {920-928},
pmid = {41807728},
issn = {2058-5276},
support = {R35 GM142460/GM/NIGMS NIH HHS/United States ; FAIN2235762//NSF | BIO | Division of Molecular and Cellular Biosciences (MCB)/ ; R35GM142460//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; },
mesh = {*Lysogeny/genetics ; *CRISPR-Cas Systems ; Prophages/genetics/physiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Bacteriophages/genetics/physiology ; *CRISPR-Associated Proteins/metabolism/genetics ; *Listeria monocytogenes/virology/genetics ; *Bacterial Proteins/metabolism/genetics ; Virus Activation ; },
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) systems present a barrier to prophage acquisition by restricting invading phages or by inducing autoimmune cleavage of integrated prophage DNA. The RNA-sensing type VI CRISPR nuclease Cas13 mediates non-specific RNA cleavage upon recognition of phage lytic transcripts, but how this system influences the temperate phage life cycle remains unknown. Here we report that the Listeria seeligeri type VI-A CRISPR system restricts the lytic cycle of temperate phages but tolerates prophage acquisition and interferes with prophage induction through a non-abortive mechanism. During attempts at induction, Cas13 activation forces prophage re-integration, thus maintaining lysogeny. We also find that during polylysogenic induction, Cas13 acts specifically, restricting only the targeted phage, in contrast to its behaviour during lytic replication. Our findings show that Cas13 elicits a unique response to each stage of the temperate phage life cycle, enabling type VI CRISPR hosts to acquire potentially beneficial prophages while mitigating lysis.},
}
@article {pmid41808208,
year = {2026},
author = {Zhang, Y and Wang, M and Bi, C and Li, M},
title = {Targeted native long-read sequencing of DNA methylation alterations following CRISPR-Cas9-induced double-strand breaks in human cells.},
journal = {BMC research notes},
volume = {19},
number = {1},
pages = {},
pmid = {41808208},
issn = {1756-0500},
support = {BAS/1/1080-01-01//King Abdullah University of Science and Technology/ ; },
mesh = {Humans ; *DNA Methylation/genetics ; *CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; 5-Methylcytosine/analogs &amp; derivatives/metabolism ; Epigenesis, Genetic ; Sequence Analysis, DNA/methods ; Nanopore Sequencing ; },
abstract = {OBJECTIVES: CRISPR-Cas9 nucleases are widely used to introduce targeted DNA double-strand breaks (DSBs) for genome engineering, but the long-term impact of these lesions on local epigenetic information remains poorly characterized. In a companion research article, we used Cas9-assisted targeted nanopore sequencing (CTS) to reveal that CRISPR-Cas9-induced DSBs can disrupt local epigenetic maintenance across multiple genomic contexts and cell systems. Here, we present a structured description of the raw and minimally processed datasets underlying the study. These datasets provide base-resolution measurements of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) at the differentially methylated regions (DMRs) of several imprinted loci, two heterochromatic regions, a cancer-associated promoter epimutation region, and the SNRPN DMR at early/late passages of a clonal line. They enable re-analysis and methodological benchmarking of DSB-associated epigenetic instability.<br><br>DATA DESCRIPTION: We provide aligned BAM files and per-CpG methylation calls for multiple genomic contexts under both CRISPR-targeted and non-targeting control conditions. Specifically, the collection includes: (i) imprinted loci in human embryonic stem cells (hESCs), including small nuclear ribonucleoprotein polypeptide N (SNRPN), paternally expressed 10 (PEG10), and KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1), (ii) heterochromatic regions in hESCs, including urothelial cancer associated 1 (UCA1), and cysteine rich C-terminal 1 (CRCT1)), (iii) the epimutation locus of MutL homolog 1 (MLH1) in RKO cells, and (iv) the DMR of SNRPN locus in early- and late-passage derivatives of a single hESC clone. For each collection, there is a dataset that includes both the raw aligned Nanopore sequencing reads (BAM) deposited in the NCBI Sequence Read Archive (SRA) and the corresponding processed per-CpG 5mC/5hmC matrices deposited in Zenodo. All higher-level analyses in the research article-such as DMR calling, haplotype-resolved analyses, and structural variant (SV) characterization-are fully reproducible using these deposited data. Additional processed analyses are comprehensively documented in the companion article and are therefore not duplicated here. Together, these datasets offer a rich resource for benchmarking long-read methylation analysis workflows and further investigation of DSB-associated epigenetic instability across diverse genomic contexts.},
}
@article {pmid41808396,
year = {2026},
author = {Zargul, A and Liu, H and Zhang, W and Wang, H and Liu, J and Chen, C},
title = {Advances in Pathogen Detection by Nanosensors: Biorecognition Strategies, Signal Amplification, and Platform Engineering.},
journal = {ACS nano},
volume = {20},
number = {11},
pages = {9007-9050},
doi = {10.1021/acsnano.5c22148},
pmid = {41808396},
issn = {1936-086X},
mesh = {*Biosensing Techniques/methods ; Humans ; *Nanotechnology/methods ; Nucleic Acid Amplification Techniques ; Animals ; },
abstract = {The escalating global threat of infectious diseases, compounded by antimicrobial resistance (AMR), calls for improved diagnostic strategies. Conventional pathogen detection techniques─culture, enzyme-linked immunosorbent assay (ELISA), and microscopy─remain hindered by prolonged turnaround times, suboptimal sensitivity for low-abundance analytes, and operational intricacy. Nanosensor technologies have emerged as powerful enablers of rapid, ultrasensitive, and field-deployable diagnostics. This review delineates the convergence of three transformative domains: (1) advanced biorecognition strategies─including monoclonal antibodies, aptamers, bacteriophages, antimicrobial peptides, molecularly imprinted polymers, and lectins─that confer high-fidelity molecular selectivity within complex biological matrices; (2) multimodal signal amplification technologies, encompassing nanomaterial-enhanced mechanisms, enzymatic cascades, and isothermal nucleic acid amplification that drive detection down to the single-cell and femtomolar regimes; and (3) integrated platform engineering, uniting clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems, artificial intelligence (AI), and microfluidics to achieve multiplexed, real-time, point-of-care deployment. Advances are critically evaluated through standardized performance metrics─limit of detection, assay time, specificity, and operational simplicity─to reveal both synergistic opportunities and enduring translational bottlenecks. Collectively, these developments define a strategic framework for next-generation nanosensor diagnostics poised to revolutionize infectious disease surveillance and enable precision-guided therapeutic intervention.},
}
@article {pmid41808567,
year = {2026},
author = {Ding, X and Liu, Y and Luo, L and Cai, Y and Wang, C and Jin, J and Chen, Y},
title = {Review Genomic Hotspot Mining and Characterization for Stable Expression of Therapeutic Protein in Chinese Hamster Ovary Cells.},
journal = {ACS synthetic biology},
volume = {15},
number = {3},
pages = {1241-1247},
doi = {10.1021/acssynbio.5c00776},
pmid = {41808567},
issn = {2161-5063},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Recombinant Proteins/genetics/biosynthesis ; CRISPR-Cas Systems/genetics ; Chromosome Mapping ; Cricetinae ; Promoter Regions, Genetic ; Genomics/methods ; Lentivirus/genetics ; },
abstract = {The development of rCHO cell lines that stably express therapeutic proteins is crucial for pharmaceutical protein industrial production. In this study, a systematic method was established to identify genomic hotspots for exogenous protein expression in CHO cells and construct stable recombinant CHO cell strains. Four stable monoclonal cell lines (1b7, 1d2, 2d9, and 2f7) were obtained by using the lentiviral random integration reporter gene. Chromosome mapping analysis found four stable integration sites: chr1_0 (7,30,83,299-7,32,45,508 bp) in 1b7, chr1_0 (17,69,68,187-17,69,68,191 bp) in 1d2, chr3 (4,08,81,262-4,08,99,858 bp) in 2d9, and chr5 (1,69,77,575-1,70,61,744 bp) in 2f7. Based on these sites, we developed recombinant CHO cells capable of long-term stable expression of foreign proteins through the combined application of CRISPR/Cas9 technology and Bxb1 recombinase-mediated cassette exchange. Utilizing "promoter capture technology", all screened LP cell monoclonal lines can express exogenous proteins, with the entire construction process completed in just 2∼3 weeks.},
}
@article {pmid41809894,
year = {2025},
author = {Yang, P and Khoshandam, M and Bhia, I and Raji, S and Soltaninejad, H and Hosseinkhani, S and Sani, M and Hamidieh, AA and Sheykhhasan, M},
title = {Integrating CRISPR/Cas technology with clinical trials: Principles, progress and challenges.},
journal = {Asian journal of pharmaceutical sciences},
volume = {20},
number = {6},
pages = {101068},
pmid = {41809894},
issn = {2221-285X},
abstract = {CRISPR represent a groundbreaking genome-editing technology that has revolutionized genetic modification. This innovative tool offers an unparalleled revolution in the future treatment of genetic disorders, neurological diseases, infectious diseases and cancer. Despite the rapid expansion of CRISPR applications, its clinical use in humans is still relatively limited, with only 69 active clinical trials and 6 completed studies reported so far. This review examined current clinical trials and their processes in addressing various diseases via the CRISPR/Cas system. While earlier literatures have focused mainly on delivery methods and materials for CRISPR/Cas9, our review emphasized innovative targeting conditions and approaches for novel and functional therapeutic designs. In addition, we reviewed recent research to increase the efficiency of CRISPR editing in the management of genetic disorders and cancer, while exploring their future challenges and potential. This review provided a unique perspective on the advancement of CRISPR technology. By addressing these aspects, we aim to contribute to ongoing efforts to improve CRISPR-based therapies and expand their clinical applications, ultimately striving to transform the future of medical treatment.},
}
@article {pmid41810060,
year = {2026},
author = {Shen, Z and Liu, Y and Hao, Y and Bo, Y and Dai, X and Wang, S and Xia, T and Su, X and Liu, H},
title = {Advances in Double-Stranded DNA Targeting Technologies.},
journal = {Exploration (Beijing, China)},
volume = {6},
number = {1},
pages = {20250065},
pmid = {41810060},
issn = {2766-2098},
abstract = {Double-stranded DNA (dsDNA) serves as a fundamental repository of genetic information and plays a pivotal role in the diagnosis and therapeutic management of diseases. However, the inherent stability of the DNA double helix under physiological conditions presents a challenge in accessing internal bases. To address this, various molecular targeting technologies have been developed, offering high specificity while destabilizing the DNA structure. This review provides a comprehensive overview of current dsDNA targeting tools, such as hybridization probes, modified nucleic acid probes, zinc finger proteins (ZFPs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system, Argonaute proteins (Agos), and the lambda exonuclease-pDNA system (λ Exo-pDNA), and some cutting-edge molecular tools. It delves into the mechanisms behind these technologies. It highlights their applications in diverse areas, including in vitro detection, in situ imaging, gene editing, and their integration with artificial intelligence (AI)-driven tools. Additionally, the review compares these techniques, discusses future technological opportunities, and identifies challenges in integrating these tools into diagnostic and therapeutic practices. By providing a holistic view of these rapidly evolving technologies, this review aims to fill a gap in the current literature and explore the future potential of dsDNA targeting innovations.},
}
@article {pmid41810315,
year = {2026},
author = {Guo, Z and Hu, R and Wang, J and Zhou, M and Zhu, K and Xu, Y},
title = {Research Progress on Point-of-Care Testing Technology for Mycoplasma Pneumonia.},
journal = {International journal of general medicine},
volume = {19},
number = {},
pages = {584824},
pmid = {41810315},
issn = {1178-7074},
abstract = {Mycoplasma pneumoniae (MP) is a significant respiratory pathogen in children, often causing refractory and severe pneumonia. Sensitive, rapid, and portable diagnostic tools are crucial for guiding clinical management. Although traditional methods like culture, ELISA, and PCR are widely used, they suffer from drawbacks such as lengthy turnaround times, complex procedures, or reliance on laboratory equipment. Consequently, Point-of-care testing (POCT) technologies, valued for their speed, portability, and ease of use, have emerged as a key research focus for MP diagnosis. This review systematically summarizes advancements in POCT platforms, covering: (1) immunological methods; (2) molecular biology methods; and (3) biosensor technologies. Their sensitivity, specificity, and clinical performance are comparatively analyzed. Colloidal gold immunochromatography delivers results within 15 minutes but exhibits limited sensitivity. Molecular methods like LAMP and CRISPR-coupled systems achieve single-copy detection limits via isothermal amplification and gene editing, with processing times under 1 hour. Biosensors enable high-sensitivity automated detection through integrated signal amplification and microfluidics. Despite these advantages, POCT development faces challenges including cost-sensitivity tradeoffs, standardization barriers, and sample matrix interference. Future directions encompass multi-modal detection, AI-assisted interpretation, multiplex pathogen screening, and dynamic drug resistance gene monitoring. These innovations will expand POCT device deployment in primary care and home settings. This will ultimately improve effectiveness in controlling respiratory infections.},
}
@article {pmid41810550,
year = {2026},
author = {Rahmanian, M and Khoshandam, M and Mousazadeh, M and Yang, P and Soltaninejad, H and Karami Dehkordi, P and Sadeghizadeh, M and Hedayati Goudarzi, MT and Azimi, AH and Sheykhhasan, M},
title = {CRISPR in Medicine: A Systematic Review of Clinical Trials and Therapeutic Applications.},
journal = {Human gene therapy},
volume = {37},
number = {5-6},
pages = {170-182},
doi = {10.1177/10430342251409714},
pmid = {41810550},
issn = {1557-7422},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Genetic Therapy/methods ; Clinical Trials as Topic ; Neoplasms/therapy/genetics ; *Hematologic Diseases/therapy/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein 9 (CRISPR/Cas9) technology has become a revolutionary tool in medicine, offering substantial potential for treating a wide range of diseases, including hematological disorders, cancers, genetic conditions, and ophthalmological diseases. This systematic review evaluates the efficacy, safety, and applicability of CRISPR/Cas9 in clinical trials. A comprehensive search of the PubMed, Scopus, Web of Science, and Cochrane databases was conducted. All studies, up to November 2024, meeting the eligibility criteria assessing the application of CRISPR for the treatment of diseases were included. A quality assessment of the included studies was conducted using the Cochrane risk of bias tool. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) 2020 statement for systematic reviews and meta-analyses was followed, and a total of 17 studies were included. This systematic review of CRISPR/Cas9 technology focused on its effectiveness and safety across various diseases. In nonmalignant hematological disorders, CRISPR successfully treated β-thalassemia and sickle cell disease, resulting in high transfusion independence and the elimination of disease crises. In malignant hematological disorders, B-cell acute lymphoblastic leukemia, CRISPR-engineered chimeric antigen receptor T (CAR-T) cells achieved an 83.3% complete remission rate. Furthermore, CRISPR-based CAR-T cells showed promising results in B-cell non-Hodgkin's lymphoma. In oncology, lung cancer and other solid tumors are among the diseases that have been safely engineered using CRISPR gene editing technology. For genetic disorders, CRISPR improved vision in retinal degeneration and reduced symptoms in hereditary angioedema and transthyretin amyloidosis with mild side effects. The results demonstrated CRISPR's potential across a wide range of conditions. In conclusion, the findings underscore the potential role of CRISPR/Cas9 technology across a wide range of diseases. However, challenges remain, including optimizing delivery systems, minimizing off-target effects, addressing immunogenicity concerns, and ethical considerations.},
}
@article {pmid41811192,
year = {2026},
author = {Shen, Y and Yeung, AT and Ditchfield, P and Korn, E and Clements, R and Chen, X and Wang, B and Huang, Z and Sheen, M and Jarman, PA and Han, C},
title = {A genome-wide MAGIC kit for recombinase-independent mosaic analysis in Drosophila.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41811192},
issn = {2050-084X},
support = {R24OD031953//NIH Office of the Director/ ; },
mesh = {Animals ; *Mosaicism ; *Drosophila/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Crossing Over, Genetic ; CRISPR-Cas Systems ; *Genome, Insect ; },
abstract = {Mosaic analysis has been instrumental in advancing developmental and cell biology. Most current mosaic techniques rely on exogenous site-specific recombination sequences that need to be introduced into the genome, limiting their application. Mosaic analysis by gRNA-induced crossing-over (MAGIC) was recently developed in Drosophila to eliminate this requirement by inducing somatic recombination through CRISPR/Cas9-generated DNA double-strand breaks. However, MAGIC has not been widely adopted because gRNA markers, a required component for this technique, are not yet available for most chromosomes. Here, we present a complete, genome-wide gRNA-marker kit that incorporates optimized designs for enhanced clone induction and more effective clone labeling in both positive MAGIC (pMAGIC) and negative MAGIC (nMAGIC). With this kit, we demonstrate clonal analysis in a broad range of Drosophila tissues, including cell types that have been difficult to analyze using recombinase-based systems. Notably, MAGIC enables clonal analysis of pericentromeric genes, deficiency chromosomes and in interspecific hybrid animals, opening new avenues for gene function study, rapid gene discovery, and understanding cellular basis of speciation. This MAGIC kit complements existing systems and makes mosaic analysis accessible to address a wider range of biological questions.},
}
@article {pmid41811196,
year = {2026},
author = {Vieira, CSD and Wang, W and Sanchez-Valdez, F and Lim, J and White, BE and Souza, CGS and Tarleton, RL and Paes, MC and Nogueira, NPA},
title = {Glycosomal Phosphoenolpyruvate Carboxykinase CRISPR/Cas9-Deletion and Its Role in Trypanosoma cruzi Metacyclogenesis and Infectivity in Mammalian Host.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {6},
pages = {e71672},
pmid = {41811196},
issn = {1530-6860},
support = {00x0ma614//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior (CAPES)/ ; 88887.311601/2018-00-2629/2018//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior (CAPES)/ ; E26/010.001706/2019//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; E26/010.100623/2018//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; E26/211.815/2021//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro (FAPERJ)/ ; 402419/2022-7//Conselho Nacional Pesquisa (CNPq) SWE (Sandu&#xed;che no Exterior)/ ; },
mesh = {Animals ; *Trypanosoma cruzi/pathogenicity/genetics/enzymology/growth &amp; development ; *CRISPR-Cas Systems ; Mice ; *Chagas Disease/parasitology ; Glucose/metabolism ; *Phosphoenolpyruvate Carboxykinase (ATP)/genetics/metabolism ; *Microbodies/enzymology/metabolism ; Mitochondria/metabolism ; *Protozoan Proteins/genetics/metabolism ; },
abstract = {Trypanosoma cruzi, the causative agent of Chagas disease, possesses glycosomes-unique organelles that house key metabolic enzymes, several of which are promising therapeutic targets. Among them, phosphoenolpyruvate carboxykinase (PEPCK) plays a central role in succinic fermentation, the main pathway for NAD[+] regeneration within the organelle. Using CRISPR/Cas9 editing, the PEPCK gene was disrupted in T. cruzi, producing single-allele knockout epimastigotes (TcPEPCK-sKO) with reduced PEPCK expression and enzyme activity. In a high glucose environment, PEPCK disruption impaired glucose consumption and mitochondrial respiration, particularly oxidative phosphorylation, reducing dependence on mitochondrial ATP production when glucose was supplied. To compensate, pyruvate phosphate dikinase was upregulated, increasing alanine production, possibly to maintain redox balance in glycosomes. Despite this metabolic adaptation, the growth of TcPEPCK-sKO epimastigotes was partially reduced compared with non-deleted parasites. In contrast, under low glucose conditions, PEPCK activity was not critical for mitochondrial bioenergetics, ATP production, or proliferation. Although TcPEPCK-sKO epimastigotes exhibited a minor reduction in growth in high glucose medium, their differentiation (metacyclogenesis) and invasion were severely compromised. However, once inside the host cell, TcPEPCK-sKO amastigotes increased their replication, leading to enhanced trypomastigote production. The same was observed in in vivo infection, where TcPEPCK-sKO infection in IFNγ-deficient mice caused uncontrolled parasitemia and severe pathology, highlighting the critical role of PEPCK in host-pathogen interactions. However, an intact immune system effectively contained TcPEPCK-sKO infection. Taken together, our findings demonstrate that glycosomal PEPCK is crucial for coupling glycolysis to mitochondrial bioenergetics, enabling the parasite differentiation within the insect vector and controlling the infection of mammalian host cells.},
}
@article {pmid41811507,
year = {2026},
author = {Thakur, MK and Pandey, S and Singh, SK and Singh, SK and Singh, A},
title = {Hybrid seed production: new paradigms and challenges in the twenty-first century.},
journal = {Planta},
volume = {263},
number = {4},
pages = {},
pmid = {41811507},
issn = {1432-2048},
mesh = {*Seeds/genetics/growth &amp; development ; *Plant Breeding/methods ; *Hybridization, Genetic ; *Crops, Agricultural/genetics ; Hybrid Vigor ; Genomics ; Climate Change ; },
abstract = {Hybrid seed technology future depends on integrating advanced genomics, AI-driven breeding, and enabling policies to sustainably delivery climate-resilient, high-performing hybrids with broad accessibility and equitable benefits worldwide. Hybrid seeds, which exploit heterosis, have driven agricultural productivity gains since the 1920s. Understanding the genetics and molecular biology of hybrid generation led to the development of modern hybrid systems. With time, modern hybrid systems integrated advanced genomic tools such as CRISPR/Cas, marker-assisted selection (MAS), and genomic selection (GS) with established technologies like cytoplasmic male sterility (CMS), restorer-of-fertility (Rf) systems, and chemical hybridizing agents (CHAs) for better hybrid production in a shorter time. Moreover, the integration of emerging approaches leveraging artificial intelligence and machine learning (AI/ML) for trait prediction, multi-parent populations to expand genetic diversity, and epigenetics to engineer climate-resilient hybrids with enhanced stress tolerance is also being explored. However, regulatory hurdles, such as divergent global policies for genetically modified (GM) hybrids, intellectual property (IP) disputes, and restricted germplasm exchange under access-and-benefit-sharing frameworks like the Nagoya Protocol, hinder innovation. Climate change exacerbates both biotic and abiotic stresses, disrupts production zones, and threatens pollinator-dependent crops, while socio-economic barriers limit the adoption of smallholder farming. Case studies of different crops demonstrate the success of hybrids, yet gaps in scalability and accessibility persist. Overall, realizing the potential of hybrid technology hinges on sustained collaboration across scientific, industrial, and policy domains to overcome technical, environmental, and socio-economic constraints. This review examines various techniques for hybrid production that incorporate genomics, future advancements, and synergies between synthetic biology, automation, and predictive breeding, as well as policies that strike a balance between intellectual property protection and germplasm accessibility for hybrid seed production.},
}
@article {pmid41811515,
year = {2026},
author = {Thiebaut, F and Urquiaga, MC and de Araújo, PM and de Carvalho Vivarini, A and Grativol, C},
title = {Small but big player: the important role of microRNAs in legume crops.},
journal = {Molecular genetics and genomics : MGG},
volume = {301},
number = {1},
pages = {},
pmid = {41811515},
issn = {1617-4623},
mesh = {*MicroRNAs/genetics ; *Crops, Agricultural/genetics/growth &amp; development ; *Fabaceae/genetics/growth &amp; development ; Gene Expression Regulation, Plant ; *RNA, Plant/genetics ; Symbiosis/genetics ; RNA Interference ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Legumes are essential components of global cropping systems due to their nutritional value and contribution to sustainable agriculture. Among the regulatory molecules, small RNAs (sRNAs), particularly microRNAs (miRNAs), play crucial roles in plant development and in responses to biotic and abiotic stresses. miRNAs regulate genes involved in diverse developmental processes, including nodule formation, which is fundamental for the nitrogen-fixing symbiosis that characterizes legumes. Functional studies have demonstrated that miRNAs are key modulators of plant defense, contributing to resistance against pathogens and environmental challenges. Moreover, miRNAs also participate in cross-kingdom communication, such as plant-bacteria interactions, influencing symbiotic efficiency. Advances in molecular biology have enabled the manipulation of miRNAs and their targets for crop improvement. Current approaches include the design of artificial miRNAs (amiRNA), modulation of miRNA expression through miRNA-encoded peptides, genome editing of non-coding genes using CRISPR/Cas9, and the application of RNA interference (RNAi) technology. Together, these strategies highlight the potential of miRNA-based tools in plant biotechnology. A deeper understanding of the molecular mechanisms governing miRNA-mediated gene silencing will provide powerful resources for optimizing legume productivity and resilience within sustainable agricultural systems.},
}
@article {pmid41811974,
year = {2026},
author = {Baba, S and Oncul, O and Aktas, Z},
title = {CRISPR-Cas based plasmid design for multidrug-resistant Klebsiella pneumoniae isolates.},
journal = {FEMS microbiology letters},
volume = {373},
number = {},
pages = {},
pmid = {41811974},
issn = {1574-6968},
support = {TDK-2020-36552//Istanbul University/ ; },
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; *Plasmids/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Humans ; Klebsiella Infections/microbiology ; beta-Lactamases/genetics ; Gene Editing ; },
abstract = {Antimicrobial resistance is a major global health concern that requires innovative therapeutic strategies. This study aimed to address this challenge by designing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (CRISPR-Cas)-based plasmid systems for potential genome editing applications in multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates. Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem were determined according to European Committee on Antimicrobial Susceptibility Testing guidelines. All isolates (n = 5) were resistant, with MIC ranges of 4-128 μg/ml for imipenem, 8-64 μg/ml for meropenem, and 8-256 μg/ml for ertapenem. Resistance gene analysis revealed blaOXA-48-like and blaCTX-M-15 in all isolates, while blaNDM-1 was detected in one isolate. Two CRISPR-based plasmid systems, CRISPR-Cas9 and CRISPR-assisted cytidine deaminase, were designed. Target genes were amplified by polymerase chain reaction, and guide RNA (gRNA) sequences were designed from selected regions. Apramycin (50 μg/ml) was identified as a suitable selection marker. The pSGKP-AmpR(Pro)-ApmR plasmid was successfully constructed, whereas Cas9 and APOBEC constructs could not be cloned. Overall, this study highlights technical challenges in developing CRISPR-based tools for MDR K. pneumoniae and emphasizes the need for isolate-specific plasmid design and gRNA optimization.},
}
@article {pmid41812798,
year = {2026},
author = {Huang, G and Zhuge, B and Du, X and Wang, M and Zong, H},
title = {TSA-ultrasound synergy enhances CRISPR-Cas9 gene editing efficiency in diploid yeast.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107449},
doi = {10.1016/j.mimet.2026.107449},
pmid = {41812798},
issn = {1872-8359},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Diploidy ; *Hydroxamic Acids/pharmacology ; Gene Knockout Techniques ; *Candida/genetics/drug effects ; Sonication/methods ; Chromatin ; },
abstract = {The CRISPR-Cas9 system is invaluable for microbial engineering. However, its efficiency remains limited in numerous microorganisms, especially in polyploid yeasts where the compact chromatin structures pose significant physical barriers, which are major constraints in microbial engineering. To address this limitation, we developed and validated a standardized synergistic protocol using the diploid industrial yeast Candida glycerinogenes as a model. This protocol combines TSA-induced chromatin decondensation with brief, low-intensity ultrasonication to enhance Cas9 accessibility. Key parameters were systematically optimized, and 200 nM Trichostatin A (TSA) (10 h) followed by 200 W sonication (3 min) were established as the optimal condition set. Validation results showed that the protocol more than doubled single-gene knockout efficiencies (GPD1, TRP1) compared with conventional methods. Importantly, it enabled complex edits that were previously unattainable in C. glycerinogenes, including the precise deletion of a 7.8-kb fragment and the editing of an 11.4-kb region for functional genomics. In summary, this study establishes a simple and effective workflow that overcomes chromatin-based barriers in the polyploid industrial yeast C. glycerinogenes, providing a practical tool for genetic engineering and functional genomics in this and potentially other recalcitrant yeasts.},
}
@article {pmid41812941,
year = {2026},
author = {Wang, M and Niu, D and Zhang, Q and Tang, Y and Zhao, Y and Chen, F},
title = {CRISPR-based correction of apolipoprotein E4 in Alzheimer's disease: Therapeutic strategies and macromolecular delivery innovations.},
journal = {International journal of biological macromolecules},
volume = {354},
number = {},
pages = {151352},
doi = {10.1016/j.ijbiomac.2026.151352},
pmid = {41812941},
issn = {1879-0003},
mesh = {*Alzheimer Disease/genetics/therapy ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Apolipoprotein E4/genetics ; Blood-Brain Barrier/metabolism ; Exosomes/metabolism ; Genetic Therapy/methods ; Nanoparticles/chemistry ; },
abstract = {Alzheimer's disease (AD) is the leading cause of dementia worldwide, with substantial unmet clinical needs. The apolipoprotein E4 (APOE4) allele is the strongest genetic risk factor for late onset AD, with each copy increasing risk approximately two- to three-fold, and homozygous carriers facing up to a 10- to 15-fold higher risk compared to APOE3 carriers. APOE4 contributes to diverse pathogenic mechanisms including lipid dysregulation, neuroinflammation, synaptic dysfunction, and vascular compromise. The precise, allele-specific correction of APOE4 therefore holds transformative therapeutic potential. CRISPR-based genome editing technologies, including nuclease disruption, base editing, and prime editing, offer unprecedented opportunities to directly modify APOE4 at its genomic source. Here, we review mechanistic underpinnings of APOE4 pathology, summarize current gene editing platforms for APOE4 correction, evaluate relevant in vitro and in vivo model systems, and assess delivery strategies with an emphasis on nanoparticle and exosome based approaches. We highlight recent breakthroughs in exosome mediated APOE4 editing while addressing ongoing technical hurdles in allele specificity and translational barriers such as Cas nuclease immunogenicity, limited delivery efficiency across the blood brain barrier (BBB), and concerns over long term genomic safety. This review concludes that overcoming BBB constraints remains the most significant challenge for clinical translation, and that innovations in exosome and nanoparticle based delivery platforms represent the most promising strategies for advancing CRISPR therapeutics for AD.},
}
@article {pmid41813543,
year = {2026},
author = {Qiao, JH and Gao, Q and Wang, XB},
title = {Virus-induced genome editing: toward crop breeding applications.},
journal = {Trends in plant science},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tplants.2026.01.007},
pmid = {41813543},
issn = {1878-4372},
abstract = {CRISPR-Cas-based genome editing has revolutionized precise genome manipulation in plants, yet its practical application is still constrained by the inefficient delivery of editing reagents across different genotypes. Plant viruses are promising vehicles for delivering genome-editing components, bypassing plant transformation and/or tissue culture. Virus-induced genome editing (VIGE) has provided powerful tools for achieving heritable edits in model plants such as Arabidopsis thaliana and Nicotiana benthamiana. VIGE has now progressed from proof-of-concept to practical applications in agricultural crops. Notably, a recent breakthrough in VIGE in tiller has successfully achieved heritable genome editing in hexaploid wheat. This review outlines the latest advances in VIGE across diverse plant species, highlights its potential for crop improvement, and discusses future research directions.},
}
@article {pmid41813887,
year = {2026},
author = {Tou, CJ and Xie, K and Ferreira da Silva, J and Kalailingam, P and Amar-Lewis, E and Rufino-Ramos, D and Sawyer, W and Eller, ML and Starzyk, J and Majumdar, I and Wang, J and Lee, D and Yang, S and Meis, RJ and Dahl, GA and Li, J and Shan, R and Artzi, N and Musolino, PL and Wu, H and Kleinstiver, BP},
title = {Immune evasive DNA donors and recombinases license kilobase-scale writing.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41813887},
issn = {1476-4687},
abstract = {Genome-editing technologies that use recombinases to insert kilobase-scale DNA sequences into mammalian genomes canonically require large double-stranded DNA (dsDNA) donors[1,2]. However, dsDNA molecules evoke problematic and toxic innate immune responses, limiting integration efficiencies and generally constraining applicability to ex vivo or immune-deficient contexts. By harnessing mechanisms of integrative prokaryotic viruses and mobile genetic elements, here we demonstrate that recombinases are compatible with immune evasive circular single-stranded DNA molecules optimally bearing a partial-duplex region that reconstitutes the recombinase recognition sequence. This approach, which we term integration through nucleus-synthesized template addition of large lengths (INSTALL), is compatible with diverse protein and RNA-guided recombinases for high-fidelity kilobase-scale human genome writing. INSTALL minimizes innate immune responses in primary human cells and in mice, improving recombinase-mediated integration efficiencies and supporting systemic in vivo non-viral DNA delivery by substantially increasing tolerability and broadening the dosing range compared with lipid nanoparticle-delivered dsDNA molecules. Together, INSTALL overcomes fundamental challenges for DNA delivery and integration methods by synergizing immune-stealth nucleic acids with recombinases to enable kilobase-scale integration strategies without viral vectors.},
}
@article {pmid41814120,
year = {2026},
author = {Simonneau, B and Baghdoyan, S and Cailleret, M and Simon, S and Ruckebusch, O and Vrablikova, B and Giraud-Triboult, K and Kassar, LE and Fanen, P and Duriez, B},
title = {CRISPR-Cas9 genome editing in the parental iPSC line PCIi033-A to introduce the homozygous mutation p.F508del (c.1521_1523del) in the CFTR gene.},
journal = {Stem cell research},
volume = {92},
number = {},
pages = {103947},
doi = {10.1016/j.scr.2026.103947},
pmid = {41814120},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Homozygote ; Cell Line ; *Mutation/genetics ; Cystic Fibrosis/genetics/pathology ; Cell Differentiation ; },
abstract = {Cystic Fibrosis (CF) is an autosomal recessive disease caused by mutations in the CFTR gene. Patients carrying the most common mutation, p.F508del, benefit from the triple therapy Kaftrio®. We genome-edited the commercially available iPSC line PCIi033-A (wild-type CFTR) to generate the subclone PCIi033-A-5, which is homozygous for the mutation c.1521_1523del (p.F508del), using CRISPR-SpCas9 tools. PCIi033-A-5 has a normal karyotype and stem cell morphology, is pluripotent, and differentiates into the three germ layers. Introducing this mutation in a parental isogenic iPSC line is essential to demonstrate the feasibility of modeling CF disease after differentiation of the iPS cells into bronchial epithelium.},
}
@article {pmid41816357,
year = {2026},
author = {Pan, Z and Xu, L and Fan, Z and Cao, Y and Ren, F},
title = {CRISPR-based diagnostics for infectious diseases: mechanisms, advancements and clinical transformation prospects.},
journal = {Frontiers in cellular and infection microbiology},
volume = {16},
number = {},
pages = {1769226},
pmid = {41816357},
issn = {2235-2988},
mesh = {Humans ; *CRISPR-Cas Systems ; *Communicable Diseases/diagnosis ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Infectious diseases continue to pose significant global public health challenges, necessitating the development of rapid, sensitive, specific, and field-deployable diagnostic platforms. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated proteins (Cas) has revolutionized genome editing and concurrently enabled a new generation of molecular diagnostic tools. Leveraging the inherent trans-cleavage activities of Cas enzymes, platforms such as SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter) have emerged, combining target recognition precision with reporter systems to achieve ultra-sensitive detection of pathogen-specific nucleic acids. This review systematically examines the mechanistic foundations of CRISPR diagnostics, synthesizes recent advancements in infectious disease applications, evaluates their advantages in sensitivity, specificity, operational simplicity, and multiplexing capacity, and critically analyzes current implementation barriers and future translational pathways.},
}
@article {pmid41816845,
year = {2026},
author = {Chen, WD and Liu, L and Cheng, L},
title = {Nitroreductase-Responsive Oligomeric crRNAs for Enzyme-Triggered Regulation of CRISPR Activity.},
journal = {The Journal of organic chemistry},
volume = {91},
number = {12},
pages = {4494-4501},
doi = {10.1021/acs.joc.5c02287},
pmid = {41816845},
issn = {1520-6904},
mesh = {*Nitroreductases/metabolism/chemistry ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism ; },
abstract = {Hypoxic tumors overexpress nitroreductase (NTR), providing an endogenous trigger for selective biomolecular activation. Here, we describe the synthesis of NTR-responsive clustered regularly interspaced short palindromic repeats (CRISPR) guide RNAs via the site-specific incorporation of a p-nitrobenzyl (p-NB) phosphoramidite at the 5' terminus of crRNAs. Click-mediated oligomerization into trimeric and tetrameric constructs effectively suppressed Cas nuclease activity. Enzymatic reduction by NTR induced linker cleavage, releasing active crRNAs and restoring DNA cleavage in vitro, establishing a strategy for enzyme-regulated CRISPR control.},
}
@article {pmid41816914,
year = {2026},
author = {Zhao, H and Zhou, T and Zhang, M and Wang, C and Wang, R and Shu, X and Cheng, F and Xue, Q and Liu, C and Xu, J and Cao, X and Du, J and Wang, L and Liu, H and Li, M},
title = {Associate toxin-antitoxin with CRISPR-Cas to harness (ATTACH) engineered microbes.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41816914},
issn = {1362-4962},
support = {2024YFA0918500//National Key Research and Development Project/ ; XDB0810000//Chinese Academy of Sciences/ ; 32370090//National Natural Science Foundation of China/ ; 32150020//National Natural Science Foundation of China/ ; 32400063//National Natural Science Foundation of China/ ; 32270092//National Natural Science Foundation of China/ ; 32200057//National Natural Science Foundation of China/ ; 32370120//National Natural Science Foundation of China/ ; 2020090//Youth Innovation Promotion Association of CAS/ ; },
mesh = {*CRISPR-Cas Systems ; Animals ; Mice ; *Toxin-Antitoxin Systems/genetics ; Escherichia coli/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Plasmids/genetics ; Gene Editing/methods ; Promoter Regions, Genetic ; },
abstract = {Robust biocontainment is essential for the safe use of engineered microbes, but existing strategies suffer from genetic instability and/or laborious construction. Here, we present ATTACH, a kill switch that associates toxin-antitoxin with CRISPR-Cas to harness engineered microbes. Our approach employs a CRISPR-repressed toxin-antitoxin (CreTA) module to make microbes addicted to the type I-F Cas effector proteins, and places both the Cas3 nuclease and the chromosome-targeting guide RNA under inducible promoters, thereby improving the genetic stability and stringency of the CRISPR-based suicidal program. Additionally, we have developed a single-plasmid, antibiotic-independent ATTACH device, which shows robust, stringent containment of a microbial chassis in murine gut, and negligible impacts on culture growth or lycopene production during batch fermentation. Our data highlight the potential of CreTA to stabilize CRISPR-based kill switches, advancing their development into more portable and reliable biocontainment tools for engineered microbes.},
}
@article {pmid41818662,
year = {2026},
author = {Wu, L and Chen, W and Huang, R and Zhou, F},
title = {EXAGO: An Argonaute-Based Primer-Free Exponential Amplification Strategy for Ultrasensitive Zero-Background Detection of Point Mutation.},
journal = {Analytical chemistry},
volume = {98},
number = {11},
pages = {8199-8208},
doi = {10.1021/acs.analchem.5c06961},
pmid = {41818662},
issn = {1520-6882},
mesh = {Humans ; *Argonaute Proteins/metabolism/genetics/chemistry ; *Nucleic Acid Amplification Techniques/methods ; *Point Mutation ; ErbB Receptors/genetics ; Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; Lung Neoplasms/genetics/diagnosis ; Limit of Detection ; },
abstract = {Ultrasensitive gene detection is crucial for precise molecular diagnostics. Conventional nucleic acid amplification methods frequently encounter nonspecific amplification triggered by exogenous primers, which limits their utility in ultrasensitive detection. Programmable gene editing tools, such as CRISPR/Cas and Argonaute (Ago), provide new avenues for developing next-generation detection technologies. Here, we develop an Ago-mediated exponential amplification strategy, EXAGO, for ultrasensitive detection of the epidermal growth factor receptor (EGFR) L858R mutation─a critical biomarker in nonsmall cell lung cancer. Leveraging the flexible programmability and single-base-resolution cleavage activity of Ago, EXAGO can specifically initiate DNA polymerase reactions at the target mutation site. The mechanism of dual-circuit operation allows the system to achieve an efficient exponential amplification under thermal cycling. By circumventing exogenous primers, it also prevents nonspecific amplification caused by primer misidentification at the source. Moreover, wild-type genes are entirely unable to trigger signal amplification, thereby underscoring their superior specificity and the potential for practical application. Experimental results demonstrated that EXAGO achieves femtomolar-level sensitivity and exhibits favorable recovery rates for detecting plasma-diluted samples. Moreover, the use of a thermostable enzyme allows direct compatibility with cell thermal lysis, enabling detection in cell lysates. In summary, EXAGO offers a robust and practical solution for accurate genetic mutation analysis in complex samples and promotes the application of Ago-based tools in molecular diagnostics.},
}
@article {pmid41819069,
year = {2026},
author = {Stephan, TL and Hoodless, PA},
title = {The ups and downs of maturing zonated hepatoctyes.},
journal = {Developmental cell},
volume = {61},
number = {3},
pages = {462-463},
doi = {10.1016/j.devcel.2026.02.009},
pmid = {41819069},
issn = {1878-1551},
mesh = {Animals ; *Hepatocytes/metabolism/cytology ; *Liver/metabolism/embryology/cytology ; Mice ; CRISPR-Cas Systems ; *Cell Differentiation ; },
abstract = {Current in vitro protocols differentiating hepatocytes fail to activate mature metabolic genes, induce zone-specific phenotypes, and suppress fetal liver signatures. In this issue, Taguchi, Magalhães et al.[1] used CRISPR-Cas9 screening in a mouse model of hepatic development to identify Nr1i3 and Nfix as regulators of hepatocyte maturation and zonation.},
}
@article {pmid41819070,
year = {2026},
author = {Wang, J and Lai, L},
title = {Repurposing Cas13's collateral cleavage activity to mitigate host cell dominance in interspecies chimera formation.},
journal = {Developmental cell},
volume = {61},
number = {3},
pages = {464-465},
doi = {10.1016/j.devcel.2026.02.010},
pmid = {41819070},
issn = {1878-1551},
mesh = {Animals ; Humans ; Mice ; *Chimera ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {The insufficient contribution of human cells is a key obstacle to interspecies chimera. In this issue of Developmental Cell, He et al. harnessed the RNA collateral cleavage activity of Cas13 to diminish the competitive advantage of host cells, increasing integration ratio of human cells to 1% in host mice.},
}
@article {pmid41819296,
year = {2026},
author = {Wang, C and Zhu, C and Liu, Q and Yang, L},
title = {Prokaryotic argonaute proteins: From ancient defense mechanisms to modern biosensing applications.},
journal = {Biotechnology advances},
volume = {89},
number = {},
pages = {108869},
doi = {10.1016/j.biotechadv.2026.108869},
pmid = {41819296},
issn = {1873-1899},
mesh = {*Biosensing Techniques/methods ; *Argonaute Proteins/genetics/metabolism/chemistry ; CRISPR-Cas Systems ; *Prokaryotic Cells/metabolism ; },
abstract = {Prokaryotic Argonaute (pAgo) proteins constitute an evolutionarily ancient nuclease family that is rapidly maturing into a versatile molecular toolkit rivaling CRISPR-Cas. This review synthesizes recent advances in pAgo biology and biotechnology, tracing their phylogeny across thermophilic, mesophilic, and psychrotolerant lineages and highlighting temperature-adapted catalytic signatures that diverge from eukaryotic Agos. In vivo studies reveal pAgo roles in gDNA-guided host defense, transcriptional silencing and recombination, all executed through programmable DNA- or RNA-guided nuclease activity. We detail how guide length, 5' nucleotide identity, divalent cations and accessory factors modulate cleavage efficiency, enabling rational optimization. The review then maps the explosion of pAgo-based biosensing platforms, including selective nucleic acid enrichment platforms, ultrasensitive pathogen detection methods, programmable DNA cloning systems, and high-resolution imaging techniques. Their independence from protospacer adjacent motifs (PAMs), stable DNA guides, multi-turnover kinetics, and broad thermal tolerance position pAgos as ideal complements to CRISPR systems. Finally, we outline current limitations and future directions, including the discovery and engineering of novel variants, elucidation of guide-generation mechanisms, and development of next-generation gene-editing tools, aiming to accelerate translation of these versatile enzymes into practical biotechnological and therapeutic translation.},
}
@article {pmid41820207,
year = {2026},
author = {Porwal, S and Malviya, R and Belagodu Srighar, S and Shareef, J},
title = {Ribosome-targeted and Adjuvant Strategies to Combat Antibiotic Resistance.},
journal = {Infectious disorders drug targets},
volume = {},
number = {},
pages = {},
doi = {10.2174/0118715265396934250805055608},
pmid = {41820207},
issn = {2212-3989},
abstract = {INTRODUCTION: This study examines ribosome-targeted and adjuvant strategies to combat the growing threat of antimicrobial resistance (AMR), with a focus on novel therapeutic approaches, including phage therapy, monoclonal antibodies, CRISPR systems, and AI-driven drug discovery. The objective is to review current challenges and evaluate innovative strategies targeting bacterial ribosomes, a primary site for antibiotic action.<br><br>METHODS: A systematic literature review was conducted using databases, such as PubMed, ScienceDirect, Scopus, and Google Scholar.<br><br>RESULTS: The study indicates that bacteria evade ribosome-targeting antibiotics through various mechanisms, including porin modification, efflux pumps, ribosomal mutations, and enzymatic degradation. Innovative strategies, including AI-enabled virtual screening, phage-antibiotic synergy, ribosomal protein-targeted monoclonal antibodies and vaccines, and CRISPR-Cas systems, have shown potential in overcoming these mechanisms and restoring antibiotic efficacy.<br><br>DISCUSSION: These advanced strategies represent a significant shift from traditional approaches, as they directly target ribosomal functions or resistance genes. While promising, limitations such as phage specificity, challenges in CRISPR delivery, and regulatory concerns must be addressed to ensure clinical translation. The integration of AI with molecular techniques enhances therapeutic precision and development speed.<br><br>CONCLUSION: Ribosome-targeted therapies and adjunctive strategies, such as AI, phage therapy, monoclonal antibodies, and CRISPR, offer precise and innovative solutions to overcome antibiotic resistance.},
}
@article {pmid41820622,
year = {2026},
author = {Padureanu, T and Cocoș, R and Matache, IM and Bucur, O},
title = {Gene-sized editing for the therapy of genetic diseases.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {},
pmid = {41820622},
issn = {1438-7948},
abstract = {Programmable genome editing technologies have reshaped the landscape of biomedical sciences, enabling the development of methods with great translational potential. CRISPR-Cas represents one of the most important and widely adopted genome editing tools, although its reliance on double-stranded DNA breaks implies inherent limitations on the precision and safety of genomic insertions. Thus, several research groups have focused on the development of new editing technologies, among which prime editing has emerged as a cutting-edge system. Ongoing advancements in prime editing, including protein engineering, have enhanced its efficiency and expanded its functionality. However, prime editing cannot achieve integration of large DNA sequences larger than 5 kilobases. To overcome this limitation, PASTE and PASSIGE methods were developed as novel genome editing methods that merge precise genome rewriting with efficient recombinase-mediated gene insertion. In this review, we investigate the mechanistic principles of these systems, compare their performance in cellular and animal models, and discuss the ongoing efforts to enhance system components and delivery. We extended our investigation to recent progress supporting their translational potential, assessing efficient delivery methods, genome site specificity, safety, and long-term efficacy, which are crucial for successful in vivo applications.},
}
@article {pmid41821244,
year = {2026},
author = {Elamin Eltom, A and Kareem, AK and Shaaban, Z and Sanaan Jabbar, H and Sharma, MK and Ghazi Al-Shawi, S and AlMohamadi, H and Abbas, Z and S Jabir, M and Ahmed AbdRabou, M},
title = {Progress of Magnetic Particles-Integrated CRISPR/Cas Biosensors for Pathogen Bacteria Detection: Design, Mechanism and Application.},
journal = {Critical reviews in analytical chemistry},
volume = {},
number = {},
pages = {1-19},
doi = {10.1080/10408347.2026.2636658},
pmid = {41821244},
issn = {1547-6510},
abstract = {Ensuring food safety through rapid, sensitive, and point-of-care (POC) detection of microbial pathogens is crucial for protecting public health and minimizing the socio-economic losses associated with foodborne diseases. Despite stringent regulatory measures, foodborne illnesses caused by microbial contamination continue to pose a significant global challenge. In this context, the emergence of CRISPR/Cas systems has significantly improved the performance of biosensors due to their programmability, high specificity, and precise recognition of target RNA and DNA sequences. Following target recognition, Cas proteins exhibit both cis- and trans-cleavage activities, enabling highly sensitive signal amplification. To achieve rapid analysis and low detection limits, recent studies have increasingly focused on integrating CRISPR/Cas system with magnetic particles (MPs). MPs offer key advantages, including superparamagnetism, biocompatibility, and facile surface functionalization, which enhance target enrichment, assay speed, and analytical sensitivity. Accordingly, substantial progress has been made in MP-conjugated CRISPR/Cas biosensors for the detection of diverse foodborne microbial pathogens. This review comprehensively summarizes recent advances in the integration strategies of magnetic particles with CRISPR/Cas-based biosensing platforms for the quantitative detection of microbial pathogens. Particular emphasis is placed on performance metrics, assay design, and the feasibility of these systems for POC applications, highlighting their potential to enhance food safety monitoring.},
}
@article {pmid41823840,
year = {2026},
author = {Loweree-Rivera, FD and Pérez-Álvarez, S and Castillo, AM and Vega Mares, JH and Leyva-Hernández, HA and Sánchez Chávez, E and Escobedo-Bonilla, CM and Uranga-Valencia, LP and Chávez Medina, JA},
title = {Pelecyphora chihuahuensis (Britton &amp; Rose) D. Aquino &amp; Dan. Sánchez: A Review on Its Taxonomy, Ecology and Conservation of an Endemic Mexican Cactus Species with Biotechnological Perspectives.},
journal = {Biology},
volume = {15},
number = {5},
pages = {},
pmid = {41823840},
issn = {2079-7737},
abstract = {The cactus Pelecyphora chihuahuensis is endemic to northern Mexico and represents an interesting subject on the integration of classical taxonomy with modern biotechnological tools to solve conservation issues. Because of its narrow ecological range and high ornamental value, the species is increasingly at risk from degradation of its habitats, climate change, and plant poaching. This review includes current knowledge on its taxonomic status, ecological distribution, and conservation needs, with a focus on biotechnological means to aid its preservation. Aspects such as molecular markers, next-generation sequencing, and previously reported GIS-based species distribution models provide valuable insights into its identity and ecological niche. Biotechnological tools for ex situ conservation include in vitro propagation and cryopreservation. Potential applications of CRISPR-Cas and synthetic biology in preserving rare cacti are also discussed. By uncovering gaps, this review opens a window of opportunity to urgently promote the sustainable management of P. chihuahuensis and related endangered cacti by merging biotechnology with ecology and taxonomy, the results presented here underscore the importance of integrating scientific findings into future research that supports the development and implementation of effective policies that prioritize the conservation and biocultural preservation of arid-land flora, ensuring that both ecological integrity and cultural values are maintained for these unique ecosystems.},
}
@article {pmid41824054,
year = {2026},
author = {Ren, C and Lin, Y and Mohamed, MSM and Liu, C and Liang, Z},
title = {A simplified RUBY reporter-enabled hairy-root system for rapid evaluation of genome editing and gene function in grapevine.},
journal = {Planta},
volume = {263},
number = {4},
pages = {},
pmid = {41824054},
issn = {1432-2048},
support = {YSBR-093//CAS Project for Young Scientists in Basic Research/ ; 32530090//National Natural Science Foundation of China/ ; 2022078//Youth Innovation Promotion Association of the Chinese Academy of Sciences/ ; },
mesh = {*Vitis/genetics/metabolism ; *Gene Editing/methods ; *Plant Roots/genetics/metabolism ; Genes, Reporter ; CRISPR-Cas Systems ; Lignin/metabolism/biosynthesis ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; Glucosyltransferases/genetics ; Gene Expression Regulation, Plant ; },
abstract = {The simplified RUBY represents an efficient reporter for monitoring plant transformation and, when combined with hairy-root system in grapevine, can facilitate the application of novel CRISPR technologies and gene functional study. Monitoring successful transformation events is essential for plant transformation and genome editing. The development and application of the RUBY reporter enable effective selection of transformation events based solely on distinct red pigmentation. Here, we report that a simplified version of RUBY (siRUBY), lacking the glucosyltransferase gene, also functions effectively as a visual selection marker for plant transformation. Furthermore, the siRUBY-assisted hairy-root system was established as a rapid and efficient platform for evaluating activity of adenine base editor (ABE) in grapevine. Targeted A-to-G base editing was achieved using ABE8e, with an average efficiency of approximately 14%. Moreover, this platform is also suitable for functional investigation of genes of interest. Knockout of the MYB4a gene promoted lignin deposition, likely through upregulating key lignin biosynthesis genes while repressing transcription of downstream flavonoid biosynthesis genes. Collectively, these results demonstrate that siRUBY coupled with grapevine hairy roots provides a robust platform for rapid and efficient genome editing in grapevine.},
}
@article {pmid41824451,
year = {2026},
author = {Yuan, L and Liu, Q and Xiao, X and Xu, L and Liang, L and Guo, Y and Yao, Y and Wang, H and Feng, Y and Hua, X and Feng, Y},
title = {AlphaFold 3-powered discovery of phage proteins that inhibit bacterial transcription initiation.},
journal = {Cell reports},
volume = {45},
number = {3},
pages = {117082},
doi = {10.1016/j.celrep.2026.117082},
pmid = {41824451},
issn = {2211-1247},
mesh = {*Viral Proteins/metabolism/chemistry ; *Bacteriophages/metabolism ; DNA-Directed RNA Polymerases/metabolism ; *Transcription Initiation, Genetic ; *Transcription, Genetic ; Protein Binding ; *Escherichia coli/genetics/metabolism/virology ; },
abstract = {Many phages encode proteins that specifically inhibit host RNA polymerase activity, thereby sabotaging and, in some cases, hijacking the host transcription machinery to serve their needs. Traditional methods for identifying new phage proteins that inhibit bacterial transcription are labor intensive and require access to live phages. To overcome these limitations, we develop a highly efficient pipeline for AlphaFold 3-guided discovery of phage proteins that inhibit bacterial transcription initiation. Using this pipeline, three phage proteins are identified and characterized. Structural and biochemical analyses demonstrate that these phage proteins bind to distinct sites on RNA polymerase and inhibit transcription initiation via different mechanisms. This study showcases the power of AlphaFold 3 in discovering novel binders of large protein complexes, and the pipeline developed here could be readily adapted to screen modulators of other large targets, such as the ribosome, proteasome, and CRISPR-Cas systems.},
}
@article {pmid41824934,
year = {2026},
author = {Arif, MA and Mubashir, F and Raffay, A and Talha, M and Zohaib, M and Khan, AR and Naseer, MU and Khan, MH and Idrees, M and Rafique, S and Afzal, S and Amin, I and Shahid, M},
title = {Recent Advances in CRISPR-Cas Systems for Dengue Virus Detection.},
journal = {Critical reviews in eukaryotic gene expression},
volume = {36},
number = {1},
pages = {19-35},
doi = {10.1615/CritRevEukaryotGeneExpr.2025062420},
pmid = {41824934},
issn = {2162-6502},
mesh = {*Dengue Virus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Dengue/diagnosis/virology/genetics ; RNA, Viral/genetics ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Dengue virus (DENV) infections persist as a significant global health threat despite decades of surveillance and control efforts. The disease may progress to severe dengue, marked by hemorrhage, plasma leakage, and vital organ impairment, contributing to substantial worldwide morbidity and mortality. The rapidly escalating DENV burden demands rapid and innovative diagnostic approaches that move beyond conventional detection methods. CRISPR-Cas-based technologies have emerged as a revolutionary approach, offering next-generation solutions for DENV diagnostics. This review outlines the recent advances in the use of CRISPR based technologies for robust and more sensitive detection of dengue virus nucleic acids, critically evaluating their advantages over conventional diagnostics, current limitations, and future prospects. The roles of Cas12 and Cas13 in DENV-RNA detection are discussed in detail. Additional key areas mentioned include field-deployable and portable CRISPR-Cas technologies, serotype-specific detection, hybrid and isothermal amplification-based approaches, and a combination of CRISPR with electrochemical sensing techniques and nanotechnology. Collectively, these advances highlight the potential of CRISPR-based diagnostics in evolving future strategies for rapid and effective dengue virus detection and control of infections.},
}
@article {pmid41825677,
year = {2026},
author = {Xu, Z and Haghighat, M and Shafiabady, N and Wu, R and Sang, R and Deng, W},
title = {Nuclear-targeted CRISPR/Cas delivery by using aptamer-modified polymer lipid nanoparticles.},
journal = {Nanomedicine : nanotechnology, biology, and medicine},
volume = {74},
number = {},
pages = {102928},
doi = {10.1016/j.nano.2026.102928},
pmid = {41825677},
issn = {1549-9642},
abstract = {Efficient nuclear delivery of CRISPR/Cas ribonucleoproteins (RNP) remains a significant hurdle for non-viral systems. To address this, we developed a polymer-lipid hybrid nanoparticle functionalized with the AS1411 aptamer, targeting nucleolin to facilitate nucleus-directed delivery. Confocal imaging confirmed the accumulation of these aptamer-modified nanoparticles within the cell nuclei. For precise quantification, we utilized an AI-assisted segmentation approach based on deep convolutional neural networks (CNN) to analyse nanoparticle and DAPI colocalization. We further evaluated in vitro gene knockout efficiency of Cas9/sgRNA by using this nucleus-targeted system. Aptamer-functionalised nanoparticles reduced GFP-positive cells to 30.0%, compared with 40.8% for untargeted nanoparticles. Further evaluation targeting the Lcn2 gene demonstrated higher knockout efficacy and a more potent inhibition of breast cancer cell proliferation. These findings indicate that aptamer-mediated nuclear targeting enhances CRISPR/Cas9 editing efficacy and may offer the potential to advance the performance of non-viral gene therapies.},
}
@article {pmid41826699,
year = {2026},
author = {Skafar, V and de Souza, I and Ghosh, B and Ferreira Dos Santos, A and Porto Freitas, F and Chen, Z and Sun, S and Donate Castillo, M and Nepachalovich, P and Seufert, L and Bothe, S and Tschuck, J and Mathur, A and Nunes-Alves, A and Buhr, J and Aponte-Santamaría, C and Schmitz, W and Mack, M and Eilers, M and Bargou, R and Chaufan, M and Kaur, M and Palma, M and Ubellacker, JM and Elling, U and Augustin, HG and Hadian, K and Meierjohann, S and Proneth, B and Conrad, M and Fedorova, M and Alborzinia, H and Friedmann Angeli, JP},
title = {Riboflavin metabolism shapes FSP1-driven ferroptosis resistance.},
journal = {Nature cell biology},
volume = {28},
number = {4},
pages = {696-706},
pmid = {41826699},
issn = {1476-4679},
mesh = {*Ferroptosis/drug effects ; Humans ; *Riboflavin/metabolism/pharmacology ; Antioxidants/metabolism ; Lipid Peroxidation/drug effects ; Cell Line, Tumor ; CRISPR-Cas Systems ; Phospholipid Hydroperoxide Glutathione Peroxidase/metabolism/genetics ; },
abstract = {Membrane protection against oxidative insults is achieved by the concerted action of glutathione peroxidase 4 (GPX4) and endogenous lipophilic antioxidants such as ubiquinone and vitamin E. More recently, ferroptosis suppressor protein 1 (FSP1) was identified as a critical ferroptosis inhibitor, acting via the regeneration of membrane-embedded antioxidants. Yet, regulators of FSP1 are largely uncharacterized, and their identification is essential for understanding the mechanisms buffering phospholipid peroxidation and ferroptosis. Here we report a focused CRISPR-Cas9 screen to uncover factors influencing FSP1 function, identifying riboflavin (vitamin B2) as a modulator of ferroptosis sensitivity. We demonstrate that riboflavin supports FSP1 stability and the recycling of lipid-soluble antioxidants, thereby mitigating phospholipid peroxidation. Furthermore, we show that the riboflavin antimetabolite roseoflavin markedly impairs FSP1 function and sensitizes cancer cells to ferroptosis. Our findings provide a rational strategy to modulate the FSP1-antioxidant recycling pathway and underscore the therapeutic potential of targeting riboflavin metabolism, with implications for understanding the interaction of nutrients, as well as their contributions to a cell's antioxidant capacity.},
}
@article {pmid41826758,
year = {2026},
author = {Deol, KK and Harris, CA and Tomlinson, SJ and Delaney, CJ and Al-Farhan, A and Mathiowetz, AJ and Doubravsky, CE and Pratt, DA and Olzmann, JA},
title = {Vitamin B2 metabolism promotes FSP1 stability to prevent ferroptosis.},
journal = {Nature structural &amp; molecular biology},
volume = {33},
number = {3},
pages = {525-536},
pmid = {41826758},
issn = {1545-9985},
mesh = {*Ferroptosis ; Humans ; *Riboflavin/metabolism ; *S100 Calcium-Binding Protein A4/metabolism/genetics/chemistry ; Protein Stability ; Flavin-Adenine Dinucleotide/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Ferroptosis, a regulated form of cell death driven by excessive lipid peroxidation, has emerged as a promising therapeutic target in cancer. Ferroptosis suppressor protein 1 (FSP1) is a critical regulator of ferroptosis resistance, yet the mechanisms controlling its expression and stability remain mostly unexplored. To uncover regulators of FSP1 abundance, we conducted CRISPR-Cas9 screens using a genome-edited, dual-fluorescent FSP1 reporter cell line, identifying both transcriptional and post-translational mechanisms that determine FSP1 levels. Notably, we identified riboflavin kinase and flavin adenine dinucleotide (FAD) synthase, enzymes that are essential for synthesizing FAD from vitamin B2, as key contributors to FSP1 stability. Biochemical and cellular analyses revealed that FAD binding is critical for both FSP1 activity and stability. FAD deficiency and mutations blocking FSP1-FAD binding triggered FSP1 degradation through a ubiquitin-proteasome pathway involving the E3 ligase RNF8. Unlike other vitamins that inhibit ferroptosis by scavenging radicals, vitamin B2 supports ferroptosis resistance through FAD cofactor binding, ensuring proper FSP1 stability and function. This study provides a rich resource detailing mechanisms that regulate FSP1 abundance and highlights a novel connection between vitamin B2 metabolism and ferroptosis resistance, with implications for therapeutic strategies targeting FSP1 in cancer.},
}
@article {pmid41827871,
year = {2026},
author = {Zheng, J and Wu, M and Wang, X and Zuo, Z and Zhou, C and Zuo, E and Lu, Y},
title = {Prime Editing Exhibits Limited Genome-Wide Off-Target Effects in Cellular and Embryonic Gene Editing.},
journal = {Cells},
volume = {15},
number = {5},
pages = {},
pmid = {41827871},
issn = {2073-4409},
support = {2023ZD0405302 and 2023ZD04074//the Biological Breeding-Major Projects/ ; 2021YFD1300100 and 2024YFC3406001//the National Key Research and Development Program of China/ ; 32371549 and W2533083//the National Natural Science Foundation of China/ ; },
mesh = {*Gene Editing/methods ; Animals ; CRISPR-Cas Systems/genetics ; Humans ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Embryo, Mammalian/metabolism ; Mice ; *Genome ; Polymorphism, Single Nucleotide/genetics ; },
abstract = {Prime editing (PE) is a precise genome-editing technology that avoids double-strand breaks, holding great promise for clinical and agricultural applications. However, its genome-wide off-target effects are not fully understood, raising safety concerns. Here, we systematically compared the safety profiles of four prime editor variants (PE2max, PE3max, PE4max, and PE5max) using PEM-seq and RNA-seq. We further applied an ultra-sensitive method, Genome-wide Off-target analysis by Two-cell embryo Injection (GOTI), to assess PE5max. Our results show that PE5max did not produce detectable sgRNA-dependent off-target single-nucleotide variants (SNVs) in the GOTI assay and induced only limited large deletions and chromosomal translocations. Collectively, this side-by-side benchmarking under matched conditions demonstrates that PE5max achieves an improved specificity profile, with no detectable increase in genome-wide off-target SNVs, advancing its potential for safer therapeutic use.},
}
@article {pmid41827889,
year = {2026},
author = {Siles, L and Ruiz-Nogales, S and Méndez-Vendrell, P and Pomares, E},
title = {Precise CRISPR/Cas9 and Cas12 Correction Using Lipoplexes in Retinal Models Derived from Patients with Inherited Retinal Dystrophies.},
journal = {Cells},
volume = {15},
number = {5},
pages = {},
pmid = {41827889},
issn = {2073-4409},
support = {Fi-201401//Fundaci&#xf3; de Recerca de l'Institut de Microcirurgia Ocular/ ; //IMO Grupo Miranza/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Retinal Dystrophies/genetics/pathology ; *Retina/pathology/metabolism ; Retinal Pigment Epithelium/metabolism/pathology ; Induced Pluripotent Stem Cells/metabolism ; Bestrophins/genetics ; Organoids/metabolism ; *Models, Biological ; },
abstract = {Gene editing, particularly CRISPR/Cas technology, represents a promising approach for the treatment of rare genetic diseases, including inherited retinal dystrophies, for which effective therapies are largely unavailable. Despite extensive research investigating gene editing across a wide range of cell types, transient delivery of CRISPR/Cas components and efficient homology-directed repair (HDR) in differentiated cells remain challenging. In this study, we employed hiPSCs derived from patients with Stargardt disease or Best disease, carrying pathogenic variants in ABCA4 or BEST1, respectively, to explore gene editing in human models. CRISPR/Cas9 and Cas12 nucleases were delivered into hiPS-derived retinal pigment epithelium (RPE) and retinal organoids using lipoplexes and compared with electroporation. We evaluated transfection efficiency, sgRNA-mediated DNA cleavage, and HDR-based correction. Precise repair of the pathogenic BEST1 variant was successfully achieved in hiPS-derived RPE cells using both nucleases, with Cas12 yielding the highest efficiency, exceeding 10% of HDR correction. Edited RPE cells preserved normal morphology and expressed specific maturity markers. In contrast, retinal organoids exhibited moderate transfection efficiency but showed no detectable CRISPR/Cas-induced DNA cleavage, highlighting the need for further optimization of gene editing in more complex cellular tissues. This study demonstrates, for the first time, precise correction of a single-nucleotide mutation in patient-derived RPE using CRISPR/Cas9 and Cas12 delivered using lipoplexes. These findings underscore the therapeutic potential of CRISPR/Cas-based strategies for inherited retinal dystrophies and provide a proof of concept for future clinical approximations.},
}
@article {pmid41828338,
year = {2026},
author = {Xu, M and Ma, Y and Liu, M and Chen, Y and Duan, Z and Shen, Z and Han, Y},
title = {Bmp16 Regulates Arterial Valve Morphogenesis Through Modulation of Notch Signaling in Zebrafish.},
journal = {International journal of molecular sciences},
volume = {27},
number = {5},
pages = {},
pmid = {41828338},
issn = {1422-0067},
mesh = {Animals ; *Zebrafish/embryology/genetics/metabolism ; *Signal Transduction ; *Heart Valves/metabolism/embryology ; *Zebrafish Proteins/metabolism/genetics ; *Receptors, Notch/metabolism/genetics ; *Morphogenesis/genetics ; *Bone Morphogenetic Proteins/metabolism/genetics ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; },
abstract = {Congenital valve defects account for a substantial proportion of cardiovascular malformations, yet the molecular mechanisms orchestrating cardiac valve development remain incompletely elucidated. While Bone morphogenetic protein (BMP) signaling is essential for valvulogenesis, the specific contributions of individual BMP ligands, particularly the teleost-specific bmp16, have not been characterized. Using the CRISPR/Cas9 system, we generated a bmp16 null knockout and delineated critical roles of this ligand in valvular morphogenesis. bmp16 knockout embryos display a significant reduction in Sox9-positive valvular cells and exhibit severely dysplastic arterial valves, characterized by increased interleaflet distance, thickened leaflets, and shortened leaflet lengths. These morphological abnormalities correlate with impaired valve function, culminating in progressive blood regurgitation, ventricular dilation, and pericardial edema. Mechanistically, loss of bmp16 or pharmacological inhibition of BMP signaling significantly downregulates notch1b expression in developing valves, while pharmacological activation of Notch signaling rescues the regurgitation phenotype in bmp16 mutants. Collectively, our findings establish bmp16 as a novel regulator of valve development and uncover a functional BMP-Notch signaling axis required for vertebrate valvulogenesis, providing new insights into the molecular mechanisms that govern cardiac valve formation and pathogenesis.},
}
@article {pmid41828461,
year = {2026},
author = {Ferrero, M and Acquadro, A and Moglia, A},
title = {From Lab to Field: CRISPRing Major Cultivated Solanaceae for Crop Improvement.},
journal = {International journal of molecular sciences},
volume = {27},
number = {5},
pages = {},
pmid = {41828461},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; *Crops, Agricultural/genetics/growth &amp; development ; *Solanaceae/genetics ; Plants, Genetically Modified/genetics ; Genome, Plant ; Plant Breeding/methods ; },
abstract = {The Solanaceae family includes some of the most economically and agronomically important crops, such as tomato, potato, pepper and eggplant. Recently, CRISPR/Cas-based genome editing has emerged as a powerful tool for functional genomics and crop improvement, enabling precise and efficient genetic modifications. This review provides an overview of CRISPR/Cas-mediated genome editing technologies and their applications in the major cultivated Solanaceae crops. The use of CRISPR/Cas9 systems for targeted gene knockout and knock-in approaches is described, together with advances in precision editing strategies such as base editing and prime editing, which allow precise nucleotide substitutions and small sequence changes. The expanding CRISPR toolbox is further explored through alternative Cas proteins, such as Cas12a and Cas13 with distinct targeting features and potential applications. Emerging delivery strategies, including ribonucleoprotein-mediated editing in protoplasts, virus-induced gene editing (VIGE), de novo induction of meristems and genome editing by grafting, represent promising approaches to generate transgene-free edited plants. In addition, the current status of field trials involving genome-edited Solanaceae crops in Europe is outlined, considering the regulatory landscape and legislative requirements for their release in the environment. Despite regulatory constraints, some genome-edited crops have reached the market, highlighting their potential to contribute to sustainable agriculture and crop improvement.},
}
@article {pmid41828635,
year = {2026},
author = {Dagdelen, B and Arikoglu, H and Erkoc-Kaya, D and Bozkurt, B},
title = {Precise CRISPR-Mediated Editing of the TGFBI R555W Mutation in Patient-Derived Peripheral Blood Mononuclear Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {5},
pages = {},
pmid = {41828635},
issn = {1422-0067},
support = {19102037//Sel&#xe7;uk University/ ; },
mesh = {Humans ; *Leukocytes, Mononuclear/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Corneal Dystrophies, Hereditary/genetics ; *Mutation ; *Transforming Growth Factor beta/genetics ; *Extracellular Matrix Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Over 70 mutations in the transforming growth factor beta-induced (TGFBI) gene are associated with corneal dystrophies that impair vision. The R555W hotspot mutation is a major cause of granular corneal dystrophy type 1 (GCD1). Here, we evaluated the technical feasibility of CRISPR/Cas9-mediated editing of the R555W mutation in peripheral blood mononuclear cells (PBMCs) obtained from a patient with GCD1. Three single guide RNAs (sgRNA1-3) and matched single-stranded oligodeoxynucleotide donors (ssODN1-3) were designed and co-transfected into PBMCs. Transfected cells were enriched by flow cytometric sorting, with GFP-positive cells representing approximately 2-4% of the total electroporated population. Editing outcomes were initially screened using high-resolution melting (HRM) analysis, and the sgRNA3-ssODN3 combination identified as the most promising candidate was subsequently validated by next-generation sequencing (NGS). Sequencing revealed a homology-directed repair efficiency of 98.2% among GFP-positive sorted cells, demonstrating efficient and precise genome editing within the enriched population. Because PBMCs are not disease-relevant corneal epithelial cells and only genomic endpoints were assessed, the clinical applicability of this study is limited and the work should be considered a technical proof-of-concept. This framework supports optimization of CRISPR-based strategies prior to studies in biologically relevant corneal models.},
}
@article {pmid41828895,
year = {2026},
author = {Gu, X and Zhou, Y},
title = {Unlocking the Potential of Macroalgae: Innovative Pretreatment Strategies for Efficient Biorefinery.},
journal = {Molecules (Basel, Switzerland)},
volume = {31},
number = {5},
pages = {},
pmid = {41828895},
issn = {1420-3049},
support = {No. 31900088//the National Natural Science Foundation of China/ ; No. ZR2025MS493//the Natural Science Foundation of Shandong Province of China/ ; },
mesh = {*Seaweed/chemistry/metabolism/genetics ; Biomass ; *Biofuels ; Fermentation ; Cell Wall/chemistry/metabolism ; Gene Editing ; Biotechnology/methods ; CRISPR-Cas Systems ; },
abstract = {Macroalgae represent a promising third-generation feedstock for biorefinery due to their high biomass productivity and non-reliance on arable land. However, their complex cell wall structure poses a significant barrier to efficient bioconversion. This review integrates current pretreatment methods, including physical, chemical, biological, and combined approaches, with a focus on their mechanisms, effectiveness, and limitations. Furthermore, it explores the conversion of pretreated macroalgal biomass into bioenergy and biochemicals, such as bioethanol, organic acid and polyhydroxyalkanoate, via microbial fermentation. The review also examines the application of genetic editing tools (e.g., CRISPR-Cas systems) for the targeted modification of macroalgae to improve their inherent characteristics for biorefinery, such as reducing biomass recalcitrance or increasing the content of target carbohydrates. Finally, future perspectives on technological innovations and integrated industrial chains of macroalgal biorefinery are discussed. This review serves as a systematic reference for deepening the understanding of macroalgal cell wall deconstruction processes and supports the development of efficient and environmentally benign pretreatment strategies to advance macroalgal biorefinery toward industrialization.},
}
@article {pmid41829816,
year = {2026},
author = {Al-Sawa'eer, AS and Al-Samydai, A and Odeh, L and Haj Ahmad, F and Obekh, R and Elqader, YMA and Khaleel, A and Al-Athamneh, AM and Gabriele, M and Di Simone, SC and Ferrante, C and Menghini, L and Ali Agha, ASA},
title = {Early Plant Development as a Systems-Level Trait: Integrating Omics, Artificial Intelligence, and Emerging Biotechnologies.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {5},
pages = {},
pmid = {41829816},
issn = {2223-7747},
abstract = {Seed germination and early seedling development are critical determinants of crop establishment, stress tolerance, and yield stability, yet these stages remain insufficiently integrated into contemporary crop improvement strategies. Recent advances across genome editing, microbiome-assisted seed treatments, nanotechnology-enabled priming, and artificial intelligence-guided phenotyping have generated substantial but fragmented insights into early developmental regulation. This review synthesizes recent advances across early plant development research. It demonstrates that seemingly diverse technologies converge on a limited set of regulatory control nodes, including abscisic acid-gibberellin balance, redox homeostasis, and root system architectural plasticity. By integrating evidence from molecular, microbial, physicochemical, and computational studies, early plant ontogeny is presented as a tunable regulatory state governed by quantitative thresholds rather than as a strictly predetermined genetic process. Advances in deep learning, reinforcement learning, and high-throughput phenotyping further enable the modeling and optimization of early developmental trajectories across genotype by environment contexts. Together, these insights establish early development as a programmable target for crop improvement and provide a mechanistic foundation for designing integrated interventions that enhance developmental uniformity, stress resilience, and yield stability across diverse agroecological systems.},
}
@article {pmid41830331,
year = {2026},
author = {Martínez-Alvarez, L and Peng, X},
title = {Diversity and evolution of archaeal immune strategies.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41830331},
issn = {1362-4962},
support = {DFF-0135-00402//Danish Council for Independent Research/ ; 10.46540/4264-00120B//Danish Council for Independent Research/ ; NNF17OC0031154//Novo Nordisk Foundation/ ; DeiC-KU-N1-2024089//Danish e-Infrastructure Consortium/ ; DFF-0135-00402//Natural Sciences/ ; 10.46540/4264-00120B//Natural Sciences/ ; },
mesh = {*Archaea/genetics/immunology/classification ; Phylogeny ; *Genome, Archaeal ; CRISPR-Cas Systems/genetics ; *Evolution, Molecular ; Bacteria/genetics/immunology ; },
abstract = {Archaeal antiviral defense systems remain poorly characterized despite recent advances in understanding prokaryotic immunity. Here, we analyze 7747 archaeal genomes, the largest and most diverse dataset to date, revealing a striking disparity in defense system prevalence and diversity compared to Bacteria. Nearly one-third of archaeal genomes have no detected systems beyond CRISPR-Cas and restriction-modification (in contrast to only 2.2% bacterial genomes), and only 50-55% contain CRISPR-Cas systems, far below previous estimates. Many known defense systems appear restricted to Bacteria, while several single-gene putative candidate systems (PDCs) recently identified through a guilt-by-embedding approach are enriched in Archaea. Phylogenetic analyses suggest that PDC-S70 and PDC-M05 likely originated in Archaea, representing rare archaeal contributions to the prokaryotic immune repertoire. Consistent with earlier studies, our findings support the existence of deep evolutionary links between archaeal and eukaryotic systems for argonautes and viperins. These analyses highlight both the underexplored nature and the evolutionary significance of archaeal immunity, calling for expanded efforts to uncover archaeal-specific systems and improve our understanding of immune evolution across domains of life.},
}
@article {pmid41830765,
year = {2026},
author = {Leandro, K and Rufino-Ramos, D and Lopes, SM and Silva, FS and Rodrigues-Santos, P and Silva, AC and Fernandes, AR and Henriques, C and Pereira, D and Lobo, D and Gabriel, GL and Faro, R and Nobre, RJ and Perdigão, PRL and Kleinstiver, BP and de Almeida, LP},
title = {Extracellular vesicles-mediated delivery of SpCas9 RNPs for therapeutic gene editing in Spinocerebellar Ataxia Type 3.},
journal = {Biomaterials},
volume = {331},
number = {},
pages = {124119},
doi = {10.1016/j.biomaterials.2026.124119},
pmid = {41830765},
issn = {1878-5905},
mesh = {*Gene Editing/methods ; *Extracellular Vesicles/metabolism ; Animals ; Humans ; *Machado-Joseph Disease/therapy/genetics ; Ataxin-3/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Ribonucleoproteins/genetics/metabolism/administration &amp; dosage ; CRISPR-Cas Systems ; Mice ; Induced Pluripotent Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Repressor Proteins ; },
abstract = {Spinocerebellar Ataxia Type 3 (SCA3) is a neurodegenerative dominantly-inherited disorder caused by an overexpansion of a CAG tract within the ATXN3 gene, conferring toxic properties to the ataxin-3 protein. Genome editing with CRISPR-Cas9 enzymes is a promising strategy to inactivate mutant ATXN3 alleles, however, in vivo delivery remains challenging. Extracellular vesicles (EVs) are promising delivery vehicles for Cas9 and single guide RNA (sgRNA) ribonucleoproteins that minimize genomic exposure to highly active endonucleases. In this study, we designed SpCas9 with a palmitoylation motif that enables SpCas9 and sgRNA enrichment into EVs. Introduction of a photocleavable linker - PhoCl - allowed the photo-inducible release of SpCas9 from the palmitoylation motif in EVs, increasing target engagement to ATXN3 in vitro. EVs loaded with SpCas9 ribonucleoproteins resulted in ATXN3 knockout in SCA3 patient-derived iPSCs and two SCA3 animal models. These findings highlight an innovative route for transient delivery of gene editing tools. This approach provides a promising therapeutic platform for the treatment of genetic diseases, including SCA3.},
}
@article {pmid41831412,
year = {2026},
author = {Liu, J and Luo, S and Chen, S and Chen, G and Zhu, Y and Lou, Y and Fan, R and Zhang, Y and Pan, J and Zhu, C and Xu, L and Li, L},
title = {Dual-function CRISPR/Cas12a assisted strand displacement reaction with RuHex-loaded DNA condensates for ultrasensitive electrochemical detection of hepatocellular carcinoma mRNA.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118610},
doi = {10.1016/j.bios.2026.118610},
pmid = {41831412},
issn = {1873-4235},
mesh = {Humans ; *Liver Neoplasms/genetics/diagnosis ; *Carcinoma, Hepatocellular/genetics/diagnosis ; *Biosensing Techniques/methods ; *RNA, Messenger/genetics/isolation &amp; purification ; CRISPR-Cas Systems/genetics ; Electrochemical Techniques/methods ; Limit of Detection ; Ruthenium/chemistry ; DNA/chemistry ; CRISPR-Associated Proteins/chemistry ; Endodeoxyribonucleases/chemistry ; Bacterial Proteins ; },
abstract = {Hepatocellular carcinoma (HCC) typically develops in a clinically silent manner, and the suboptimal sensitivity and specificity of currently available diagnostic biomarkers remain significant obstacles to its accurate and early detection. To improve molecular diagnostic performance, we developed a dual-function CRISPR/Cas12a assisted strand displacement reaction (dCas12a-SDR) with hexaammine ruthenium(III) chloride (RuHex)-loaded DNA condensate for ultrasensitive and highly specific detection of HCC-associated mRNAs. Upon target recognition, the previously sequestered Cas12a activation site within the electrode-immobilized capture probe is exposed, thereby inducing hybridization between the accessible single-stranded domains retained in RuHex-loaded DNA condensates (RuDC) and the displacement strand (Ds), which ultimately leads to the release of electroactive Ds-RuDC condensates and the effective activation of Cas12a. Activated Cas12a then removes the activation site via cis-cleavage, releasing the target to enter subsequent reaction cycles; concurrently, the activated system initiates trans-cleavage of adjacent capture probes and Ds-RuDC assemblies on the electrode that harbor trans-cleavage motifs, thereby promoting the release of RuDC from the electrode interface. This cascade ultimately leads to a pronounced reduction in the electrochemical signal. Owing to this target-triggered dual cis- and trans-cleavage mechanism mediated by Cas12a, the biosensor achieves highly efficient signal amplification. The platform affords a limit of detection as low as 39.2 aM for PD-L1 mRNA and exhibits excellent specificity, stability, and reproducibility. Moreover, by jointly detecting a panel of HCC-associated mRNAs (PD-L1, GPC3, EpCAM, and FGA), the platform successfully discriminated healthy individuals from patients with early-stage hepatocellular carcinoma in clinical serum samples. Collectively, this platform provides a powerful tool for molecular diagnosis of hepatocellular carcinoma.},
}
@article {pmid41831437,
year = {2026},
author = {Yang, J and Wang, T and Liu, Z and Wu, W and Sun, Y and Zhan, Y and Zhang, S and Chen, H and Liu, B and Yue, C and Yin, Z and Shan, Z and Li, X and Li, Z and Yuan, Z and Yin, H and Zhang, H},
title = {Molecular basis for dual-spacer-guided target cleavage by the TIGR-TasH system.},
journal = {Molecular cell},
volume = {86},
number = {7},
pages = {1217-1229.e6},
doi = {10.1016/j.molcel.2026.02.017},
pmid = {41831437},
issn = {1097-4164},
mesh = {*RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry/ultrastructure ; Cryoelectron Microscopy ; *CRISPR-Cas Systems ; *Bacteriophages/genetics/enzymology ; Models, Molecular ; Gene Editing ; *Viral Proteins/genetics/metabolism/chemistry ; Nucleic Acid Conformation ; Protein Domains ; DNA/metabolism/genetics/chemistry ; Protein Binding ; },
abstract = {The RNA-directed programmable nuclease systems, exemplified by the CRISPR-Cas system, have been widely used in genome editing. In contrast to the single-spacer configuration of CRISPR RNA (crRNA), the guide RNA (tigRNA) of the tandem interspaced guide RNA (TIGR) system features a dual-spacer arrangement, thereby directing the TIGR-associated (Tas) protein to engage both strands of the target double-stranded DNA (dsDNA). Here, we determine six cryo-electron microscopy structures of the Salicola phage TIGR-TasH complex. The central coiled-coil region of TasH mediates dimerization, while the C-terminal nucleolar protein (Nop) domain is able to autonomously process precursor tigRNA. Upon target binding, the dynamic N-terminal HNH nuclease domain is recruited for cleavage through a β-hairpin, which also determines the target preference. More interestingly, the conserved box C motif of tigRNA stabilizes this β-hairpin in an adenine-specific manner, enabling us to rationally design a guide RNA-defined nickase, distinct from conventional protein-based nickase strategies used in genome editing.},
}
@article {pmid41831575,
year = {2026},
author = {Park, E and Lee, S and Kim, D and Choi, Y and Choung, S and Kim, H and Kim, SG},
title = {Virus-induced genome editing enables functional genomics across diverse plant species.},
journal = {Molecules and cells},
volume = {49},
number = {5},
pages = {100348},
pmid = {41831575},
issn = {0219-1032},
mesh = {*Gene Editing/methods ; *Genomics/methods ; *Genome, Plant ; CRISPR-Cas Systems ; *Plant Viruses/genetics ; *Plants/genetics/virology ; },
abstract = {Virus-induced genome editing (VIGE) is expanding plant functional genomics by enabling precise and heritable genome modification across diverse species. While classical model systems such as Arabidopsis thaliana have provided foundational genetic insights, many ecologically, agriculturally, and chemically important traits reside in species that remain difficult to manipulate genetically. By coupling CRISPR-Cas systems with plant viral vectors, VIGE bypasses key limitations of conventional transformation and enables rapid mutagenesis without repeated tissue culture and plant regeneration. This approach enables researchers to examine gene function in species selected for biological relevance rather than technical convenience. Here, we review the conceptual framework, technical considerations, and applications of VIGE, and discuss its current limitations and future prospects in ecological, evolutionary, and crop research.},
}
@article {pmid41831871,
year = {2026},
author = {Li, J and Cai, W and Wang, L and He, J and Meng, Y and Liu, J and Xu, L and Feng, K and Shen, G and Wei, P and He, L},
title = {Functional analysis of inducible choline/carboxylesterase CCE01 associated with fenpropathrin and abamectin detoxification in Tetranychus urticae (Koch).},
journal = {Pesticide biochemistry and physiology},
volume = {219},
number = {},
pages = {107003},
doi = {10.1016/j.pestbp.2026.107003},
pmid = {41831871},
issn = {1095-9939},
mesh = {Animals ; *Ivermectin/analogs &amp; derivatives/metabolism/toxicity/pharmacology ; *Pyrethrins/metabolism/toxicity ; *Tetranychidae/drug effects/enzymology/genetics/metabolism ; *Acaricides/metabolism/toxicity/pharmacology ; Drosophila melanogaster/genetics ; Inactivation, Metabolic ; *Carboxylesterase/metabolism/genetics ; Arthropod Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The two-spotted spider mite, Tetranychus urticae, represents a globally significant and highly destructive polyphagous pest, notorious for its capacity to develop severe resistance to numerous acaricides. Our previous investigative work identified a specific esterase gene, designated TuCCE01, which was consistently found to be over-expressed in multiple resistant mite populations. This study systematically investigated the functional role of the esterase gene TuCCE01 in metabolizing acaricides. Quantitative real-time PCR revealed that TuCCE01 expression in a susceptible strain was significantly induced by sublethal doses of fenpropathrin and abamectin, showing 3.6-fold and 3.8-fold increases after 24 h, respectively. RNA interference-mediated knockdown of TuCCE01 increased mite susceptibility to both acaricides. Critically, CRISPR/Cas9-mediated gene editing was successfully employed to generate homozygous TuCCE01 knockout mutant lines. Bioassays demonstrated that this specific knockout strain exhibited significantly increased sensitivity to both fenpropathrin and abamectin, with LC50 values markedly reduced. Conversely, transgenic Drosophila melanogaster overexpressing TuCCE01 exhibited enhanced tolerance. Importantly, heterologous expression and in vitro incubation assays confirmed that recombinant TuCCE01 could effectively deplete both acaricides. Homology modeling and molecular docking demonstrated that both acaricides bind to the substrate-binding pocket of TuCCE01, indicating direct enzymatic metabolism, which was consistent with the metabolic assays. This study enhances our understanding of CCE-mediated acaricide acute detoxification and provides a candidate molecular marker for future resistance monitoring.},
}
@article {pmid41832076,
year = {2026},
author = {Gundra, SR and Jiang, W and Aouida, M and Wang, Q and Kazlak, AM and Elbehery, AHA and Saleh, A and Masood, M and Ghouneimy, A and Mahfouz, M},
title = {Characterization and engineering of highly efficient Cas12j genome editors.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.02.001},
pmid = {41832076},
issn = {1879-3096},
abstract = {The large size of widely used CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) enzymes limits their delivery for therapeutic applications. Cas12j nucleases offer a hypercompact alternative but show modest editing efficiency. To overcome this limitation, we identified eight novel Cas12j orthologs from viral metagenomes, which in their native form exhibit low editing activity in mammalian cells. We therefore engineered T5 exonuclease-Cas12j fusions, resulting in substantially enhanced genome-editing activity across multiple mammalian cell types, reaching levels comparable to established compact CRISPR-Cas editors. Intriguingly, robust cellular editing occurred in the presence of a previously unrecognized trinucleotide sequence context within the target DNA. Furthermore, we developed Cas12j-based adenine base editors by coupling catalytically inactive Cas12j orthologs with adenine deaminase, enabling efficient A-to-G base conversion in mammalian cells. This study expands the CRISPR toolbox by establishing engineering principles that convert compact Cas12j nucleases into efficient and modular genome-editing platforms well suited for delivery-constrained therapeutic applications.},
}
@article {pmid41832226,
year = {2026},
author = {Lee, SY and Park, HH},
title = {AcrIIA7 hijacks tracrRNA to block CRISPR-Cas system.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-70749-w},
pmid = {41832226},
issn = {2041-1723},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; },
abstract = {The CRISPR-Cas9 system provides adaptive immunity against invading genetic elements through a dual-RNA-guided DNA cleavage mechanism. This system relies on the precise assembly of a ribonucleoprotein (RNP) complex composed of the Cas9 endonuclease, a CRISPR-derived RNA (crRNA), and a trans-activating CRISPR RNA (tracrRNA). Around 100 anti-CRISPR proteins that inhibit CRISPR-Cas systems have been identified, and the mechanisms by which they act are increasingly being elucidated. However, the inhibitory mechanisms of many Acrs, including AcrIIA7, remain poorly understood. Here, we present the structure of AcrIIA7 and uncover a previously unrecognized mechanism by which it inhibits Cas9 function. Structural and biochemical analyses reveal that AcrIIA7 specifically binds to tracrRNA, preventing its association with crRNA and thereby blocking formation of the active Cas9 RNP complex. This tracrRNA hijacking mechanism represents a unique strategy for CRISPR inhibition, in which an anti-CRISPR protein targets an RNA scaffold essential for Cas9 activation rather than interacting directly with the Cas9 protein. Our findings provide the first structural insight into tracrRNA-targeted anti-CRISPR activity and highlight RNA-RNA interaction interfaces as vulnerable nodes in CRISPR-Cas immunity.},
}
@article {pmid41833778,
year = {2026},
author = {Quintana, AJ and García-Suárez, R and Prieto, A and Sánchez, J and Gómez, I and Verduzco-Rosas, LA and doNascimento, N and Lopez-Molina, S and Zhang, J and Soberón, M and Bravo, A and Pacheco, S},
title = {Genome editing of ABCB6 transporter confers resistance to cypermethrin in the major pest of corn, Spodoptera frugiperda.},
journal = {Insect biochemistry and molecular biology},
volume = {190},
number = {},
pages = {104537},
doi = {10.1016/j.ibmb.2026.104537},
pmid = {41833778},
issn = {1879-0240},
mesh = {Animals ; *Spodoptera/genetics/drug effects/metabolism/growth &amp; development ; *Insecticide Resistance/genetics ; *Insecticides/pharmacology ; Gene Editing ; *Pyrethrins/pharmacology ; *Insect Proteins/genetics/metabolism ; *ATP-Binding Cassette Transporters/genetics/metabolism ; CRISPR-Cas Systems ; Bacillus thuringiensis Toxins ; Larva/genetics/growth &amp; development/drug effects/metabolism ; Bacillus thuringiensis ; Hemolysin Proteins ; Bacterial Proteins ; },
abstract = {Spodoptera frugiperda is a major global pest that affect multiple crops, mainly corn and rice. Unfortunately, this pest has evolved resistance to various chemical and biological pesticides. ATP-binding cassette (ABC) transporters, particularly members of the B subfamily, are associated with detoxification by exporting xenobiotics and plant-derived metabolites from the intoxicated insect cells. In addition, some are involved in the mode of action of Bacillus thuringiensis biopesticide Cry toxins, functioning as receptors for these proteins. In this study, we analyzed transcriptomic data from the midgut tissue of S. frugiperda and identified the ABCB6 as one of the most highly expressed transporters within the ABCB subfamily. To explore its functional role, we generated a CRISPR-Cas9 knockout (KO) mutation. Strikingly, loss of SfABCB6 conferred resistance to the chemical pyrethroid insecticide cypermethrin, while the susceptibility to B. thuringiensis Cry1Ab, Cry1Fa and Vip3Aa toxins remained unchanged. Consistently, the ABCB6 CRISPR-Cas9 KO in S. frugiperda derived Sf9 cells conferred resistance to cypermethrin, reiterating the observed larval phenotype. In contrast, the overexpressing of ABCB6 in Sf9 cells exhibited increased susceptibility to cypermethrin. However, SfABCB6 KO showed fitness costs in the insect, as this mutation drastically reduced fertility. Our results provide evidence that SfABCB6 transporter facilitates cypermethrin toxicity participating in insecticide resistance and pointing out its potential role as a novel target for pest management strategies.},
}
@article {pmid41834687,
year = {2026},
author = {Yang, J and Huang, Q},
title = {Cas13a/crRNA trans-cleavage triggered primer exchange reaction based self-priming chain extension for sensitive and label-free infantile pneumonia related microRNA analysis.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {12},
pages = {2524-2531},
doi = {10.1039/d6ay00090h},
pmid = {41834687},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Pneumonia/genetics/diagnosis ; Limit of Detection ; *RNA, Circular/genetics ; },
abstract = {Accurate and sensitive detection of microRNAs (miRNAs) is crucial for both pathophysiological studies and clinical diagnostics. Conventional amplification methods often face limitations such as dependence on thermal cycling, susceptibility to contamination, and insufficient specificity to discriminate among closely related miRNA family members. To address these challenges, we developed a label-free isothermal detection platform that integrates the precise RNA-targeting ability of the CRISPR/Cas13a system with a self-priming amplification cascade driven by the primer exchange reaction (PER). In this assay, target miRNA binding directly activates the trans-cleavage activity of the Cas13a/crRNA complex, which subsequently cleaves a uracil-rich toehold region on a stem-loop DNA primer (H1). Following dephosphorylation, the cleaved primer initiates a PER-mediated self-priming amplification process, generating long tandem double-stranded DNA products that can be sensitively detected using the fluorescent dye SYBR Green I. The proposed method demonstrates several key advantages: (i) high specificity enabled by the programmable Cas13a/crRNA complex, allowing clear distinction between the target miRNA and sequences with single-base mismatches or high homology; (ii) exceptional sensitivity, achieving a detection limit of 406 aM and a dynamic range spanning six orders of magnitude, through coupling Cas13a collateral cleavage with exponential isothermal amplification; (iii) excellent reproducibility, reflected by low relative standard deviations and a coefficient of variation of 3.65% in spiked serum samples; and (iv) strong concordance with the reference RT-qPCR method in mock clinical specimens, highlighting its reliability for potential clinical use. In summary, this CRISPR/Cas13a-coupled self-priming amplification strategy provides a robust, accurate, and highly sensitive means for miRNA quantification, offering a promising alternative for point-of-care molecular diagnostic applications.},
}
@article {pmid41834690,
year = {2026},
author = {Marpaung, DSS and Yap Sinaga, AO and Damayanti, D and Utari, NWA and Harmiansyah, and Karangan, A and Kusmali, M},
title = {Integration of CRISPR/Cas12a and Toehold-Mediated Strand Displacement for Alternative Conventional miRNA Detection.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {27},
number = {6},
pages = {e202500932},
doi = {10.1002/cbic.202500932},
pmid = {41834690},
issn = {1439-7633},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {MicroRNAs (miRNAs) are short, noncoding RNAs that regulate gene expression and serve as powerful biomarkers for cancer and other diseases. Conventional detection methods such as RT-qPCR, Northern blotting, microarrays, and next-generation sequencing provide robust analytical capabilities but remain limited by complexity, cost, and poor suitability for point-of-care diagnostics. CRISPR/Cas12a has emerged as a versatile nucleic acid detection platform with high specificity and sensitivity. However, its intrinsic preference for DNA substrates restricts direct application to miRNA sensing. Early CRISPR/Cas12a-based assays relied on enzymatic amplification, direct RNA-induced activation, or split-component designs, each offering proof-of-concept feasibility but facing trade-offs in sensitivity, workflow complexity, or robustness. Toehold-mediated strand displacement (TSD) provides a powerful alternative by converting miRNA inputs into DNA activators or crRNAs that efficiently trigger Cas12a. This integration enables enzyme-free amplification, programmable logic operations, and enhanced sensitivity, while reducing reliance on multienzyme cascades. This review critically evaluates conventional, enzymatic, direct, and split-based CRISPR/Cas12a strategies and emphasizes emerging TSD-assisted platforms as next-generation solutions for sensitive, specific, and portable miRNA detection.},
}
@article {pmid41834849,
year = {2026},
author = {Tenea, GN},
title = {Comprehensive genomic and metabolomic profiling of Weissella confusa UTNCys2-2 highlights bioactive potential.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1779198},
pmid = {41834849},
issn = {1664-302X},
abstract = {INTRODUCTION: The genus Weissella comprises a diverse group of lactic acid bacteria (LAB) widely distributed across plant- and food-associated ecosystems and recognized for their functional and technological versatility. Weissella confusa UTNCys2-2, a plant-derived strain isolated from Amazonian spiral ginger (Costus sp.), that produces exopolysaccharides (EPS) with documented antioxidant activity and promising probiotic properties.<br><br>METHODS: Whole-genome sequencing of UTNCys2-2 was performed to establish its taxonomic assignment, phylogenomic analysis, while genome mining was conducted to evaluate safety, metabolic potential, and biosynthetic capabilities. Carbohydrate-active enzymes (CAZymes), Kyoto Encyclopedia of Genes and Genomes (KEGG), and MetaCyc pathways were analyzed for functional insights. Moreover, the metabolite composition of the cell-free supernatant (CFS) was examined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with Sequential Windowed Acquisition of all Theoretical Fragment Ion Mass Spectra (SWATH-MS).<br><br>RESULTS: The genome consists of a 2.32&#x202f;Mb circular chromosome (44.59% GC) encoding 2,194 proteins, 76 tRNAs, and 10 rRNAs, with no plasmids. Phylogenomic analyses assigned the strain to the W. confusa clade, clustering closely with the reference strain DSM 20196. UTNCys2-2 harbors a complete Type II-A CRISPR-Cas system, intact prophages, and mobile elements, while lacking virulence determinants and transferable antimicrobial resistance genes. Functional annotation revealed 118 CAZymes supporting EPS biosynthesis, polysaccharide utilization, and carbohydrate metabolism. KEGG and MetaCyc pathways highlighted glycogen and riboflavin biosynthesis, stress tolerance, and metabolic versatility. Genome mining identified a Type III polyketide synthase (T3PKS) gene cluster with low similarity to known pathways, suggesting potential for novel secondary metabolites. Pangenome analysis showed extensive strain-specific genes linked to carbohydrate metabolism and EPS production. Metabolomic profiling of the CFS detected alkaloids, bioactive peptides, functional carbohydrates, and phenolics, supporting antimicrobial, probiotic, and host-interactive activities.<br><br>CONCLUSION: W. confusa UTNCys2-2 represents a biosafe and metabolically versatile strain with strong genomic capacity for EPS production, potential for novel secondary metabolite biosynthesis, and diverse bioactive properties, supporting its applicability in food fermentation, probiotic development, and microbial biotechnology.},
}
@article {pmid41834871,
year = {2026},
author = {Abdul Rehman, Y and Fayyaz, A and Alblooshi, AS and Muhammad, K and Mundra, S and Alam, MT},
title = {Molecular adaptations and engineering of extremophiles for synthetic biology and biotechnological applications.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1754802},
pmid = {41834871},
issn = {1664-302X},
abstract = {Extremophiles are microorganisms that thrive in environments previously thought to be uninhabitable, including extreme temperature, salinity, pH, pressure, and radiation. These organisms, found in Archaea, Bacteria, and Eukarya, exhibit distinct structural, metabolic, and genetic adaptations, such as enhanced enzyme stability, efficient DNA repair mechanisms, and robust stress-response systems that enable survival under extreme conditions. Understanding these adaptation mechanisms is key to engineering similar traits in mesophilic organisms. This review discusses the diversity of extremophiles and presents phylogenetic and comparative genomic insights which may provide insights into the origins and evolution of early life on Earth We highlight recent advances in CRISPR/Cas-based genome editing, genome-scale metabolic modeling (GEM), and synthetic biology that have expanded the use of extremophiles in sustainable industrial biotechnology. The exceptional stability and catalytic efficiency of extremozymes under harsh conditions underscore their potential in various biotechnological applications. Finally, we discuss the ecological significance of extremophiles in climate change mitigation and outline current challenges and future directions in extremophile research.},
}
@article {pmid41836275,
year = {2026},
author = {Otero, CP and Qi, LS},
title = {Rewriting the epigenome: CRISPR tools for biological discovery and therapeutics.},
journal = {Current opinion in biomedical engineering},
volume = {38},
number = {},
pages = {},
pmid = {41836275},
issn = {2468-4511},
support = {R21 AG077193/AG/NIA NIH HHS/United States ; },
abstract = {The eukaryotic epigenome plays a central role in regulating gene expression, cellular identity, and development through dynamic, multilayered biochemical modifications to DNA, histones, and chromatin architecture. Disruption of these regulatory mechanisms contributes to a wide range of human diseases, including cancer, neurodegenerative disorders, and immunological conditions. Targeted epigenome editing offers promising discovery and therapeutic strategies by enabling the correction of aberrant epigenetic states without the need for permanent changes to the DNA sequence. The catalytically inactive CRISPR-Cas (dCas) molecule fused to epigenetic effector domains has emerged as a versatile platform for programmable, locus-specific modulation of chromatin states. These CRISPR-based epigenetic editors can deposit or remove desired epigenetic marks and alter three-dimensional genome organization to fine-tune gene expression with high specificity. Recent developments have expanded the CRISPR epigenome editing toolbox by introducing new effector domains, improving multiplexing capabilities, and enabling large-scale genetic screening, leading to novel insights into the functional genomics across various cellular contexts. However, clinical translation remains challenged by inefficient delivery and suboptimal editing efficacy in vivo. This review highlights recent advances in CRISPR-based epigenetic editing, with a focus on applications in primary cells, new tool development, and the translational potential of epigenome modulation for safe, durable, and precise therapies.},
}
@article {pmid41836767,
year = {2026},
author = {Luo, L and Yang, Y and Zhang, Y and Mao, G},
title = {Advances in nanozyme-assisted CRISPR diagnostic technology.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1796403},
pmid = {41836767},
issn = {2296-4185},
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) system has significant potential in biological diagnostics because of its precise nucleic acid identification abilities. Traditional CRISPR diagnostics, however, have limitations such as insufficient signal output, dependence on exogenous enzymes, and high equipment demands. Nanozymes, as nanomaterials with enzyme-mimetic catalytic activity, integrate the catalytic efficiency of natural enzymes with the stability and modifiability of nanomaterials, providing a viable resolution to the limitations in CRISPR diagnostics. This article comprehensively evaluates the advancements in nanozyme-enhanced CRISPR diagnostic technologies. Furthermore, it delineates the fundamental attributes of the CRISPR diagnostic system and nanozymes, as well as the necessity of their integration. Moreover, the coupling mechanisms between the CRISPR/Cas system and nanozymes, including the regulation of nanozyme catalytic activity by Cas protein function and CRISPR signal amplification facilitated by nanozymes, were also comprehensively evaluated. The application of this technique in detecting nucleic acid and non-nucleic acid targets was assessed. Further, this study discusses the current limitations of this technology, such as complex separation of heterogeneous systems, laborious reaction protocols, and slow detection rates. The future advancements, such as the establishment of homogenous systems, the creation of integrated devices, and the utilization of single-atom nanozymes, have also been discussed in this review. The results of this study will provide references for the comprehensive integration of nanozymes and CRISPR technology, together with their diagnostic applications.},
}
@article {pmid41837829,
year = {2026},
author = {Bao, J and Ju, X and Zhang, H and Tang, D and Yan, J and Cui, C and Gao, SS},
title = {Synergistic CRISPR-Cas9 Host Engineering and Enzyme Evolution for Enantioselective Synthesis of a Vibegron Pyrrolidine Intermediate.},
journal = {Organic letters},
volume = {28},
number = {12},
pages = {3695-3700},
doi = {10.1021/acs.orglett.6c00065},
pmid = {41837829},
issn = {1523-7052},
mesh = {Stereoisomerism ; *Pyrrolidines/chemistry/chemical synthesis/metabolism ; Molecular Structure ; *CRISPR-Cas Systems ; Biocatalysis ; *Oxidoreductases/metabolism/chemistry ; Directed Molecular Evolution ; },
abstract = {The stereoselective synthesis of chiral pyrrolidine motifs is essential to vibegron production but remains challenging using conventional chemical routes. Here we report an imine reductase (IRED) catalyzed asymmetric imine reduction to access a key vibegron intermediate. Directed evolution afforded a highly efficient variant delivering 94% conversion and >99% d.e. Combined enzyme and host engineering enabled clean whole cell catalysis, establishing a robust and scalable biocatalytic platform.},
}
@article {pmid41840760,
year = {2026},
author = {Karchner, SI and Aluru, N and Franks, DG and Mandl, CA and Goldstone, JV and Burke, T and Champlin, D and La Du, JK and Perone, DM and Stinson, S and Truong, L and Clark, BW and Nacci, D and Tanguay, RL and Hahn, ME},
title = {Using fish models to understand the role of aryl hydrocarbon receptor (AHR)-interacting protein (AIP) in controlling sensitivity and resistance to dioxin-like compounds in vivo.},
journal = {Toxicological sciences : an official journal of the Society of Toxicology},
volume = {209},
number = {3},
pages = {},
pmid = {41840760},
issn = {1096-0929},
support = {P42 ES007381/ES/NIEHS NIH HHS/United States ; R01 ES032323/ES/NIEHS NIH HHS/United States ; P30 ES030287/ES/NIEHS NIH HHS/United States ; S10 OD032203/OD/NIH HHS/United States ; R01 ES033888/ES/NIEHS NIH HHS/United States ; P42 ES016465/ES/NIEHS NIH HHS/United States ; R01ES033888//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; R01ES032323//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; P42ES007381//National Institute of Environmental Health Sciences (NIEHS) of the National Institutes of Health (NIH)/ ; P42ES016465//Superfund Research Program at Boston University/ ; P30ES030287//Superfund Research Program at Oregon State University/ ; 1S10OD032203-01//Superfund Research Program at Oregon State University/ ; //Tufts University Core Facility Genomics Core/ ; //NIH/ ; //NIH Public Access Policy/ ; //PubMed Central upon the Official Date of Publication/ ; },
mesh = {Animals ; *Receptors, Aryl Hydrocarbon/metabolism/genetics ; *Fundulidae/genetics/embryology/metabolism ; *Dioxins/toxicity ; Polychlorinated Dibenzodioxins/toxicity ; *Intracellular Signaling Peptides and Proteins/genetics/metabolism ; Polychlorinated Biphenyls/toxicity ; Zebrafish/genetics ; CRISPR-Cas Systems ; Zebrafish Proteins/genetics ; Embryo, Nonmammalian/drug effects ; *Fish Proteins/genetics/metabolism ; },
abstract = {Humans are exposed to chemicals such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) that cause toxicity through activation of the aryl hydrocarbon receptor (AHR). There is inter-individual variation in sensitivity to the effects of AHR ligands, but it is not fully explained by variation in the AHR. A clue to the genetic mechanisms underlying differential sensitivity to AHR agonists has emerged from studies of Atlantic killifish (Fundulus heteroclitus) populations with evolved tolerance to PCBs, TCDD, and PAHs. Genomic studies of these populations identified AHR-interacting protein (AIP/Ara9/XAP2) as the strongest candidate resistance gene. However, the precise role of AIP in the mechanism of resistance is unknown. To understand the role of AIP in the toxicity of dioxin-like compounds in vivo, we used CRISPR-Cas9 to generate AIP loss-of-function alleles in killifish and zebrafish (Danio rerio). Homozygous mutant killifish and zebrafish die during larval development-by 30 and 12 d postfertilization, respectively-whereas heterozygous mutants develop, survive, and reproduce normally. During embryonic and early larval stages, homozygous mutant zebrafish exhibit reduced sensitivity to embryotoxic effects of exposure to 3,3',4,4',5-pentachlorobiphenyl (PCB126) and TCDD. Gene expression profiling of aip-deficient larvae revealed hundreds of differentially expressed genes. PCB126 induced similar sets of well-known AHR-regulated genes in mutant and wild-type larvae, although with reduced magnitude overall in AIP mutants. This study highlights the important role of AIP in fish larval development and demonstrates that AIP status can influence the response of vertebrate embryos to dioxin-like compounds in vivo.},
}
@article {pmid41840824,
year = {2026},
author = {Thalib, HI and Khan, S and Hanin Shaikh, A and Alawi, KM and Alabdrabalrasol, ZH and Mehveen, S and Haidar, S and ElSayed Hassan, F and Shaik, NA},
title = {CRISPR Gene Editing for Nucleotide Repeat Expansion Disorders: A Systematic Review of Preclinical and Clinical Evidence.},
journal = {Genetic testing and molecular biomarkers},
volume = {30},
number = {3},
pages = {71-80},
doi = {10.1177/19450265261434900},
pmid = {41840824},
issn = {1945-0257},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Myotonic Dystrophy/genetics/therapy ; Genetic Therapy/methods ; *Trinucleotide Repeat Expansion/genetics ; Muscular Dystrophy, Duchenne/genetics/therapy ; Huntington Disease/genetics/therapy ; Animals ; },
abstract = {BACKGROUND: Incurable hereditary diseases such as Duchenne muscular dystrophy (DMD), Huntington's disease (HD), and myotonic dystrophy type 1 (DM1) fall into the nucleotide repeat expansion disorder (NRED) category. The discovery of CRISPR-Cas genome editing has paved the way toward hopeful strategies for accurate DNA-level repair. This systematic review presents preclinical data on the efficacy, molecular effects, and limitations of CRISPR-based treatments for NREDs.<br><br>METHODS: As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses 2020 guidelines, systematic PubMed, Scopus, and Embase searches up to June 2025 identified studies that evaluated CRISPR-Cas systems in human-derived in vitro models of NREDs. Methodological Index for Non-Randomized Studies tool was used to score eligible studies by methodological quality. CRISPR platforms, delivery systems, gene targets, molecular endpoints, and functional rescue data were extracted and synthesized descriptively.<br><br>RESULTS: Twenty-four out of 6510 records screened were included. They employed most of them to target specific DMD (n = 9), HD (n = 6), and DM1 (n = 3) with patient-derived induced pluripotent stem cells or differentiated myogenic/neuronal cells. Streptococcus pyogenes CRISPR-associated protein 9 as a nuclease was the most frequently used, although engineered Cas9 enzymes and dCas9 fusion proteins were also utilized to control transcription. Delivery was achieved through viral vectors (adeno-associated virus, lentivirus) and nonviral routes (plasmid, lipofection, electroporation). Uniform genomic editing, transcript rescue, and protein restoration were seen in CRISPR-mediated editing studies, and functional restoration was demonstrated for splicing correction and dystrophin restoration. Methodological flaws such as the absence of blinding, failure to follow up, and lack of full reporting of off-target effects limited robustness.<br><br>CONCLUSION: CRISPR-Cas systems exhibit reproducible molecular and functional correction in NRED models with their translational potential. Methodological strength, whole safety profiling, and in&#xa0;vivo verification remain a necessity, however, before clinical translation.},
}
@article {pmid41841492,
year = {2026},
author = {Li, Z and Kong, J and Wu, W and Duan, Y and Zhu, Z and Hua, C and Yan, P and Cao, C and Cao, X and Xiao, Y and Lu, M and Chen, M},
title = {Structural and functional insights into the adenosine deaminase of the type III-B CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41841492},
issn = {1362-4962},
support = {2023YFC3402300//National Key Research and Development Program of China/ ; 2021ZD0203400//National Key Research and Development Program of China/ ; 82473977//National Natural Science Foundation of China/ ; 32271330//National Natural Science Foundation of China/ ; 32471316//National Natural Science Foundation of China/ ; 82304614//National Natural Science Foundation of China/ ; 82373892//National Natural Science Foundation of China/ ; SBK2024010634//Basic Research Program of Jiangsu/ ; BK20250200//Basic Research Program of Jiangsu/ ; //State Key Laboratory of Natural Medicines/ ; SKLNMZZ2024JS31//China Pharmaceutical University/ ; 2632025TD02//Fundamental Research Funds for the Central Universities/ ; 2023YFC3402300//National Key Research and Development Program of China/ ; 2021ZD0203400//National Key Research and Development Program of China/ ; 82473977//National Natural Science Foundation of China/ ; 32271330//National Natural Science Foundation of China/ ; 32471316//National Natural Science Foundation of China/ ; 82304614//National Natural Science Foundation of China/ ; SBK2024010634//Basic Research Program of Jiangsu/ ; BK20250200//Basic Research Program of Jiangsu/ ; //Project Program of State Key Laboratory of Natural Medicines/ ; SKLNMZZ2024JS31//China Pharmaceutical University/ ; 2632025TD02//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Adenosine Deaminase/chemistry/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Adenine Nucleotides/metabolism ; Adenosine Triphosphate/metabolism ; },
abstract = {Type III CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) systems confer antiviral immunity via cyclic oligoadenylate (cOA) signaling. Here, we elucidate a cooperative bacterial defense strategy involving two cOA-activated CRISPR-associated Rossmann fold (CARF)-containing effectors, adenosine deaminase CAAD and ribonuclease Csx1, in Thermoanaerobaculum aquaticum. Genomic analyses indicate widespread co-occurrence of CRISPR-associated adenosine deaminase (CAAD) with ancillary CARF-containing effectors in type III CRISPR systems, suggesting that multiple CARF-containing proteins may contribute to a coordinated cOA-dependent defense. Biochemical and structural studies reveal the intrinsic dynamics of CAAD hexamer, and demonstrate that cA4/cA6 binding stabilizes CAAD hexamers, triggering metal-ion-dependent conversion of ATP into inosine triphosphate. Concurrently, the downstream Csx1 is exclusively activated by cA4 to cleave single-stranded RNA. Strikingly, we found that both effectors are capable of degrading cA4, suggesting that this CAAD-Csx1 pair may be cross-regulated and achieve immunity through a dual-targeting mechanism: in response to infection, Csx1 degrades viral RNA while CAAD disrupts nucleotide metabolism via ATP deamination, which can be relieved via cA4 degradation when infection has been eliminated. This study proposes an enhanced defense mechanism through coordinated activation and regulation of multiple CRISPR effectors by a single signaling molecule, unveiling unprecedented complexity in CRISPR immunoregulation.},
}
@article {pmid41841497,
year = {2026},
author = {Nusawardhana, A and Hale, A and Straka, J and Nicolae, CM and Moldovan, GL},
title = {Genome-wide CRISPR screens identify the EXO1-CAF-1 pathway suppressing R-loop-associated DNA damage.},
journal = {Nucleic acids research},
volume = {54},
number = {5},
pages = {},
pmid = {41841497},
issn = {1362-4962},
support = {R01ES026184/GF/NIH HHS/United States ; R01GM134681/GF/NIH HHS/United States ; R01CA244417/GF/NIH HHS/United States ; F31CA294862/GF/NIH HHS/United States ; 4D01_2024_1002//Four Diamonds Transformative Patient-Oriented Cancer Research/ ; /NH/NIH HHS/United States ; //Penn State University/ ; },
mesh = {*Exodeoxyribonucleases/genetics/metabolism ; *DNA Damage ; Cisplatin/pharmacology ; DNA Repair/genetics ; Humans ; *R-Loop Structures/genetics ; CRISPR-Cas Systems ; *DNA Repair Enzymes/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Synthetic Lethal Mutations ; },
abstract = {DNA repair is critical for cellular homeostasis under both normal conditions as well as in response to genotoxic agents such as chemotherapeutics. EXO1 is a 5'-3' exonuclease with multiple roles in DNA biology. To better understand these roles, we employed CRISPR loss-of-function genome-wide screening to identify genes required for proliferation and cisplatin sensitivity in EXO1-deficient cells. We uncovered differential regulators of cisplatin sensitivity between wildtype (WT) and EXO1-deficient cells. By analyzing the genetic networks that these regulators belong to, we found that DNA repair was the main biological process suppressing cisplatin sensitivity in WT cells, but this was not the case in EXO1-deficient cells, indicating that EXO1 is critical for the repair of cisplatin-induced DNA damage. Moreover, synthetic lethality screens identified a genetic interaction between EXO1 and the histone chaperone CAF-1. Mechanistically, we show that EXO1 and CAF-1 are independently recruited to R-loops and participate in separate, synergistic pathway of R-loop suppression. Even in the absence of DNA damage treatment, concomitant loss of EXO1 and CAF-1 causes R-loop accumulation and increased R-loop-associated DNA damage. Our work sheds light on the critical roles of EXO1 in genomic stability.},
}
@article {pmid41841731,
year = {2026},
author = {Xiong, L and Yadav, V and Sun, S and Heitman, J},
title = {Dissecting the homeodomain MAT locus and engineering novel tripolar and bipolar mating systems in Cryptococcus amylolentus.},
journal = {mBio},
volume = {17},
number = {4},
pages = {e0005926},
pmid = {41841731},
issn = {2150-7511},
mesh = {*Genes, Mating Type, Fungal ; *Cryptococcus/genetics/physiology ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Gene Deletion ; Reproduction/genetics ; },
abstract = {Sex in fungi is governed by the mating-type (MAT) locus, which exists as a bipolar, pseudobipolar, or tetrapolar system. The significance and impact of MAT on sexual reproduction, however, remain understudied. Furthermore, the evolution of fungal MAT loci shares features with the evolution of plant and animal sex chromosomes. Pathogenic Cryptococcus species harbor a bipolar system with a large contiguous MAT locus, whereas closely related species, such as the non-pathogen C. amylolentus, possess a tetrapolar system with unlinked P/R and HD loci. The P/R locus encodes pheromones and pheromone receptors that mediate partner recognition and cell-cell fusion, while the HD locus encodes homeobox domain-containing proteins (Sxi1 and Sxi2) that play important and evolutionarily conserved roles in sexual reproduction. Here, we explored the roles of HD genes in sexual reproduction and determined the implications of a tetrapolar to bipolar MAT transition. Using a CRISPR-Cas9 system we developed for C. amylolentus, we generated gene deletion mutants and demonstrated that a single compatible pair of heteroallelic Sxi1 and Sxi2 is both necessary and sufficient for mating. By relocating the HD genes to the P/R locus, we found that the artificially generated bipolar configuration led to defective sexual development, which could be partially alleviated through additional rounds of sexual reproduction. Transcriptomic profiling revealed the Sxi1-Sxi2 heterodimeric complex drives expression of genes required for DNA replication and ergosterol biosynthesis during sexual reproduction. These findings provide the first experimental demonstration of a tetrapolar-to-bipolar transition in a tetrapolar species, illuminating MAT locus evolution and homeodomain protein functions.IMPORTANCESexual reproduction is critical for fungal survival and adaptation, yet the mechanisms driving transitions between mating systems remain unclear. With Cryptococcus amylolentus, we provide the first experimental validation of a mating system transition from its original tetrapolar state, through an intermediate tripolar state, to a derived bipolar state in a tetrapolar species. We show that homeodomain (HD) protein heterodimers phenotypically govern dikaryotic filamentation and also transcriptionally modulate DNA replication. These findings establish a mechanistic basis for how MAT locus reorganization drives bipolar evolution from an ancestral tetrapolar state and reinforce that fertility depends on the coordinated control of MAT locus architecture and regulatory functions.},
}
@article {pmid41843435,
year = {2026},
author = {Wang, W and Lin, S and Luo, Y and Shao, C and Shi, Q and Shao, J and Chen, Y and Hu, H and Wan, S and Song, X and Jin, D and Jin, Y},
title = {Genomic and phenotypic insight into Clostridioides difficile RT027 isolates from China reveals diverse virulence associated with clinical symptoms.},
journal = {Emerging microbes &amp; infections},
volume = {15},
number = {1},
pages = {2637287},
pmid = {41843435},
issn = {2222-1751},
mesh = {*Clostridioides difficile/genetics/pathogenicity/isolation &amp; purification/classification/drug effects ; *Clostridium Infections/microbiology/epidemiology ; China/epidemiology ; Humans ; Virulence/genetics ; *Genome, Bacterial ; Phylogeny ; Phenotype ; Anti-Bacterial Agents/pharmacology ; Whole Genome Sequencing ; Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Virulence Factors/genetics ; Genomics ; Ribotyping ; },
abstract = {Clostridioides difficile (C. difficile) ribotype 027 (RT027) has caused severe outbreaks in North America and Europe over the past 20 years. However, RT027 infections are rare in Asia, particularly in China, with limited severe cases. To clarify its molecular and phenotypic features, we investigated 11 RT027 isolates collected from Shandong, China. Whole-genome sequencing, comparative transcriptomics, CRISPR-Cas analysis, and pan-genome profiling were combined with phenotypic assays of toxin production, sporulation, antimicrobial resistance, and motility. Evolutionary analysis demonstrated that isolates from China clearly diverged from the FQR2 lineage and were phylogenetically closer to FQR1, forming a distinct sublineage. All isolates from Shandong, China encoded a complete tcd and cdt locus and harboured rifamycin and aminoglycoside resistance genes. However, transcriptomic profiling demonstrated significantly reduced expression of binary toxin genes (cdtAB, cdtR), decreased spo0A transcription, and downregulation of flagellar pathways. Phenotypic assays confirmed impaired sporulation, motility and cytotoxicity, while TcdB production and adhesion was comparable to reference strains. CRISPR-Cas elements were conserved but showed reduced transcriptional activity, suggesting diminished host-pathogen interactions. The pan-genome revealed high genomic conservation, consistent with limited functional diversity. Together, these data indicate that RT027 isolates circulating in China possess unique evolutionary trajectories and attenuated virulence traits, helping to explain the rarity of severe RT027 infections in Asia. These findings provide important insights into the regional epidemiology of C. difficile and inform strategies for diagnosis, treatment, and prevention.},
}
@article {pmid41843552,
year = {2026},
author = {Leix, K and Serrano-Zayas, C and Vyas, HS and Graham, SE and Emmer, BT},
title = {Functional interrogation of candidate cis-regulatory elements at the LDLR locus.},
journal = {PLoS genetics},
volume = {22},
number = {3},
pages = {e1012082},
pmid = {41843552},
issn = {1553-7404},
mesh = {*Receptors, LDL/genetics/metabolism ; Humans ; Enhancer Elements, Genetic/genetics ; Gene Expression Regulation ; Promoter Regions, Genetic/genetics ; Genome-Wide Association Study ; *Regulatory Sequences, Nucleic Acid/genetics ; Introns/genetics ; CRISPR-Cas Systems ; Cholesterol, LDL/genetics ; },
abstract = {Regulation of LDLR gene expression plays an important role in the development of atherosclerotic diseases including heart attack and stroke. Although LDLR regulation by sterol response elements has been well characterized, the functional significance of other noncoding regions at the LDLR locus remains poorly defined. In this study, we developed and applied a high throughput CRISPR screen to test the functional importance of candidate LDLR cis-regulatory elements (CREs) in their native genomic context. In total, we found 25 discrete regions to exhibit a significant impact on LDLR expression. For one of these regions with particularly strong activity in the first intron, we validated the presence of an enhancer by confirming that its disruption reduced endogenous LDLR expression while its insertion upstream of a minimal promoter augmented reporter gene expression. We then applied a massively parallel reporter assay to fine map enhancer activity within this region to a 129 bp interval that is highly conserved among vertebrates, exhibits biochemical hallmarks of enhancer activity, is enriched for transcription factor binding motifs, and contains a common genetic variant (rs57217136) that has been associated with human LDL cholesterol levels by genome-wide association studies. Overall, these findings demonstrate the power of CRISPR screening to interrogate candidate CREs and clarify the functional landscape of noncoding sequences at the LDLR locus.},
}
@article {pmid41845915,
year = {2026},
author = {Liu, S and Geng, S and Dreisigacker, S},
title = {Genomics in wheat improvement: Progress and perspectives.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70176},
pmid = {41845915},
issn = {1940-3372},
support = {32201878//National Natural Science Foundation of China/ ; 2024SFGC0404//Key R&amp;D Program of Shandong Province, China/ ; 2023ZD0406802//Biological Breeding-National Science and Technology Major Project/ ; 202403250025//China Scholarship Council (CSC)/ ; },
mesh = {*Triticum/genetics ; *Genomics ; *Genome, Plant ; Plant Breeding ; Gene Editing ; CRISPR-Cas Systems ; },
abstract = {Bread wheat (Triticum aestivum) remains a major source of food and calories globally, yet its vast genome, polyploidy, and high number of repetitive sequences make genomic research challenging in this crop. In this review, we discuss the progress and future perspectives of genome research in wheat. Current efforts focus on the establishment of genome assemblies, advances in functional genomics, advances in epigenetics, translational genetics, and CRISPR-Cas9 genome editing offers a powerful tool for site-specific genome editing for wheat improvement and functional genetic analysis. These approaches have elucidated the genetic basis of many important agronomic traits such as grain yield, biotic and abiotic stress, and wheat quality. Future aims are expected to expand to pan-genomics, the mechanism of wheat domestication, funnel the outputs of functional genomics for deployment in wheat breeding, multi-omics studies facilitate genetic dissection, and the era of big data: creation, integration and utilization, and artificial intelligence breeding.},
}
@article {pmid41847198,
year = {2026},
author = {D, J and Mulavagili, S and Vijayasimha, M},
title = {Commentary: CRISPR-Cas systems against carbapenem resistance-from proof-of-concept to clinical translation.},
journal = {Frontiers in microbiology},
volume = {17},
number = {},
pages = {1773181},
pmid = {41847198},
issn = {1664-302X},
}
@article {pmid41849360,
year = {2026},
author = {Lin, H and Wang, S and Xie, Y and Cheng, C and Jin, J and Song, X and Zhang, H},
title = {Single-tube two-step RPA-CRISPR/Cas12b platform for detection of Pseudomonas aeruginosa.},
journal = {PloS one},
volume = {21},
number = {3},
pages = {e0340856},
pmid = {41849360},
issn = {1932-6203},
mesh = {*Pseudomonas aeruginosa/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; Bacterial Proteins/genetics ; Pseudomonas Infections/diagnosis/microbiology ; DNA, Bacterial/genetics ; },
abstract = {Pseudomonas aeruginosa is a ubiquitous opportunistic pathogen of significant clinical and public health concern, necessitating the development of rapid and reliable detection methods. Traditional diagnostic approaches, which rely on culture-dependent techniques and biochemical identification, are often labor-intensive, time-consuming, and technically demanding. This study describes a novel single-tube, two-step, rapid detection platform that integrates recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats-associated protein Cas12b technology. Through systematic experimental optimization, the study identified an optimal RPA primer pair (F2-R1) and single-guide ribonucleic acid 553 that targets the lasR gene of P. aeruginosa, with reaction conditions optimized at 42°C and a primer concentration of 10 μM. The RPA-clustered regularly interspaced short palindromic repeats/Cas12b fluorescence detection system (RPA-Cas12b-Fluo) demonstrated a sensitivity threshold of 10 copies of deoxyribonucleic acid (DNA) per reaction and a bacterial detection limit of 50 colony-forming units (CFU) per reaction. When coupled with a lateral flow strip (RPA-Cas12b-LFS), the sensitivity was slightly reduced but remained robust, achieving detection limits of 10[2] copies and 200 CFU per reaction. Specificity assays confirmed a high discriminatory capacity for P. aeruginosa with no cross-reactivity observed against P. fluorescens, P. putida, or six common foodborne pathogens, thereby validating the specificity profile of the platform. The applicability of the method was further validated by analyzing 20 water samples, which demonstrated 100% concordance with the national standard culture method. These findings have significant implications for improving outbreak surveillance and mitigating the risk of foodborne transmission associated with P. aeruginosa.},
}
@article {pmid41849610,
year = {2026},
author = {Dai, Y and Abudujielili, Z and Ding, Y and Huang, W and Yin, J and Ou, L and Hu, J and Zheng, S and Li, C},
title = {Self-inactivating AAV-CRISPR at different ages enables sustained amelioration of Huntington's disease deficits in BAC226Q mice.},
journal = {Science advances},
volume = {12},
number = {12},
pages = {eaea8052},
pmid = {41849610},
issn = {2375-2548},
mesh = {Animals ; *Huntington Disease/genetics/therapy/pathology ; Mice ; *Gene Editing/methods ; *Huntingtin Protein/genetics/metabolism ; *CRISPR-Cas Systems ; Humans ; Disease Models, Animal ; *Dependovirus/genetics ; Genetic Therapy/methods ; Genetic Vectors/genetics ; },
abstract = {Huntington's disease (HD) is a monogenic autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in exon 1 of the HTT gene, yielding a gain-of-toxic-function mutant Huntingtin protein (mHTT). CRISPR-Cas9 is a potentially powerful therapeutic strategy for HD by eliminating mutant HTT (mHTT) gene. We developed a specific SaCas9 guide RNA to target human mHTT and a self-inactivating gene editing system that abolishes SaCas9 after a short transient expression for high gene editing efficiency and maximal safety to prevent off-target effects. Both conventional and the self-inactivating gene editing systems successfully eliminated mHTT gene, 60 to 90% mHTT protein and 90% of mHTT aggregation in BAC226Q mouse brains, which resulted in significant long-term rescue of neuropathology, motor deficits, weight loss, and shortened life span. These beneficial effects were observed when gene editing was applied before, at, and well after the onset of pathological and behavioral abnormalities. These proof-of-concept data demonstrate that gene editing can be a highly effective therapeutic approach for HD.},
}
@article {pmid41849748,
year = {2026},
author = {Wang, Q and Chen, D and Berr, A and Shen, WH},
title = {CRISPR gene editing of AtRING1 unravels a critical role of RAWUL domain in PRC1 repression of transcription.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70794},
pmid = {41849748},
issn = {1365-313X},
support = {ANR-12-BSV2-0013-02//Agence National de la Recherche/ ; //Centre National de la Recherche Scientifique/ ; },
mesh = {*Arabidopsis Proteins/genetics/metabolism ; *Arabidopsis/genetics/metabolism ; Gene Editing ; *Polycomb Repressive Complex 1/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Histones/metabolism ; Protein Domains ; Glucosyltransferases ; },
abstract = {Polycomb Group (PcG) proteins, including members of Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2), regulate many key developmental processes through transcriptional gene repression. While the molecular mechanisms of PRC2 and its histone methyltransferase involved in depositing histone 3 lysine 27 trimethylation (H3K27me3) are well understood, the components and E3 ubiquitin ligase functions of PRC1 in plants remain largely elusive. In Arabidopsis, AtRING1 is a key PRC1 component, containing an N-terminal RING-finger domain and a C-terminal RAWUL domain. Previous studies have relied on T-DNA insertion mutants in the investigation of AtRING1 function. By editing AtRING1A using CRISPR/Cas9 technology in the atring1b-1 background, here we have generated and characterized one N-terminal stop mutant atring1[ko] and two C-terminal deletion mutants atring1[▵C-terminal] lacking the RAWUL domain. We show evidence that atring1[ko] represents the strongest loss-of-function mutant, exhibiting embryonic callus-like structures, demonstrating the essential role of AtRING1 in cell differentiation. Remarkably, the atring1[▵C-terminal] mutants exhibit mild developmental defects, suggesting that the RING domain alone retains partial function, while the RAWUL domain fine-tunes PRC1 activity. Our molecular analyses support a model in which AtRING1/PRC1-mediated H2A monoubiquitination (H2Aub1) often precedes PRC2-mediated H3K27me3 deposition at some target loci. Strikingly, the RAWUL domain is required for efficient H2Aub1 enrichment and influences H3K27me3 deposition in a locus-specific manner. Taken together, our study provides new insights into the molecular mechanism underlying PRC1 E3 ligase activity, supporting that PRC1 function facilitates PRC2 activity in epigenetic gene silencing.},
}
@article {pmid41850286,
year = {2026},
author = {Jin, S and Zhu, Z and Li, Y and Zhang, S and Liu, Y and Li, D and Li, Y and Luo, Y and Cheng, Z and Zhao, KT and Gao, Q and Yang, G and Li, H and Liang, R and Zhang, R and Qiu, JL and Zhang, YE and Gogo Liu, JJ and Gao, C},
title = {Functional RNA splitting drove the evolutionary emergence of type V CRISPR-Cas systems from transposons.},
journal = {Cell},
volume = {189},
number = {7},
pages = {2189-2193},
doi = {10.1016/j.cell.2026.03.014},
pmid = {41850286},
issn = {1097-4172},
}
@article {pmid41850995,
year = {2026},
author = {Liu, Y and Niu, X and Wei, W},
title = {Decoding disease-relevant variants with base and prime editors at scale.},
journal = {Trends in biochemical sciences},
volume = {51},
number = {4},
pages = {392-409},
doi = {10.1016/j.tibs.2026.02.001},
pmid = {41850995},
issn = {0968-0004},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Variation ; CRISPR-Cas Systems ; *Genomics/methods ; Genome, Human ; },
abstract = {Interpreting variants of uncertain significance remains a central challenge in human genomics. Base and prime editors have launched a new era of precision functional genomics, enabling programmable, double-strand break-free introduction of point mutations and small indels directly within the genome. Here, we review the technological evolution of these editors and their transformative application in high-throughput functional screens. We highlight how base and prime editing platforms systematically annotate clinical variants, reveal mechanisms of drug resistance and immune evasion, and dissect fundamental biological processes at single-nucleotide resolution. Crucially, we address current challenges and future perspectives for precision editing screens. By enabling causal genotype-to-phenotype mapping, precision editing screens are redefining genomic variation interpretation and accelerating its translation into precision diagnostics and therapeutics.},
}
@article {pmid41851456,
year = {2026},
author = {Nyberg, WA and Bernard, PL and Ngo, W and Wang, CH and Ark, J and Rothrock, A and Borgo, GM and Kimmerly, GR and Jung, JH and Allain, V and Hamilton, JR and Baldwin, A and Stickels, R and Wyman, S and Khan, SH and Lang, S and Marsh, D and Almudhfar, N and Novick, C and Mortazavi, Y and Zhang, S and AbdElwakil, MM and Sandoval, LR and Hwang, S and Chu, SN and Jung, H and Liu, C and Sharma, D and McCreary, T and Li, Z and Satpathy, AT and Carnevale, J and Rutishauser, RL and Cromer, MK and Roybal, KT and Dodgson, SE and Doudna, JA and Asokan, A and Eyquem, J},
title = {In vivo site-specific engineering to reprogram T cells.},
journal = {Nature},
volume = {652},
number = {8110},
pages = {712-721},
pmid = {41851456},
issn = {1476-4687},
support = {P30 DK063720/DK/NIDDK NIH HHS/United States ; S10 1S10OD021822-0//NIH S10 Instrumentation grant/ ; },
mesh = {Animals ; Mice ; *T-Lymphocytes/metabolism/cytology/immunology ; Receptors, Chimeric Antigen/genetics/metabolism/immunology ; CRISPR-Cas Systems/genetics ; Humans ; Transgenes/genetics ; *Gene Editing/methods ; Dependovirus/genetics ; Female ; *Cellular Reprogramming/genetics ; Genetic Vectors/genetics ; *Cell Engineering/methods ; Immunotherapy, Adoptive/methods ; Male ; },
abstract = {Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune and infectious diseases. Enhancing T cell function through genome editing, either by disrupting endogenous genes or precisely inserting DNA payloads, has shown considerable promise[1]. However, the ex vivo manufacturing process is lengthy and costly, limiting accessibility of these therapies. In vivo generation of CAR T cells could overcome these barriers, but current methods rely either on transient expression with limited durability, or on random integration of DNA payloads that lack specificity. Here we demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR-Cas9 ribonucleoproteins and a DNA donor template, using enveloped delivery vehicles and adeno-associated viruses, respectively. We optimized both vectors for T cell-specific delivery and gene-targeting efficiency. By integrating a CAR transgene into a T cell-specific locus, we generate therapeutic levels of CAR T cells in vivo in humanized mouse models of B cell aplasia, and haematological and solid malignancies. These findings offer a pathway to more efficient, precise and widely accessible T cell therapies.},
}
@article {pmid41851990,
year = {2026},
author = {Guo, Y and Li, J and Haque, A and Chen, J and Yang, X and Wang, Y and Yao, L and Zhuo, C and Wang, J and He, N and Lin, Y and Xiao, S and Liu, B and Zhuo, C},
title = {Subtypes of Type I-E CRISPR-Cas Systems Distribution in Human Escherichia coli Isolates from China.},
journal = {The CRISPR journal},
volume = {},
number = {},
pages = {25731599261430828},
doi = {10.1177/25731599261430828},
pmid = {41851990},
issn = {2573-1602},
abstract = {The correlation between CRISPR-Cas systems and plasmid-mediated bacterial antibiotic resistance is increasingly growing attention. However, currently no reports exist on the relationship between the CRISPR-Cas systems and the carriage of blaNDM or plasmids in E.coli. Here, molecular characterization and phylogenetic analysis of 639 E.coli isolated from humans in China were carried out. Depending on similarity in sequence, the type I-E CRISPR-Cas systems in E.coli can be grouped into two distinct clades, which we refer to for descriptive purposes within this study as the type I-E-S1 and I-E-S2, whereas the type I-E-S2 CRISPR-Cas system is further divided into I-E-S2a and I-E-S2b systems based on the presence of cas8e and cas11. ST167 (phylogroup A) and ST410 (phylogroup C) E. coli were observed bearing the type I-E-S1 and I-E-S2b systems, respectively. Compared with strains carrying the I-E-S1 type CRISPR-Cas system, the blaNDM carrying rate, the positive rate of IncX3 plasmid, and the positive rate of IncF plasmid of strains with the I-E-S2a type CRISPR-Cas system were evidently lower (p < 0.05); the blaNDM carrying rate and the positive rate of IncF plasmid of strains with the I-E-S2b type CRISPR-Cas system were evidently higher (p < 0.05). The blaNDM positive rate and IncF plasmid positive rate of strains carrying the I-E-S2a type CRISPR-Cas system were significantly lower than those of strains carrying the I-E-S2b type CRISPR-Cas system (p < 0.001). It proves that the I-E-S1, I-E-S2a, and I-E-S2b type CRISPR-Cas systems are beneficial for spreading blaNDM and IncX3 plasmids. We found significant differences in the cas gene sequences of the I-E-S1 and I-E-S2 type CRISPR loci. The type I-E CRISPR-Cas systems in E. coli isolated from Chinese sources are classified further for the first time, revealing their high correlation with blaNDM, phylogenetic groups, and multilocus sequence typing. This work paves the way for a deeper understanding of the role that CRISPR-Cas systems play in the rise of resistant E.coli ST167 and ST410.},
}
@article {pmid41854269,
year = {2026},
author = {Fernandes, LGV and Putz, EJ and Nally, JE},
title = {The Leptospira immunoglobulin-like protein LigB from Leptospira borgpetersenii serovar Arborea is not required for either acute or chronic infection.},
journal = {Infection and immunity},
volume = {94},
number = {4},
pages = {e0066225},
pmid = {41854269},
issn = {1098-5522},
mesh = {Animals ; *Leptospirosis/microbiology/immunology ; *Leptospira/genetics/pathogenicity/immunology ; *Bacterial Proteins/genetics/metabolism ; Virulence Factors/genetics/metabolism ; Rats ; Virulence ; Persistent Infection/microbiology ; CRISPR-Cas Systems ; },
abstract = {Leptospirosis is a globally important zoonotic disease caused by more than 40 pathogenic Leptospira spp. that are responsible for more than 1 million human cases and almost 60,000 deaths annually. The disease also affects many companion, domestic, and wild animal species. Leptospiral immunoglobulin-like proteins (Lig), particularly LigA and LigB, are well-established surface membrane proteins that have been extensively studied due to their interactions with the host immune system. Silencing expression of both ligA and ligB in L. interrogans serovar Copenhageni results in attenuation of virulence, confirming their role as virulence factors. To examine the role of Lig proteins in other pathogenic species of Leptospira, we applied the CRISPR-Prime Editing (CRISPR-PE) strategy to engineer a one-nucleotide frameshift deletion in ligB, generating a knockout mutant in L. borgpetersenii serogroup Ballum serovar Arborea strain LR131, a pathogenic species that lacks ligA. Despite a complete loss of LigB expression, the mutant strain maintained its ability to cause acute lethal infection in hamsters and the ability to establish renal colonization in rats. These findings demonstrate that ligB alone is dispensable for both acute and chronic infection in the L. borgpetersenii strain LR131 background. This work represents the first targeted disruption of ligB in L. borgpetersenii as facilitated by CRISPR-PE and prompts a reevaluation of LigB as a universal virulence determinant in different genetic backgrounds. These insights are critical for advancing our understanding of leptospiral pathogenesis and guiding the design of broadly protective subunit vaccines.},
}
@article {pmid41854513,
year = {2026},
author = {Han, JH and Kang, YJ and Lee, SY and Jin, HB and Lee, CS and Park, HH},
title = {AcrVA3 Is a Double Strand DNA-Cleaving Anti-CRISPR That Indirectly Inhibits Cas12.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {6},
pages = {e71705},
pmid = {41854513},
issn = {1530-6860},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2026-25470081//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry/antagonists &amp; inhibitors ; *Viral Proteins/metabolism/chemistry/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA Cleavage ; Bacteriophages/genetics/metabolism ; },
abstract = {CRISPR-Cas12 systems protect bacteria from foreign DNA, but are themselves targeted by anti-CRISPR (Acr) proteins evolved by phages. Among the eight known AcrV proteins that inhibit Cas12-based type V CRISPR-Cas systems, the mechanisms of all except AcrVA3 have been structurally and biochemically characterized. Here, we report the high-resolution structure of AcrVA3 and examine its inhibitory function in vitro. Unexpectedly, AcrVA3 does not directly work on Cas12. Instead, it exhibits double-stranded DNA (dsDNA) cleavage activity, suggesting an indirect mechanism of CRISPR inhibition through DNA degradation. This unique DNA-centric strategy contrasts with previously known Acr mechanisms and expands our understanding of how mobile genetic elements evade CRISPR immunity.},
}
@article {pmid41854526,
year = {2026},
author = {Gao, Z and Liu, G},
title = {Advancing Point-of-Care Testing for Helicobacter pylori toward CRISPR-Cas-Enabled Diagnostics.},
journal = {ACS sensors},
volume = {},
number = {},
pages = {},
doi = {10.1021/acssensors.6c00089},
pmid = {41854526},
issn = {2379-3694},
abstract = {Helicobacter pylori (H. pylori) chronically infects nearly half of the global population and is a major risk factor for gastric cancer. Timely and accurate diagnosis is critical to enable targeted eradication therapy and prevent disease progression. However, current gold-standard methods, such as invasive endoscopy and laboratory-based polymerase chain reaction, are costly, time-consuming, and logistically impractical for large-scale screening, particularly in resource-limited settings. Point-of-care testing (POCT) emerges as a transformative solution, offering rapid, user-friendly, and minimally invasive detection at the point of need. In this review, we systematically trace the evolution of H. pylori POCT, with a focus on revolutionary CRISPR-Cas-based diagnostic systems, cutting-edge advancements in substrate engineering (e.g., paper, polymer, hydrogels) and multi-modal signal transduction (e.g., optical, electrochemical). We further outline key design principles for next-generation POCT platforms that strictly align with the World Health Organization's ASSURED criteria (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, Deliverable), aiming to accelerate early detection, reduce healthcare disparities, and improve global clinical management of H. pylori infection.},
}
@article {pmid41855944,
year = {2026},
author = {Miao, Y and Wang, C and Peng, Y and Sun, X and Zheng, Z and Zhang, Q and Cheng, W and Li, J},
title = {PAM-assembled CRISPR-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for detecting prostate cancer exosomes.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118635},
doi = {10.1016/j.bios.2026.118635},
pmid = {41855944},
issn = {1873-4235},
mesh = {Humans ; *Biosensing Techniques/methods ; Male ; *Prostatic Neoplasms/diagnosis/blood/genetics ; *Exosomes/chemistry/genetics/pathology ; Colorimetry/methods ; CRISPR-Cas Systems/genetics ; *Glutamate Carboxypeptidase II/isolation &amp; purification ; Aptamers, Nucleotide/chemistry ; Antigens, Surface ; Limit of Detection ; Prostate-Specific Antigen ; Tetraspanin 30/chemistry ; Fluorescent Dyes/chemistry ; },
abstract = {Prostate specific membrane antigen (PSMA)-positive exosomes hold significant potential for the diagnosis and risk assessment of prostate cancer. However, accurate detection is severely hindered by their low abundance in blood and interference from similarly sized particles. In this study, we have developed a protospacer adjacent motif (PAM)-assembled clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a activation-based fluorescent and colorimetric dual-modal biosensor for the highly sensitive detection of PSMA-positive exosomes. In this work, two split strands respectively containing CD63 and PSMA aptamers are utilized to bind CD63 and PSMA on the exosome surface, forming a template that induces the opening of a hairpin DNA (HP DNA). A PAM site forms via hairpin-to-double-stranded structure transition. CRISPR-Cas12a recognizes PAM, activates to cleave FAM-labeled probes for fluorescence, while cerium dioxide nanozyme (CeO2 NZ) (with phosphatase-mimicking activity) hydrolyzes cleavage products. Hydroxyl radicals from hydrolysis oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to induce colorimetry. CRISPR-Cas12a-nanozyme dual recognition significantly improves prostate cancer exosome detection selectivity and sensitivity. Under optimized conditions, the limits of detection for the fluorescence and colorimetric modes reach 49 particles/μL and 63 particles/μL, respectively. By mutually validating dual detection modes, this biosensing technology accurately distinguishes prostate cancer patients from healthy individuals, holding great promise for early diagnosis.},
}
@article {pmid41855972,
year = {2026},
author = {Bai, M and Li, Y and Hu, Q and Qing, M and Bai, L},
title = {Enzymatic crRNA stabilization strategy for enhanced CRISPR/Cas12a detection.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129674},
doi = {10.1016/j.talanta.2026.129674},
pmid = {41855972},
issn = {1873-3573},
mesh = {*CRISPR-Cas Systems/genetics ; Mycobacterium tuberculosis/genetics/isolation &amp; purification ; Humans ; Influenza A virus/genetics ; CRISPR-Associated Proteins ; *Endodeoxyribonucleases/metabolism/genetics ; RNA Stability ; RNA, Viral/genetics ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system enables precise and rapid nucleic acid recognition through the programmable targeting capability of CRISPR RNA (crRNA). However, the intrinsic instability of crRNA limits the robustness and sensitivity of CRISPR-based molecular diagnosis in practical applications. Herein, we present a simple and general enhancement strategy that suppresses RNase-mediated crRNA degradation. This strategy, termed RNase inhibitor (RI)-assisted CRISPR (RI-CRISPR), leverages RI to specifically prevent crRNA degradation, thereby improving its stability and enhancing the detection performance and anti-interference capability of the CRISPR system. Using influenza A virus (IAV) and mycobacterium tuberculosis (MTB) as model targets, RI-CRISPR improves detection sensitivity by nearly twentyfold compared to conventional CRISPR/Cas12a. Clinical validation using 40 MTB samples, combined with recombinase polymerase amplification (RPA), achieves 100% specificity and 96% sensitivity compared with the GeneXpert assay. Overall, this work provides a practical strategy to enhance sensitivity and robustness of CRISPR-based diagnostics, and is expected to promote further biomedical applications of CRISPR technology.},
}
@article {pmid41855973,
year = {2026},
author = {Yang, M and Hu, J and Zhang, Z and Huang, L and Yu, Y and Qiu, D and Zu, Y and Liu, Y and Lin, Z},
title = {Homogeneous electrochemical sensor for sensitive detection of HBV DNA based on magnetic separation and CRISPR/Cas12a protein trans-cleavage.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129676},
doi = {10.1016/j.talanta.2026.129676},
pmid = {41855973},
issn = {1873-3573},
mesh = {*Hepatitis B virus/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism ; *Bacterial Proteins/metabolism/chemistry ; Humans ; *Endodeoxyribonucleases/metabolism ; Magnetic Phenomena ; },
abstract = {Infection with hepatitis B virus (HBV) represents a major challenge to global health, especially in areas of inadequate healthcare infrastructure, rapid and on-site detection plays a crucial role in effective disease management and control. Consequently, it is necessary to develop some simple but sensitive HBV screening techniques. In this work, a homogeneous electrochemical biosensor was constructed for sensitive and specific HBV DNA detection through the integration of CRISPR/Cas12a target recognition and magnetic bead-based separation. The sensor exploits the collateral cleavage activity of CRISPR/Cas12a protein upon recognition of target double-stranded DNA, enabling the degradation of methylene blue-labeled single-stranded DNA captured on magnetic beads (MBs). Then the MBs are separated and redissolved in the solution and the electrochemical response of the system can be tested in homogeneous solution. The electrochemical signal has relationship with the concentration of HBV DNA within a range of 10 fM - 10 nM, and the LOD is 3.74 fM. The sensor demonstrates excellent selectivity and biological stability, which has potential application in clinical diagnosis, especially in resource-limited environments.},
}
@article {pmid41855975,
year = {2026},
author = {Yong, Q and Ou, X and Zhao, Y and Kang, X and Gao, H and Liu, H and Li, K and Guo, Y},
title = {CRISPR/Cas and isothermal amplification in Pathogen Detection: Applications and future perspectives.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129662},
doi = {10.1016/j.talanta.2026.129662},
pmid = {41855975},
issn = {1873-3573},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Conventional pathogen detection methods are often limited by prolonged turnaround times and laboratory dependency. The integration of CRISPR/Cas systems with isothermal amplification (IA) has emerged as a promising approach to enable rapid, accurate, and field-deployable molecular diagnostics. This review systematically outlines the principles, optimization strategies, and recent advances in CRISPR-Cas and IA-integrated platforms. It highlights how synergistic mechanisms enhance detection sensitivity and examines innovative integration strategies-such as physical compartmentalization, chemical regulation, and intelligent system design-that address key compatibility challenges. The role of nanomaterials in enhancing signal amplification and facilitating system integration is thoroughly discussed. Furthermore, the suitability of various readout modalities-including fluorescence, lateral flow assays, electrochemical sensing, and digital detection-is critically evaluated. While challenges remain in terms of stability, cost, and standardization, future advances in intelligent design, portable device development, and quantitative methodologies are expected to establish this technology as a versatile platform for public health control, food safety monitoring, and related fields. This review provides a comprehensive perspective and methodological reference for researchers engaged in point-of-care testing and diagnostic technology development.},
}
@article {pmid41856106,
year = {2026},
author = {Yuan, S and Zhu, H and Yu, M and Jia, H and Peng, S and Ma, Y},
title = {Discovery of human gut phage-encoded anti-CRISPR proteins unveils diverse mechanisms for phages to evade type II CRISPR immunity.},
journal = {Cell host &amp; microbe},
volume = {34},
number = {4},
pages = {708-719.e5},
doi = {10.1016/j.chom.2026.02.017},
pmid = {41856106},
issn = {1934-6069},
mesh = {Humans ; *Bacteriophages/genetics/metabolism ; *CRISPR-Cas Systems ; *Gastrointestinal Microbiome ; *Viral Proteins/genetics/metabolism ; Bacteria/genetics/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Computational Biology ; },
abstract = {Phages encode diverse anti-CRISPR (Acr) proteins to counteract bacterial CRISPR-Cas systems. However, gut phage Acrs remain poorly characterized. Using an integrated bioinformatics and high-throughput functional screening approach, we identify 651 phage-encoded positive Acr candidates that target type II CRISPR systems, which predominate in the human gut. Among these, a subset of Acrs is verified through plasmid interference assays, with plaque assays confirming CRISPR-Cas inhibitory activity for 36 Acr candidates. Mechanistic characterization of five Acrs, including the Acr against subtype II-B systems (AcrIIB-1), reveals distinct inhibition strategies. Remarkably, 213 positive Acr candidates, designated here as GutAcraca, exhibit structural convergence by adopting similar folds and exhibit dual functionality: transcription regulation to support their production and inhibition of CRISPR-Cas systems. These GutAcraca are widely distributed across microbial species (detected in 26% of species). Our work uncovers the extensive diversity of phage-encoded Acrs in the human gut and highlights their potential as biotechnology tools.},
}
@article {pmid41856855,
year = {2026},
author = {Tsai, FY and Sternberg, SH},
title = {Memory on demand: how RNA-free Cas9 recharges CRISPR immunity.},
journal = {Trends in biochemical sciences},
volume = {51},
number = {4},
pages = {310-312},
doi = {10.1016/j.tibs.2026.01.004},
pmid = {41856855},
issn = {0968-0004},
mesh = {*CRISPR-Cas Systems/immunology/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics/immunology ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Bacteria/genetics/immunology ; Archaea/immunology/genetics ; *CRISPR-Associated Protein 9/metabolism ; },
abstract = {Bacteria and archaea acquire immune memories by integrating foreign DNA into clustered regularly interspaced short palindromic repeats (CRISPR) arrays. Zhou et al. reveal that Cas9-thought to act only with guide RNAs-also functions in its RNA-free form, stimulating spacer acquisition. Rising CRISPR RNA levels shift the equilibrium toward the RNA-bound state, attenuating acquisition and minimizing autoimmunity.},
}
@article {pmid41859774,
year = {2026},
author = {Fang, Z and Hao, Y and Zuo, X and Wang, S},
title = {Target-Gated Ratiometric pH Sensing via Tetrahedral DNA Framework-Based Dual-CRISPR System.},
journal = {Analytical chemistry},
volume = {98},
number = {12},
pages = {9208-9218},
doi = {10.1021/acs.analchem.5c07666},
pmid = {41859774},
issn = {1520-6882},
mesh = {Hydrogen-Ion Concentration ; *DNA/chemistry/metabolism ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Fluorescent Dyes/chemistry ; Extracellular Vesicles/metabolism/chemistry ; },
abstract = {Extracellular vesicle (EV)-mediated communication is tightly regulated by local pH, which governs vesicle biogenesis, cargo release, and membrane fusion. Accurate and context-specific pH sensing is therefore crucial for elucidating EV function and disease-associated microenvironmental regulation. Here, we present a tetrahedral DNA framework (TDF)-orchestrated dual-CRISPR system that integrates orthogonal Cas12a and Cas13a nucleases for target-activated, ratiometric pH detection at lipid membranes. By exploiting the distinct pH-activity profiles of Cas12a (optimal pH ∼ 8.5) and Cas13a (optimal pH ∼ 7.2), combined with their complete substrate orthogonality, we constructed a self-calibrating nanosensor featuring equimolar coassembly of both nucleases and their corresponding fluorogenic reporters at the four vertices of a TDF. The well-defined tetrahedral geometry ensured reproducible molecular organization and stable fluorescence output, eliminating variability inherent to conventional single-fluorophore probes. The sensor exhibited quantitative assembly fidelity and robust pH responsiveness across physiological ranges. Importantly, the Cas module can be programmed for conditional activation, enabling pH sensing only upon recognition of disease-associated biomarkers. Using miR-146a, a regulatory microRNA enriched in EVs implicated in inflammation and cancer progression, as a model target, we demonstrated target-gated pH monitoring on cell-derived exosomes and during liposome fusion events. This work establishes a versatile and generalizable platform for programmable, ratiometric sensing at biomembrane interfaces, offering new opportunities to probe EV-mediated intercellular communication and dynamic microenvironmental regulation.},
}
@article {pmid41859925,
year = {2026},
author = {Tibebu, R and Gamo, ME and Ellison, EE and Myers, EA and Sahoo, R and Winecke, SR and Tan, GD and Fischer, JM and Leakey, ADB and Voytas, DF},
title = {Virus-induced gene editing of stomatal regulators in Nicotiana benthamiana enables rapid functional genomics.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70805},
pmid = {41859925},
issn = {1365-313X},
support = {SC0023160//U.S. Department of Energy/ ; SC0018277//U.S. Department of Energy/ ; },
mesh = {*Nicotiana/genetics/virology ; *Gene Editing/methods ; *Plant Stomata/genetics/growth &amp; development ; Genomics/methods ; *Plant Viruses/genetics/physiology ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {Virus-induced gene editing (VIGE) holds promise as a rapid and scalable approach for functional genomics in plants. Here, we apply a tobacco rattle virus (TRV)-based single-guide RNA (sgRNA) delivery system to target key regulators of stomatal development in Nicotiana benthamiana using transgenic Cas9-expressing lines. sgRNAs fused to a mobile RNA element and co-delivered with TRV enabled both somatic and heritable genome editing across orthologs of STOMAGEN, EPF2, YODA, and SPEECHLESS. Somatic editing frequencies reached up to 95%, and heritable tetra-allelic mutations were recovered in multiple target genes. Mutants exhibited significant, gene-specific changes in stomatal density, with corresponding effects on leaf temperature indicative of altered evaporative cooling. Additionally, sgRNAs fused to an AmCyan reporter enabled visualization of virus-infected tissues, allowing stomatal phenotyping in edited M0 sectors. This TRV-based platform facilitates functional assessment of genes influencing stomatal patterning and offers a powerful tool for dissecting gene function in a developmentally and physiologically relevant context.},
}
@article {pmid41860163,
year = {2026},
author = {Wang, B and Liu, W and Li, Y and Ouyang, Q and Wang, Y and Xu, X and Li, B and Xiu, R and Zhang, X and Liu, M},
title = {Consequences of CRISPR-Cas9-Mediated Stromelysin-1 Knockout in Pancreatic Islet Microvascular Endothelial Cells.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {6},
pages = {e71098},
doi = {10.1111/jcmm.71098},
pmid = {41860163},
issn = {1582-4934},
support = {7252093//Beijing Municipal Natural Science Foundation/ ; 81900747//National Natural Science Foundation of China/ ; },
mesh = {*Endothelial Cells/metabolism ; Animals ; *CRISPR-Cas Systems/genetics ; *Islets of Langerhans/blood supply/metabolism/cytology ; Cell Movement/genetics ; Mice ; Cell Proliferation/genetics ; *Microvessels/metabolism/cytology ; Gene Knockout Techniques ; Glucose/toxicity ; Neovascularization, Physiologic ; },
abstract = {The integrity of the pancreatic islet microvasculature is critical for endocrine function, yet it is progressively compromised by glucotoxicity in diabetes. While matrix metalloproteinases are implicated, the role of stromelysin-1 as a potential upstream driver of endothelial dysfunction remains poorly defined. The aim of our study was to elucidate the role of stromelysin-1 in mediating glucotoxic injury to islet microvascular endothelial cells (IMECs). To this end, we employed a CRISPR/Cas9-mediated knockout of stromelysin-1 in IMECs. Cellular functions, including proliferation, migration, and angiogenesis, were assessed using IncuCyte ZOOM live-cell imaging, while endothelial barrier integrity was quantified via a 40 kDa dextran flux assay. Additionally, the secretome was profiled using a cytokine antibody array. We found that genetic ablation of stromelysin-1 conferred protection against glucotoxicity. Stromelysin-1 KO IMECs exhibited significantly enhanced proliferation, migration, and angiogenic capacity compared to wild-type controls. Furthermore, stromelysin-1 deficiency restored endothelial monolayer integrity by attenuating high-glucose-induced hyperpermeability. These functional improvements were linked to a remodelling of the secretome, characterised by decreased secretion of the pro-degradative MMP-2 and increased secretion of the anti-inflammatory cytokine IL-10 and the endogenous inhibitor TIMP-2. Overall, our findings establish stromelysin-1 as a crucial mediator of glucotoxic injury in islet microvascular endothelial cells.},
}
@article {pmid41860589,
year = {2026},
author = {Walsh, DJ and Hynes, R and Guo, W and Surgenor, C and Prodöhl, PA and Parle-McDermott, A},
title = {Development and Laboratory Validation of a Field-Deployable CRISPR-Cas12a eDNA Assay for Phylogeographic Lineage Detection in Arctic Char (Salvelinus alpinus).},
journal = {Molecular ecology resources},
volume = {26},
number = {3},
pages = {e70125},
pmid = {41860589},
issn = {1755-0998},
support = {//Higher Education Authority/ ; },
mesh = {Animals ; *Trout/genetics/classification ; Phylogeography/methods ; *CRISPR-Cas Systems ; *DNA, Environmental/genetics/isolation &amp; purification ; Ireland ; Sensitivity and Specificity ; },
abstract = {Environmental DNA (eDNA) tools are increasingly used for biodiversity monitoring, with most existing assays targeting species-level identification. However, the use of eDNA to resolve intraspecific genetic variation remains rare and methodologically underdeveloped. This study presents the development and laboratory validation of a novel molecular assay capable of detecting specific phylogeographic lineages, advancing eDNA applications by enabling resolution below the species level. The assay combines Recombinase Polymerase Amplification (RPA) and CRISPR-Cas12a technologies with a lateral flow platform for field-ready, on-site detection. Irish Arctic char (Salvelinus alpinus) was selected as the model due to its conservation relevance and post-glacial lineage diversity in Ireland. Mitochondrial genome sequencing of known Irish lineages identified a Protospacer Adjacent Motif (PAM) site unique to the Atlantic Subclade 1 lineage, allowing clear discrimination from co-occurring lineages. Two assays were optimised: a species-specific assay detecting all Arctic char lineages and a lineage-specific assay targeting Lineage 1. Both showed high sensitivity and specificity under laboratory conditions, with LbCas12a outperforming AsCas12a at optimised buffer concentrations. The lateral flow adaptation, utilising a dual-labelled FAM-Biotin probe, enabled portable and rapid detection with minimal equipment. Field validation using eDNA from Irish lakes highlighted the need for improved sampling protocols, as lake-edge surface samples failed to yield detections. This assay represents the first reported example of a CRISPR-based eDNA tool for phylogeographic lineage detection in the field. It offers a novel, non-invasive, and scalable approach to fine-scale ecological monitoring and establishes a foundation for future conservation tools targeting intraspecific diversity.},
}
@article {pmid41860948,
year = {2026},
author = {Hong, Y and Si, X and Liu, W and Mai, X and Zhang, Y},
title = {Ex vivo and in vivo CRISPR/Cas9 screenings identify the roles of protein N-glycosylation in regulating T-cell activation and functions.},
journal = {eLife},
volume = {14},
number = {},
pages = {},
pmid = {41860948},
issn = {2050-084X},
support = {2021YFA1101002//National Key Research and Development Program of China/ ; 81773304//National Natural Science Foundation of China/ ; 81572795//National Natural Science Foundation of China/ ; 2019A39//the "Hundred, Thousand and Ten Thousand Talent Project" by Beijing municipal government/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Glycosylation ; Mice ; *Galactosyltransferases/metabolism/genetics ; *CD8-Positive T-Lymphocytes/immunology ; *Lymphocyte Activation ; Mice, Inbred C57BL ; Humans ; Receptors, Antigen, T-Cell/metabolism ; },
abstract = {Cytotoxic CD8[+] T-cells play central roles in tumor immunotherapy. Understanding the mechanisms that regulate development, differentiation, and functions of cytotoxic CD8[+] T-cells leads to the development of better immunotherapies. By combining primary T-cell culture and a syngeneic mouse tumor model with both genome-wide and custom CRISPR/Cas9 screenings, we systematically identified genes and pathways that regulate PD-1 expression and functions of CD8[+] T-cells. Among them, inactivation of a key enzyme in glycoconjugate biosynthesis, beta 1,4-galactosyltransferase 1 (B4GALT1), leads to significantly enhanced T-cell receptor (TCR) activation and functions of CD8[+] T-cell. Interestingly, suppression of B4GALT1 enhances functions of TCR-T-cells, but has no effect on chimeric antigen receptor T (CAR-T) cells. We systematically identified the substrates of B4GALT1 on CD8[+] T-cell surface by affinity purification and mass spectrometry analysis, which include protein components in both TCR and its co-receptor complexes. The galactosylation of TCR and CD8 leads to reduced interaction between TCR and CD8 that is essential for TCR activation. Artificially tethering TCR and CD8 by a TCR-CD8 fusion protein could bypass the regulation of B4GALT1 in CD8[+] T-cells. Finally, the expression levels of B4GALT1 normalized to tumor-infiltrated CD8[+] T-cells in tumor microenvironment are significant and negatively associated with prognosis of human patients. Our results reveal the important roles of protein N-glycosylation in regulating functions of CD8[+] T-cells and prove that B4GALT1 is a potential target for tumor immunotherapy.},
}
@article {pmid41861390,
year = {2026},
author = {Huang, Y and Yi, X and Yang, X and Li, C and Li, Y and Ye, Z and He, J},
title = {Characterization of CRISPR-Cas systems in the Haemophilus genus CRISPR-Cas in Haemophilus spp.},
journal = {Genetics and molecular biology},
volume = {49},
number = {1},
pages = {e20250166},
pmid = {41861390},
issn = {1415-4757},
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) system constitutes a crucial adaptive defense mechanism in prokaryotes against foreign genetic elements. Although CRISPR-Cas systems have been characterized in numerous bacteria, the architecture and function of these systems in the Haemophilus genus remain poorly understood. This study aims to analyze CRISPR-Cas systems in 138Haemophilusstrains and investigate their function, particularly in relation to virulence factors. Results revealed that CRISPR-Cas systems were identified in 31.88% of the Haemophilusstrains. Subtype I-C was the most prevalent, followed by subtypes II-C and III-A. Repeat sequences and thecas1gene were highly conserved within the same subtype. 29.62% of spacer sequences exhibited homology to plasmids or bacteriophages. phiMHaA1 was an important target of the CRISPR-Cas system in Haemophilusgenus. The protospacer adjacent motif sequences (PAM) were determined to be 5'-TTC-3' for subtype I-C and 5'-TTT-3' for subtype II-C. Comparative analysis of virulence genes showed that CRISPR-positive strains carried more ompP2 than CRISPR-negative strains, while the distribution of hmw2C and hmw1C exhibited an opposite trend. These findings provide novel insights into the diversity and function of CRISPR-Cas systems inHaemophilusgenus and propose potential strategies for attenuating the impact ofHaemophilusvirulence factors.},
}
@article {pmid41863808,
year = {2026},
author = {Louis, EM and Fu, L and Luu, N and Sachs, LM and Shi, YB},
title = {Protocol for streamlining genotyping of germline-transmissible mutants from genome editing by using a parallel qPCR-based index and R analysis.},
journal = {STAR protocols},
volume = {7},
number = {2},
pages = {104454},
doi = {10.1016/j.xpro.2026.104454},
pmid = {41863808},
issn = {2666-1667},
abstract = {Targeted genome editing using CRISPR-Cas, ZFNs, or TALENs enables precise gene function studies but often produces point mutations or insertions or deletions (indels) that are difficult to detect by conventional PCR. We developed a parallel qPCR assay with an iGenotype index for simple, reliable genotyping. iGenotype values (1, 0, -1) remained constant across allele-specific primers. qPCR data can be analyzed via an R program, enabling large-scale or automated genotyping. For complete details on the use and execution of this protocol, please refer to Fu et al.[1].},
}
@article {pmid41863928,
year = {2026},
author = {Chang, Z and Zhu, B and Wang, Y and Taylor, JA and Dong, H and Zhu, X and Hao, Y and Zhou, Y and Xu, M and Travas-Sejdic, J},
title = {Disposable laser-induced graphene-based biosensor strip for the detection of N-protein using CRISPR activation and ratiometric electrochemical readout.},
journal = {Talanta},
volume = {305},
number = {},
pages = {129667},
doi = {10.1016/j.talanta.2026.129667},
pmid = {41863928},
issn = {1873-3573},
mesh = {*Graphite/chemistry ; *Biosensing Techniques/methods/instrumentation ; *Electrochemical Techniques/methods/instrumentation ; Humans ; *SARS-CoV-2/isolation &amp; purification ; Lasers ; *CRISPR-Cas Systems ; Gold/chemistry ; COVID-19/diagnosis/virology ; Electrodes ; *Coronavirus Nucleocapsid Proteins/analysis ; Metal Nanoparticles/chemistry ; Limit of Detection ; Methylene Blue/chemistry ; Metallocenes/chemistry ; Ferrous Compounds/chemistry ; Phosphoproteins ; },
abstract = {Pathogen detection is important for infectious diseases prevention and control. This study presents a novel positive-response, ratiometric electrochemical biosensor constructed on disposable laser-induced graphene (LIG) electrodes. The platform provides dual electrochemical signals for ultrasensitive detection of the SARS-CoV-2 N-protein by harnessing target-activated CRISPR-Cas12a trans-cleavage activity. The developed sensor incorporates a hairpin oligonucleotide (ON) functionalized on gold nanoparticles-deposited LIG electrode surface. Methylene blue (MB) molecules bind to guanine (G) bases of the hairpin ON through inherent affinity, generating the first electrochemical signal. Upon target-induced CRISPR-Cas12a activation, ferrocene (Fc)-labelled indicator ON hybridises with the hairpin ON, displacing the MB molecules and concurrently introduces the Fc as a secondary electrochemical signal reporter. Such displacement triggers a quantifiable decrease in MB electrochemical current and an increase in Fc electrochemical current, generating an Fc/MB ratiometric signal that enhances with increasing target levels and serves as the robust sensor response. The developed ratiometric biosensor achieves a linear response from 0.01 pM to 100 pM of N-protein with a detection limit of 1.3 × 10[-3] pM and with excellent selectivity. The clinical feasibility of the developed ratiometric electrochemical biosensor was confirmed by detecting N-protein in the inactivated cell-cultured SARS-CoV-2. This work demonstrates significant potential in rapid pathogen detection and point-of-care diagnostics.},
}
@article {pmid41863957,
year = {2026},
author = {Yang, Y and Huang, Z and Peng, X and Cui, F and Hu, X and Ren, H},
title = {Single-microsphere biosensors based on UiO-66 confinement-enhanced fluorescence emission for sensitive detection of antibiotics and pathogenic bacteria.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118632},
doi = {10.1016/j.bios.2026.118632},
pmid = {41863957},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Staphylococcus aureus/isolation &amp; purification/pathogenicity ; *Anti-Bacterial Agents/analysis/isolation &amp; purification ; Limit of Detection ; *Chloramphenicol/analysis/isolation &amp; purification ; Fluorescent Dyes/chemistry ; Microspheres ; Food Contamination/analysis ; Food Microbiology ; CRISPR-Cas Systems ; Fluorescence ; Humans ; Polystyrenes/chemistry ; },
abstract = {To meet the urgent need for sensitive detection of foodborne pathogens and antibiotic residues-key to preventing outbreaks and curbing antimicrobial resistance-we developed a stable fluorescent probe, H4TCPE@UiO-66 (H@U). The probe retains strong fluorescence under harsh conditions (18.70% intensity change at pH 3, 11.36% at 80 °C) and exhibits a 6.90-fold signal amplification over H4TCPE. In addition, H@U is able to be a sensitive probe integrated with millimetre-scale polystyrene microspheres (mPS) and realize rapid detection of chloramphenicol within 20 min with a detection limit of 33.52 pg/mL, representing a 26.61-fold improvement in sensitivity over the enzyme-linked immunosorbent assay. Building on this, we harnessed the trans-cleavage activity of CRISPR/Cas12a to develop a CRISPR/Cas12a-enhanced H@U mPS (H@U-C mPS) biosensor for the quantitative detection of Staphylococcus aureus, achieving a linear range of 10-10[5] CFU/mL (two orders of magnitude lower than qPCR) and a detection limit of 6.97 CFU/mL. Furthermore, the practical applicability of H@U-C mPS biosensors was successfully demonstrated through validation using 30 real-world samples. Together, these platforms offer a robust, rapid, and highly sensitive strategy for monitoring chemical hazards and microbial in food safety and clinical diagnostics.},
}
@article {pmid41864147,
year = {2026},
author = {Zhang, L and Tang, J and Feng, M and Chen, S},
title = {Development of a plasmid-free Escherichia coli strain for high-yield production of ergothioneine.},
journal = {Enzyme and microbial technology},
volume = {197},
number = {},
pages = {110850},
doi = {10.1016/j.enzmictec.2026.110850},
pmid = {41864147},
issn = {1879-0909},
mesh = {*Ergothioneine/biosynthesis ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; Plasmids/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; },
abstract = {Ergothioneine (ERG), a sulfur-containing amino acid derivative known for its antioxidant activity, has a wide range of applications in healthcare and nutrition. Escherichia coli has been extensively studied as a platform for ERG production due to its rapid growth and well-established genetic tools. However, most engineered strains rely on plasmid-based expression system, which are genetically unstable. Additionally, the requirement for antibiotics to maintain plasmid stability further limits the feasibility of plasmid-based systems for industrial-scale production. Here, we established a plasmid-free E. coli platform for ERG biosynthesis using a multi-copy chromosomal integration CRISPR-associated transposase (MUCICAT) system. We first integrated a three-gene ERG biosynthetic pathway into the E. coli genome at varying copy numbers, resulting in a five-copy strain (P5) that exhibited the highest ERG titer of 222.5 ± 5.0 mg/L. Subsequently, we reinforced the two key catalytic modules-histidine methylation and SAM biosynthesis-through iterative genomic integration of the corresponding genes, yielding a plasmid-free strain P18 that produced 370.0 ± 7.0 mg/L ERG. The engineered strain P18 exhibited excellent genetic stability, as confirmed by serial passaging. When scaled up in a 5-L bioreactor under fed-batch condition, an ERG titer of 10.1 g/L was achieved. This study demonstrates a plasmid-free ERG production strategy based on stable, multi-copy chromosomal integration of the ERG biosynthetic pathway in E. coli, highlighting its potential as an efficient platform for scalable ERG production.},
}
@article {pmid41865126,
year = {2026},
author = {Marei, HE},
title = {Recent Advances in the Non-viral Delivery of Genes to Central Nervous System Disorders.},
journal = {Cellular and molecular neurobiology},
volume = {46},
number = {1},
pages = {},
pmid = {41865126},
issn = {1573-6830},
mesh = {Humans ; *Central Nervous System Diseases/therapy/genetics ; Animals ; *Gene Transfer Techniques/trends ; *Genetic Therapy/methods/trends ; Blood-Brain Barrier/metabolism ; Nanoparticles ; Genetic Vectors ; Gene Editing ; },
abstract = {Disorders of the central nervous system (CNS), neurological disorders, neurodegenerative disorders, genetic disorders) constitute a significant burden on global health, and current treatment options remain challenging. As treatment for CNS disorders is primarily palliative, the underlying causes of disease progression are not addressed through conventional pharmacologic therapies. Gene therapy has the potential to address these root causes of disease progression; however, many of the vectors used in gene therapy (e.g., adeno-associated viruses (AAVs)) have limitations such as immunogenicity, low cargo capacity, and crossing the blood-brain barrier (BBB). These limitations have led to significant progress in the development of non-viral gene delivery systems. Compared with viral vectors, non-viral platforms offer improved safety profiles, greater design flexibility, lower production costs, and superior suitability for repeated administration. This review reports recent advancements in the development of non-viral platforms for CNS gene delivery and focuses on lipid-based nanoparticles, polymeric nanoparticles, exosome-based techniques, and new hybrid technologies. Particular emphasis is placed on nanoparticle modification approaches to enhance BBB penetration and enable delivery of genome-editing technologies (CRISPR/Cas systems). The review provides explanations of clinical trials, regulatory considerations, and manufacturing issues that result from the recent developments noted above. It also explores the emerging role of artificial intelligence in supporting carrier design and enhancing delivery efficiency. Both artificial intelligence and non-viral platforms have the potential to facilitate the advancement of safe, effective, and repeatably administered gene therapies for patients with CNS disorders.},
}
@article {pmid41865271,
year = {2026},
author = {Kahr, J and Diaz-Peregrino, R and Sandalcioglu, IE and John, P and Mawrin, C},
title = {RagC and Map4K3 deficiency in high-grade gliomas drives proliferation and modulates mTORC1-dependent cellular functions.},
journal = {Journal of neuropathology and experimental neurology},
volume = {},
number = {},
pages = {},
doi = {10.1093/jnen/nlag010},
pmid = {41865271},
issn = {1554-6578},
abstract = {Cellular growth and homeostasis via amino acid-responsive pathways are mediated by the mTOR signaling pathway. Rag GTPases and Map4K3 modify mTOR signaling as amino acid sensors. Altered mTOR signaling in relation to amino acid sensors might represent factors that modify proliferation and treatment responses in astrocytic tumors. To investigate this hypothesis, RagC and Map4K3 expression was studied in human gliomas, glioma cells (U87MG/U138MG), and nonglial cells (MCF-7, IOMM-Lee). RagC and Map4K3 knockout in glioma cells was generated using CRISPR-Cas and shRNA. High-grade astrocytomas had significantly reduced immunoreactivity for RagC and Map4K3 compared to low-grade astrocytomas. RagC- and Map4K3-deficient glioma cells had significantly increased proliferation and showed altered morphology and motility. Induced amino acid deficiency (leucine deprivation) reduced proliferation in Map4K3- but not in RagC-deficient cells. mTOR signaling in RagC- and Map4K3-deficient U87 cells was altered with increased phosphorylation of p70S6K and increased expression of RagD and transcription factor EB. In this context, uncoupled, exaggerated autophagy occurred in Map4K3-deficient U87 cells. In contrast, RagC-deficient U87 cells showed increased senescence but no autophagy induction. These data show that losses of RagC and Map4K3 in malignant gliomas have proliferation-inducing effects and differentially modulate key mTOR signaling-dependent cellular mechanisms.},
}
@article {pmid41865648,
year = {2026},
author = {Marková, K},
title = {Affinity-based nanostructured platforms for the selective pretreatment, enrichment and detection of miRNA biomarkers.},
journal = {Journal of chromatography. B, Analytical technologies in the biomedical and life sciences},
volume = {1276},
number = {},
pages = {125017},
doi = {10.1016/j.jchromb.2026.125017},
pmid = {41865648},
issn = {1873-376X},
mesh = {*MicroRNAs/analysis/isolation &amp; purification/blood ; Humans ; *Nanostructures/chemistry ; Biomarkers/analysis/blood ; Spectrum Analysis, Raman/methods ; Animals ; },
abstract = {MicroRNAs (miRNAs) are clinically relevant liquid-biopsy biomarkers, yet their reliable quantification is still limited by low abundance, matrix complexity, and frequent association with protective carriers such as Argonaute-2 and exosomes. This review focuses on affinity-based nanostructured platforms as superior tools for the selective pretreatment and enrichment of miRNAs, bridging the gap between raw clinical samples and high-performance analysis. We summarize key nanomaterial architectures, including functional nanoparticles, electrospun nanofibers, 2D nanomaterial platforms, and hybrid nanocomposites, together with surface functionalization strategies that enable the liberation of miRNAs from protein complexes and their subsequent sequence-specific capture. Emphasis is placed on how these enrichment workflows address target accessibility in protein-rich biofluids and mitigate matrix-induced interference. Beyond sample preparation, we evaluate the integration of these platforms with advanced detection modalities, including Surface-Enhanced Raman Spectroscopy (SERS) using inverse molecular sentinel (iMS) nanoprobes, enzyme-free hybridization chain reactions (HCR) and CRISPR/Cas-based assays. These innovative strategies circumvent the limitations of enzymatic amplification, offering high sensitivity and specificity. Finally, we address challenges in automation and standardization, highlighting the need for integrated enrichment-to-detection workflows that accelerate the translation of nanomaterial innovation and next-generation, point-of-care miRNA diagnostics.},
}
@article {pmid41867107,
year = {2026},
author = {Zhuang, S and Li, J and Fang, Z and Zhou, H and Zhang, R and He, J and Zhu, L and Xu, Y and Xu, D and Gu, D and Wang, J},
title = {Phosphorothioate Modification-Regulated One-Pot CRISPR Assay for Arbovirus Detection.},
journal = {ACS infectious diseases},
volume = {12},
number = {4},
pages = {1423-1433},
doi = {10.1021/acsinfecdis.6c00013},
pmid = {41867107},
issn = {2373-8227},
mesh = {*CRISPR-Cas Systems ; *Chikungunya virus/genetics/isolation &amp; purification ; Humans ; Nucleic Acid Amplification Techniques/methods ; *Chikungunya Fever/diagnosis/virology ; *Arboviruses/genetics/isolation &amp; purification ; RNA, Viral/genetics ; *Phosphorothioate Oligonucleotides/chemistry ; CRISPR-Associated Proteins/metabolism/genetics ; Endodeoxyribonucleases/metabolism/genetics ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; },
abstract = {The ongoing arthropod-borne Chikungunya virus (CHIKV) highlights the requirements of rapid and accurate diagnostic methods to enhance the epidemic control. CRISPR diagnostic (CRISPR-Dx) technology holds promise, but the development of a highly efficient one-pot diagnostic system usually requires fine-tuning of the balance between isothermal amplification and Cas cleavage procedures. Here, we describe a simple method (psHOLMES) to create one-pot, two-step CRISPR-Dx systems, using photocleavable partially phosphorothioate-modified DNA (ppPS-DNA) to regulate Cas12a activity. Cas12a activity is first inactivated via binding of ppPS-DNA during the target sequence amplification procedure, which is then reactivated by ultraviolet (UV)-mediated photolysis of ppPS-DNA after amplification, triggering Cas12a trans-cleavage reactions. psHOLMES demonstrates attomolar sensitivity for CHIKV RNA detection and zero cross-reactivity against other related arboviruses. When applied to clinical samples, psHOLMES achieved 100% (50/50) accuracy and could detect CHIKV within 30 min. As traditional efforts for fine-tuning Cas cis-cleavage activity can be omitted, psHOLMES thus enables rapid development of one-pot CRISPR-Dx systems for clinical applications.},
}
@article {pmid41867779,
year = {2026},
author = {Wester, M and Lim, J and Van, AB and Koprowski, K and Valera, E and Bashir, R},
title = {Kinetic Modeling of Target-Amplification-Free CRISPR-Cas-Based Autocatalysis Reactions.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41867779},
issn = {2692-8205},
abstract = {CRISPR-Cas-based diagnostics utilize the Cas enzyme's trans-cleavage activity to generate signal and have become popular platforms for sensitive nucleic acid detection. Recently, autocatalytic systems have been demonstrated to improve the time to response and sensitivity in some cases. However, mechanistic description of these assays is limited and optimization relies on simple trial-and-error. In this work, we present the first comprehensive kinetic model that integrates all major biochemical processes involved in these assays, including cleavage reactions, nucleic acid equilibrium kinetics, inhibition of trans-cleavage by single-stranded DNA, and degradation of single-stranded reaction components. We discuss the biochemical foundations and implementation of the ordinary differential equation model, which is built for adaptation to different reaction schemes. We use the full model to investigate the role of nucleic acid stability in assay performance for a typical nucleic acid design and show that our model demonstrates inhibition effects consistent with experimental data. We describe the reaction behavior, derive a simplified analytical model and compare its performance to the full analytical model. Finally, we demonstrate tools developed for rapid in silico optimization to guide the rational design of future target-amplification-free CRISPR-Cas-based autocatalysis assays.},
}
@article {pmid41869310,
year = {2026},
author = {Petri, K and Ferrari, S and Cianciotti, BC},
title = {Editorial: Safety and efficacy of CRISPR/Cas-based genome editing tools: applications and considerations in cell and gene therapy.},
journal = {Frontiers in immunology},
volume = {17},
number = {},
pages = {1809809},
doi = {10.3389/fimmu.2026.1809809},
pmid = {41869310},
issn = {1664-3224},
}
@article {pmid41869962,
year = {2026},
author = {Yang, L and Tan, H and Wang, Y and Zhang, J and Meng, X and Liu, X and Hou, T and Chen, W and Li, F},
title = {Fluidly Confined CRISPR-Magnetic Microbots Empowered Homogeneous Electrochemical Biosensor for Amplified Detection and Discrimination of Cancer-Derived Extracellular Vesicle Subtypes.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {10103-10111},
doi = {10.1021/acs.analchem.6c00448},
pmid = {41869962},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Extracellular Vesicles/chemistry/metabolism ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Doxorubicin/chemistry ; DNA/chemistry ; *Neoplasms ; Biomarkers, Tumor/analysis ; },
abstract = {Accurate identification and profiling of multiple protein biomarkers on tumor-derived extracellular vesicles (tEVs) are crucial for noninvasive cancer subtyping diagnosis but remain technically challenging due to their high heterogeneity, low abundance in biofluids, and preisolation/purification processes. Herein, we developed a homogeneous electrochemical biosensor empowered by fluidly confined CRISPR-magnetic microbots for the amplified detection and sensitive discrimination of tEV subtypes. The CRISPR-magnetic microbots were constructed by engineering CRISPR/Cas12a and DNA icosahedra/doxorubicin (DNA-ICOS/DOX) on intracellularly gelated magnetic cells (IGMCs). Benefiting from the synergistic effects of spatial confinement and membrane fluidity to elevate the local concentration and collision efficiency, the activity of CRISPR/Cas12a was found to be greatly enhanced on IGMCs. For selective sorting of tEVs, a logic-gated aptamer system was used to orthogonally label tEV subpopulations, which further triggers the trans-cleavage activity of CRISPR/Cas12a, resulting in the release of massive DNA-ICOS/DOX into solution. After magnetic separation, the liberated DOX molecules generate a strong electrochemical signal. Particularly, the CRISPR-magnetic microbots could efficiently reduce the background signal, endowing a significantly improved signal-to-noise ratio. Therefore, by combining the CRISPR-magnetic microbots with the dual-target-guided orthogonal barcoding strategy in a homogeneous electrochemical biosensor, precise identification and sensitive detection of tEVs were successfully achieved. More significantly, this assay achieves accurate cancer subtyping in clinical samples, demonstrating its potential as a robust, noninvasive tool for high-accuracy disease screening, classification, and progression monitoring.},
}
@article {pmid41870078,
year = {2026},
author = {Bayurova, E and Kostyushev, D and Tikhonov, A and Chulanov, V and Gordeychuk, I},
title = {Broad-acting antivirals: the pursuit of pan-viral therapeutics in the era of pandemics.},
journal = {Journal of virology},
volume = {},
number = {},
pages = {e0007726},
doi = {10.1128/jvi.00077-26},
pmid = {41870078},
issn = {1098-5514},
abstract = {The ever-present threat of new viral epidemics makes the scientific community relentlessly work on the development of universal methods of antiviral therapy. The development of broad-spectrum antivirals (BSAs) focuses either on substances acting directly on viral proteins (direct-acting antivirals [DAA]) or on substances directed at the cell's own proteins (host-targeting antivirals [HTA]). Decades of development have led to the market entry of a number of DAAs with a wide range of antiviral activities; however, their clinical approval has been obtained for individual infections. HTAs have a number of advantages over DAAs, such as a wider range of antiviral activities and a high genetic barrier to viral resistance, which is undoubtedly important when preparing for a battle with an unknown pathogen. The COVID-19 pandemic has allowed for multiple clinical trials for repurposed HTAs, previously licensed for the treatment of other diseases, including cancer. Despite the enormous work done, the arsenal of BSAs capable of protecting against future pandemics caused by pathogen X is very limited. In this review, we described data on the most studied DAAs and HTAs, effective against at least two unrelated viral pathogens, focusing on those that have been studied in late preclinical and clinical trials. In the end, we highlighted alternative new approaches such as CRISPR-Cas therapy.},
}
@article {pmid41870471,
year = {2026},
author = {Zhao, X and Wang, Y and Wang, L and Liao, S and Gong, T and Xiong, M and Yu, B and Song, ZL},
title = {Direct RNA Triggering of Cas12a through the Native crRNA Architecture Enables Clinical Nucleic Acids Diagnostics.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9982-9992},
doi = {10.1021/acs.analchem.5c08220},
pmid = {41870471},
issn = {1520-6882},
mesh = {Humans ; Manganese/chemistry/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry ; *RNA/metabolism/chemistry ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism/chemistry/genetics ; *MicroRNAs/blood/genetics ; Lung Neoplasms/diagnosis/blood ; *Bacterial Proteins/metabolism/chemistry ; },
abstract = {CRISPR/Cas12a has emerged as a powerful platform for nucleic acid diagnostics, yet its activity is widely considered to be restricted to DNA targets, limiting its applicability for direct RNA detection. Here we report a manganese-ion (Mn[2+])-empowered Cas12a (MEC) platform that overcomes this constraint by allowing the robust RNA-mediated activation of Cas12a. Structural analyses reveal that Mn[2+] strengthens RNA engagement and reorganizes the catalytic center by coordinating RNA phosphates, resulting in an enhancement of trans-cleavage efficiency by 60-fold relative to the Mg[2+] conditions, without compromising sequence specificity. This Mn[2+]-dependent activation mechanism is conserved across multiple Cas12a orthologues (LbCas12a, AsCas12a, FnCas12a), permitting amplification-free detection of RNA with femtomolar sensitivity across diverse targets, particularly the ultrashort abortive transcripts (7 nt). Analysis of clinical serum samples further demonstrates that MEC quantitatively measures circulating miR-21 with performance concordant with reference clinical assays and effectively distinguishes lung cancer patients from healthy individuals. These results reveal an unrecognized role for Mn[2+] in Cas12a biochemistry and establish a simple, versatile, and highly sensitive framework for RNA diagnostics.},
}
@article {pmid41870756,
year = {2026},
author = {Gowtham, K and Shanmugaraj, B and Thangavel, LS and Srinivasan, A and Malla, A},
title = {Advancing the frontier of plant-based therapeutics: critical innovations in molecular farming and bioprocess Integration.},
journal = {Biotechnology letters},
volume = {48},
number = {2},
pages = {},
pmid = {41870756},
issn = {1573-6776},
support = {PSGCAS/IRSG/2024-2025/Biotechnology/004//PSG/ ; },
mesh = {*Molecular Farming/methods ; Gene Editing ; CRISPR-Cas Systems ; *Plants, Genetically Modified/genetics/metabolism ; *Plants/genetics/metabolism ; Synthetic Biology ; Biotechnology/methods ; },
abstract = {Plant molecular farming (PMF) has emerged as a promising strategy for producing biopharmaceuticals and high-value biomolecules in plant systems. In this review, we present a comprehensive synthesis of current methodologies while introducing novel approaches to genetic transformation, protein expression, glycan engineering, and downstream processing. We offer in-depth analyses of recent advancements such as CRISPR/Cas9-mediated pathway editing, synthetic biology frameworks for optimizing protein yield and quality, and integrated bioprocessing solutions that enhance purification efficiency. Further, detailed case studies are discussed to illustrate actionable strategies, and future research directions are proposed to bridge current gaps. By focusing on transformative techniques and critical problem-solving perspectives, this review aims to guide researchers toward more effective and scalable PMF applications.},
}
@article {pmid41871065,
year = {2026},
author = {Huang, Y and Liang, W and Huang, M and Deng, Y and Huang, Z and Ai, C and Tan, W and Jiang, L},
title = {Application of CRISPR/Cas13a system on the rapid detection of Salmonella spp.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {3},
pages = {e0014150},
pmid = {41871065},
issn = {1935-2735},
mesh = {Humans ; *CRISPR-Cas Systems ; *Salmonella/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *Salmonella Infections/diagnosis/microbiology ; Prospective Studies ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Molecular Diagnostic Techniques/methods ; Diarrhea/microbiology/diagnosis ; Female ; Male ; Adult ; Nucleic Acid Amplification Techniques/methods ; Middle Aged ; },
abstract = {BACKGROUND: Salmonella spp. infections can manifest in various clinical symptoms, from asymptomatic carriage to gastroenteritis, and even severe sepsis. Given the rapid progression of the disease and its potential to cause severe outcomes or trigger cluster outbreaks, making the detection of Salmonella spp. critically important. Although broth enrichment culture is considered the gold standard, it is time-consuming and involves multiple steps, making it difficult to meet urgent diagnostic needs. Hence, prompt and precise detection of Salmonella spp. is crucial not only for early diagnosis and effective treatment, but also for preventing transmission, controlling outbreaks, and screening asymptomatic Salmonella carrier.<br><br>METHODS: This study developed a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated (Cas) -SE assay that integrated the advantages of the recombinase polymerase amplification (RPA) and CRISPR/Cas13a system for detecting Salmonella spp. The clinical performance of CRISPR/Cas13a-SE assay was evaluated by a cohort of 94 inpatients with diarrhea and three prospective studies.<br><br>RESULTS: The CRISPR/Cas13a-SE assay can be completed within 60 minutes, and its limit of detection was 100 fg/&#x3bc;L. Compared to the broth enrichment culture, the CRISPR/Cas13a-SE assay demonstrated a sensitivity of 87.5% and a specificity of 98.8% in a cohort of 94 inpatients with diarrhea. In our prospective studies involved three distinct cohorts: 1,662 food handlers, 211 outpatients with diarrhea, and 154 inpatients with Gram-negative bacteremia. Compared with broth enrichment culture, CRISPR/Cas13a-SE assay had a high concordance rate of 98.79% (1,642/1,662), 99.52% (210/211), and 100.00% (154/154) respectively.<br><br>CONCLUSIONS: We demonstrated that the CRISPR/Cas13a-SE system showed excellent detection performance for infectious diarrhea caused by Salmonella spp. The combined use of CRISPR/Cas13a-SE with the blood culture method enhances the rapid diagnosis of invasive salmonellosis, which is crucial for early target-based therapy. Additionally, screening of asymptomatic Salmonella carrier will be benefit for disease prevention and control.},
}
@article {pmid41871234,
year = {2026},
author = {Liao, H and Xie, H and Ye, H and Liu, X and Chen, Y and Zhong, R and He, S and Xiao, X and Xie, Z and Shao, Z and Yu, L and Chen, Z},
title = {One-Pot CRISPR/Cas12a Assay Based on Ultrashort HDA for Ultrasensitive and Universal Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {10004-10014},
doi = {10.1021/acs.analchem.5c08249},
pmid = {41871234},
issn = {1520-6882},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Influenza A virus/genetics/isolation &amp; purification ; *Nucleic Acids/analysis ; Staphylococcal Infections/diagnosis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Isothermal amplification techniques, such as helicase-dependent amplification (HDA) combined with CRISPR, are cutting-edge approaches for nucleic acid detection. In this work, we developed a novel ultrashort mesophilic HDA (termed usHDA) for rapid, highly sensitive nucleic acid amplification at 37 °C and constructed a one-pot usHDA-CRISPR/Cas12 assay. The usHDA is specifically designed for rapid amplification of ultrashort sequences (about 40 nt) at 37 °C within 30 min. This usHDA-CRISPR/Cas12a detection can be completed within 1 h, achieving a limit of detection (LOD) of 5 aM. When tested on 58 clinical specimens from patients infected with respiratory pathogens, this assay identified 41 positive and 17 negative samples for influenza A virus. This assay achieved 100% sensitivity, 100% specificity, and a perfect receiver operating characteristic curve (area under the curve value = 1.00; n = 58) compared with PCR analysis. Furthermore, 24 samples of Staphylococcus infection were detected using usHDA-CRISPR/Cas12a, and the same 100% sensitivity and specificity were achieved. These findings highlighted the strong applicability of our proposed assay for universal nucleic acid detection.},
}
@article {pmid41872458,
year = {2026},
author = {Lkhagvadorj, K and Okamura, E and Taki, T and Suzuki, H and Kuno, A and Itoh, Y and Mizuno, S and Woltjen, K and Ema, M},
title = {Optimizing CRISPR precision in mouse embryos via microhomology-mediated end joining-dominant targeting.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {},
pmid = {41872458},
issn = {2399-3642},
support = {25K02195//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; 24K18045//Ministry of Education, Culture, Sports, Science and Technology (MEXT)/ ; JP223fa627008//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {Animals ; Mice ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *DNA End-Joining Repair ; RNA, Guide, CRISPR-Cas Systems/genetics ; Mouse Embryonic Stem Cells/metabolism ; *Embryo, Mammalian/metabolism ; Female ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Fibroblast Growth Factor 10/genetics ; },
abstract = {CRISPR/Cas9 technology enables efficient gene editing in mice, but its reliance on non-homologous end joining often leads to unpredictable and mosaic mutations in founder (F0) animals. Here, we present a hybrid genome editing strategy that combines in silico prediction software with in vitro validation using mouse embryonic stem cells (mESCs). Although the software was trained on mESC datasets, actual editing outcomes in mESCs more accurately reflected mutation patterns observed in blastocysts and post-implantation embryos. Using this information to develop an integrated pipeline, we pre-selected guide RNAs (gRNAs) predicted to promote microhomology-mediated end joining (MMEJ)-dominant repair and validated them in mESCs prior to embryo injection. Applied to the Tyr and Fgf10 genes, this approach enabled efficient generation of F0 mice with highly uniform genotypes. Our strategy enhances the predictability and reproducibility of CRISPR-based genome editing in mice and may help reduce animal usage in gene editing studies.},
}
@article {pmid41872465,
year = {2026},
author = {Ahmed, MF and Sarkar, MMH and Mehzabin, K and Hossain, MI and Bhim, M and Chowdhury, SF and Naser, SR and Mumtaz, T and Faruk, MO},
title = {Genomic convergence of multidrug resistance, virulence-associated loci, and phage defense systems in Klebsiella pneumoniae from pharmaceutical wastewater in Bangladesh.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-45102-2},
pmid = {41872465},
issn = {2045-2322},
abstract = {Klebsiella pneumoniae strains that combine multidrug resistance and enhanced virulence pose a growing global public health threat. Understanding the genetic basis of these high-risk lineages is critical for surveillance and mitigation. We isolated K. pneumoniae JU-BAEC-01 from treated effluent of antibiotic-manufacturing pharmaceutical facilities in Bangladesh and performed whole-genome sequencing with comparative genomic analyses to characterize its phylogeny, resistome, virulence-associated loci, mobile genetic elements, and predicted antiviral defense systems. JU-BAEC-01 belongs to a phylogenetically distinct lineage, serotype O3b: KL150 with resistance to nearly all clinically relevant antibiotic classes except carbapenems and colistin, mediated by an extensive acquired resistome, including tmexCD3-toprJ3 (tigecycline), armA, aac(6')-Ib-cr, qnrB4, oqxAB, blaDHA-1, blaSHV-182, and blaTEM-1B, mostly carried on conjugative IncC, IncFIB, IncHI1B, and IncR plasmids. Classical hypervirulence markers are present: complete aerobactin (iucABCD-iutA) and salmochelin (iroBCDEN) clusters, rmpA2, type 1 and type 3 fimbriae, T6SS, and pgaABCD. Notably, the strain encodes one of the most elaborate anti-phage defense arsenals reported in Klebsiella to date, comprising functional Type I-E, III-A, and IV-A CRISPR-Cas systems, multiple restriction-modification systems, BREX Type I, abortive infection systems (AbiE, AbiU), and additional novel defenses that coexist with phage-derived anti-CRISPR (AcrIE9) and anti-restriction (ArdA) proteins. K. pneumoniae JU-BAEC-01 is a "perfect storm" pathogen that combines multi-drug resistance (MDR), hypervirulence, and a multilayered, highly developed defense against bacteriophages. Together, these findings highlight the environmental emergence of a genetically distinct, multidrug-resistant K. pneumoniae with substantial virulence potential and complex phage-host interaction capacity, underscoring the need for genomic surveillance of pharmaceutical wastewater systems.},
}
@article {pmid41873079,
year = {2026},
author = {Luan, M and Liu, S and Yang, Q and Zhang, Y and Wang, M and Liu, R and Niu, G},
title = {[Metabolic engineering of Streptomyces for production of hyaluronic acid].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {42},
number = {3},
pages = {1242-1260},
doi = {10.13345/j.cjb.250734},
pmid = {41873079},
issn = {1872-2075},
support = {2023YFD1700700//the National Key Research and Development Program of China/ ; },
mesh = {*Metabolic Engineering/methods ; *Streptomyces/metabolism/genetics ; *Hyaluronic Acid/biosynthesis ; Streptomyces coelicolor/metabolism/genetics ; Hyaluronan Synthases ; },
abstract = {Hyaluronic acid (HA) is a glycosaminoglycan with significant biological activities, which render it widely applicable in the cosmetics and pharmaceutical industries. The development of safe and efficient chassis cells to enhance HA synthesis efficiency has thus emerged as a key factor in HA production. Our study aims to construct a high-performance HA biosynthesis system using Streptomyces as the chassis cell, thereby providing technical support for the efficient microbial production of HA. Thus, our study focused on the metabolic engineering of Streptomyces for strengthening the HA synthesis pathway and then optimized the culture conditions for efficient HA synthesis. First, the HA-synthesizing capabilities of four hyaluronate synthases from different sources were evaluated in two host strains: Streptomyces coelicolor M1146 and Streptomyces albus J1074. The results indicated that the hyaluronate synthases derived from Streptococcus pyogenes exhibited the strongest HA synthesis capability. Notably, the HA yield in S. albus J1074 was higher. Building on this finding, S. albus J1074 was selected as the starting strain to construct a chassis strain tailored for HA synthesis: key genes in the competitive metabolic pathway of HA synthesis were knocked out, while the expression levels of genes associated with the bypass pathway were down-regulated. Furthermore, different combinations of key genes involved in the HA precursor synthesis pathway were designed, and their expression levels were enhanced via a constitutive strong promoter. The recombinant strain obtained therefrom achieved a maximum HA yield of 2.62 g/L. Finally, the synthetic capacity of this high-yield engineered strain was further unleased through the optimization of culture conditions, leading to a final HA yield of 4.63 g/L. The recombinant strain constructed in this study not only lays a foundation for the development of engineered Streptomyces but also provides an excellent chassis strain for the microbial production of HA and other related bioproducts.},
}
@article {pmid41873591,
year = {2026},
author = {Jürgens, K and Menzel, L and Klinke, N and Schäper, L and Ratnavadivel, S and Walter, S and Milting, H and Meyer, H and Paululat, A},
title = {An ARVC-5 Drosophila knock-in model reveals new functions of Tmem43 in lipid homeostasis.},
journal = {Biology open},
volume = {15},
number = {4},
pages = {},
doi = {10.1242/bio.062326},
pmid = {41873591},
issn = {2046-6390},
support = {PA517/16-1//DFG/ ; MI1146/6-1//DFG/ ; MI1146/6-1::PA517/16-1//Deutsche Forschungsgemeinschaft/ ; //Osnabr&#xfc;ck University/ ; },
mesh = {Animals ; *Lipid Metabolism/genetics ; *Homeostasis ; Disease Models, Animal ; Gene Knock-In Techniques ; Mitochondria/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; Energy Metabolism ; Lipidomics ; Mutation ; Proteomics ; CRISPR-Cas Systems ; },
abstract = {Arrhythmogenic right ventricular cardiomyopathy type 5 is caused by the missense mutation S358L in the gene TMEM43 in humans. To date, the molecular mechanisms underlying the disease remain poorly understood. We established a CRISPR/Cas9 knock-in Drosophila model carrying the orthologous Tmem43p.S333L mutation to investigate these mechanisms in vivo. The resulting flies were viable but displayed reduced lifespan, smaller body size, lipid droplet accumulation, and mitochondrial defects. Proteomic and lipidomic profiling revealed a dosage-dependent misregulation of the energy metabolism, concomitant with reduced fatty acid synthesis and ß-oxidation rates, altered peroxisomal pathways, and changes in membrane phospholipid composition. Notably, phosphatidylethanolamine (PE) and phosphatidylinositol (PI) levels were elevated, while triacylglycerols were reduced. Ultrastructural analyses confirmed mitochondrial degradation in the muscle tissue of corresponding mutants. These findings establish Tmem43p.S333L knock-in flies as a robust in vivo model of ARVC-5 and support a role for TMEM43 in linking lipid homeostasis to mitochondrial energy metabolism and integrity. Mutation-derived impairments in these processes result in cardiomyopathy.},
}
@article {pmid41873757,
year = {2026},
author = {Yu, Z and Bao, Y and Alcalá-Lalinde, A and Andrade Dos Ramos, Z and Koroglu, M and Berkhout, B and Herrera-Carrillo, E},
title = {Elucidating the kinetics of CRISPR-SaCas9 action to obtain effective HIV DNA excision with two gRNAs.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41873757},
issn = {1362-4962},
support = {1R01AI145045IH//NIH RO1/ ; DGP_EMEC_2023_00154//Junta de Andaluc&#xed;a/ ; PID2024-162385OB-I00//Spanish State Research Agency/ ; },
mesh = {*HIV-1/genetics ; *CRISPR-Cas Systems ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Gene Editing/methods ; Kinetics ; *DNA, Viral/genetics/metabolism ; Proviruses/genetics ; *CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; Genome, Viral ; HIV Infections/virology ; },
abstract = {The persistence of integrated human immunodeficiency virus (HIV) proviral DNA poses a major barrier to viral eradication, as the viral reservoir enables rapid rebound upon treatment interruption, despite effective virus inhibition. CRISPR-Cas-based editing strategies, especially those using double-site cleavage, show promise in excising proviral DNA, yet the rate and determinants of excision efficiency remain poorly understood. In this study, we systematically evaluated both single- and dual-SaCas9/gRNA approaches for HIV-1 inactivation. Sequence analysis revealed that SaCas9 can eliminate all wild-type HIV-1 genomes with a single gRNA, unlike other CRISPR-Cas systems. Dual-gRNA strategies improved antiviral efficacy, with the Gag3 + Pol5 combination achieving 97% excision efficiency. Kinetic analysis showed that excision efficiency correlates with the kinetic compatibility of paired gRNAs. Pairs of gRNAs with fast and similar kinetics achieved the highest excision efficiency. In contrast, the Gag3 + Env4 pair exhibited discordant kinetic characteristics (fast and slow), resulting in the failure to induce excision as the cut DNA will be repaired before the second cut is realized. Consequently, no excision but regular editing occurred at the two target sites. These findings provide a mechanistic framework for optimizing CRISPR-Cas-mediated excision, highlighting the critical role of both antiviral activity and kinetic synergy in guiding gRNA selection.},
}
@article {pmid41873844,
year = {2026},
author = {Wang, Q and Sheng, M and Zheng, Y and Zhang, B and Jin, Z and Zhang, T and Li, Z and Huang, J and Yang, X},
title = {Electrochemiluminescence Biosensing Platform Based on CRISPR/Cas12a and DNA Nanotweezer-Mediated Catalytic Hairpin Assembly Amplification.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9832-9841},
doi = {10.1021/acs.analchem.5c07500},
pmid = {41873844},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; Humans ; *DNA/chemistry/genetics ; Catalysis ; Nucleic Acid Amplification Techniques ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The detection of microRNAs (miRNAs) biomarkers has great potential in the early diagnosis of acute myocardial infarction (AMI). Herein, we constructed an electrochemiluminescence biosensing platform based on DNA nanotweezer (DNT)-mediated catalytic hairpin assembly (CHA) and CRISPR/Cas12a system for detecting potential AMI biomarker miRNA-133a. DNT, as a programmable molecular scaffold, can precisely organize molecules at the nanoscale and output high signal-to-background ratio detection signals, which is introduced into the construction of sensing platforms. When the target miRNA was presented, the hairpin in DNT was opened, which altered the DNT structure from a closed state to an open state and exposed the catalytic sequence for CHA. Subsequently, a large number of F/A-F duplexes were generated after the addition of fuel strands (F) and antifuel strands (A-F), which served as the target for activating the CRISPR/Cas12a system. The activated Cas12a collaterally cleaved the signal probe (H1) on the electrode surface, causing the labeled Ru(bpy)3[2+] to detach from the electrode surface, resulting in a weakened ECL signal. We found that compared with the general CHA reaction, the DNT-mediated CHA reaction significantly lowers the leakage of the circuit; thus, a high signal-to-background ratio and detection sensitivity can be obtained. Therefore, we developed a highly sensitive biosensing platform for detecting miRNA-133a with a detection limit of 0.12 fM. This sensing strategy provides a new approach for nucleic acid detection and disease diagnosis.},
}
@article {pmid41874403,
year = {2026},
author = {Zhang, H and Liu, L and Wang, D and Yang, X and Kang, Y and Huang, J and Ouyang, Y and Yu, H and Zhang, Y},
title = {CRISPR-Cas gene editing technology in biomanufacturing to enhance stress tolerance of microbial strains.},
journal = {FEMS microbiology letters},
volume = {373},
number = {},
pages = {},
doi = {10.1093/femsle/fnag030},
pmid = {41874403},
issn = {1574-6968},
support = {2023M742776//China Postdoctoral Science Foundation/ ; 2023BSHEDZZ12//Postdoctoral Research Program of Shaanxi/ ; GZB20240598//Postdoctoral Fellowship Program of CPSF/ ; 2025JC-YBQN-293//Natural Science Basis Research Plan in Shaanxi Province of China/ ; S202510698576//National Training Program of Innovation and Entrepreneurship for Undergraduates/ ; 202406280137//CSC/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Escherichia coli/genetics/metabolism ; *Stress, Physiological/genetics ; Bacillus subtilis/genetics ; Corynebacterium glutamicum/genetics/metabolism ; Industrial Microbiology/methods ; },
abstract = {In response to the loss of microbial efficiency caused by environmental stress in biomanufacturing, CRISPR-Cas gene editing technology has become a core tool for enhancing stress tolerance by accurately targeting genomic loci. This article systematically reviews the progress of its application. By optimizing engineered nucleases, gRNA design, and innovative delivery strategies, this technology successfully regulates key pathways in oxidative stress responses. It integrates functional genome screening with dynamic regulation to examine the networks of multi-gene collaborative tolerance. In the construction of high-stress-tolerant industrial chassis cells, the stress survival rate (>90% in Bacillus subtilis under thermal stress) and product synthesis ability (such as cellulose producing ethanol up to 4.5 g/l) of strains such as Escherichia coli and Corynebacterium glutamicum were significantly improved. Current challenges focus on delivery efficiency, off-target risks, and complex regulatory bottlenecks. In the future, the development of new editing tools and intelligent circuits will promote their industrial application in sustainable biomanufacturing.},
}
@article {pmid41875153,
year = {2026},
author = {Magarditchian, G and Berest, I and Ziogou, A and Matsushita, M and Reid, M and Othman, A and Kopf, M},
title = {The phospholipid profile of T cells shapes ACSL4 dependency and ferroptosis sensitivity of naive, effector, and memory T cells.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {13},
pages = {e2528153123},
pmid = {41875153},
issn = {1091-6490},
support = {ETH-30-20-1//ETH grant/ ; },
mesh = {*Ferroptosis/immunology ; *Coenzyme A Ligases/metabolism/genetics ; *Phospholipids/metabolism ; Animals ; Mice ; *CD8-Positive T-Lymphocytes/metabolism/immunology ; *Memory T Cells/metabolism/immunology ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Humans ; Fatty Acids, Unsaturated/metabolism ; Mice, Knockout ; Iron/metabolism ; },
abstract = {Iron-dependent phospholipid (PL) peroxidation, which is reduced by glutathione peroxidase 4, is recognized as the hallmark of cells undergoing ferroptosis. Although studies have attempted to elucidate the molecular mechanisms underlying ferroptosis in cancer cells, the regulation of ferroptosis in effector and memory T cells remains largely unknown. Here, using genome-wide CRISPR-Cas9 knockout screens, we demonstrate that acyl-CoA synthetase long-chain family member 4 (ACSL4) is the predominant ferroptosis inducer in primary T cells cultured in vitro, while other identified iron- and lipid metabolism-related genes only slightly modulate their sensitivity to ferroptosis. However, ACSL4 dependency relies on the PL composition of the cells. In vitro cultured T cells treated with polyunsaturated fatty acids (PUFAs), as well as effector CD8[+] T cells that are enriched in PUFA-containing PLs (PUFA-PLs), undergo ferroptosis in the absence of ACSL4. In contrast to effector T cells, naive and memory T cells share a similar PL profile, characterized by a scarcity of PUFA-PLs, and are resistant to ferroptosis. Overall, the PL composition is a central feature and determines the differential susceptibility of effector and memory T cells to ferroptosis and its molecular mechanism.},
}
@article {pmid41875383,
year = {2026},
author = {Wootan, CM and Lutterman, J and Springer, N and Xu, X and Zhang, F},
title = {DNA repair under heat: DNA polymerase λ modulates heat stress-induced mutagenesis in plants.},
journal = {The Plant cell},
volume = {38},
number = {4},
pages = {},
pmid = {41875383},
issn = {1532-298X},
support = {//University of Minnesota Multifunctional Agriculture Initiative/ ; IOS-2040218//National Science Foundation/ ; IOS-2206920//National Science Foundation/ ; //University of California, Davis/ ; CA-D-PLB-2850-H//California Agricultural Experiment Station/National Institute of Food and Agriculture/ ; },
mesh = {*DNA Repair/genetics ; *Heat-Shock Response/genetics ; *Mutagenesis/genetics ; *Arabidopsis/genetics/physiology ; *DNA Polymerase beta/metabolism/genetics ; Hot Temperature ; Gene Expression Regulation, Plant ; *Arabidopsis Proteins/metabolism/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Mutation rates often rise under environmental stress, a process known as stress-induced mutagenesis. Among abiotic factors, heat stress is a potent driver that elevates mutation rates and enhances genetic variation, yet the underlying mechanisms in eukaryotes remain unclear. Here, we investigated how heat stress influences DNA repair and mutagenesis both locally and globally in Arabidopsis thaliana using CRISPR-Cas9-induced DNA breaks, whole genome sequencing, and single-cell transcriptomic analysis. Heat stress greatly enhanced CRISPR editing efficiency across different chromatin contexts, reaching up to a 29.9-fold increase in heterochromatic regions. Moreover, heat stress consistently shifted repair outcomes toward one base pair insertions, independent of chromatin state. We identified a heat-inducible, error-prone DNA polymerase, DNA polymerase λ (DNA Polλ), as the key mediator of these repair profile changes. Genome-wide analyses of somatic mutations further revealed that the heat-induced increase in mutagenesis also depends on DNA Polλ. Single-cell transcriptomic profiling showed that DNA Polλ expression is tightly regulated and enriched in the central zone of the shoot apical meristem. Such tissue-specific patterns suggest that DNA Polλ serves dual functions: maintaining genome integrity in essential stem cell populations while also enabling stress-induced mutagenesis that can be transmitted to progeny.},
}
@article {pmid41875952,
year = {2026},
author = {Li, C and Zhang, L and Xu, Q},
title = {Extraction-free, rapid LAMP-CRISPR/Cas12a assay for detection of pseudorabies virus.},
journal = {Journal of virological methods},
volume = {343},
number = {},
pages = {115387},
doi = {10.1016/j.jviromet.2026.115387},
pmid = {41875952},
issn = {1879-0984},
mesh = {*Herpesvirus 1, Suid/isolation &amp; purification/genetics ; Animals ; Swine ; *Pseudorabies/diagnosis/virology ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Swine Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; DNA Primers/genetics ; DNA, Viral/genetics ; },
abstract = {This study developed a LAMP-CRISPR/Cas12a detection system for rapid and visual identification of porcine pseudorabies virus (PRV). Optimal sgRNA and LAMP-specific primers were designed based on the conserved sequences of the viral pathogenic gene gG. The combined detection system demonstrated superior sensitivity compared to PCR-CRISPR/Cas12a and qPCR methods, achieving a detection limit of 1.0 × 10[-4] copies/μL for the target plasmid DNA. Specificity testing confirmed the selective identification of PRV without cross-reactivity to other porcine pathogens. Parallel comparison of 26 serum samples between LAMP-CRISPR/Cas12a and PCR-CRISPR/Cas12a systems showed 100% concordance for positive results, with both detecting 12 positive samples. The method eliminates the need for viral nucleic acid extraction and requires only a constant temperature device and/or basic fluorescence detection equipment. Results are obtainable within one hour and are readable by the naked eye. This simple, sensitive, and equipment-independent approach is ideal for on-site rapid diagnosis of pseudorabies in pigs, offering significant applications in clinical diagnosis, epidemiological surveillance, and field testing.},
}
@article {pmid41876528,
year = {2026},
author = {Lin, YH and Kompa, J and Sun, DE and Mao, R and Koch, B and Hinnah, K and Wilhelm, J and Franz, N and Kühn, S and Menche, T and Adow, A and Breuer, P and Hiblot, J and Johnsson, K},
title = {A high-affinity split-HaloTag for live-cell protein labeling.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41876528},
issn = {2041-1723},
mesh = {Humans ; Microscopy, Fluorescence/methods ; *Staining and Labeling/methods ; Fluorescent Dyes/chemistry ; HEK293 Cells ; CRISPR-Cas Systems ; Peptides/chemistry/metabolism ; *Proteins/metabolism ; },
abstract = {We introduce a high-affinity split-HaloTag comprised of a short peptide tag (Hpep, 14 residues) and a large, inactive fragment (cpHaloΔ3). Hpep binds to cpHaloΔ3 spontaneously with nanomolar affinity, enabling subsequent labeling with fluorescent HaloTag ligands. The small size of Hpep facilitates cloning-free endogenous protein tagging using CRISPR/Cas9 and the complementation of Hpep-tagged proteins can be achieved in live cells through co-expression with cpHaloΔ3 and in fixed cells through incubation with cpHaloΔ3. The approach is compatible with advanced microscopy techniques such as expansion microscopy and live-cell STED imaging. Additionally, variants of Hpep that modulate the spectral properties of labeled fluorophores enable simultaneous imaging of two different Hpep-tagged proteins via fluorescence lifetime microscopy. In summary, our high-affinity split-HaloTag is a robust and versatile tool for live-cell imaging and diverse applications in chemical biology.},
}
@article {pmid41876645,
year = {2026},
author = {Ren, F and Liu, D and Ren, H and Zhang, H and Zhang, P and Lin, H and Li, C and Dong, J and An, S and Ge, X and Cheng, L and Yang, F and Liu, J and Fu, J and Tang, F and Wang, F and Liu, T and Pan, H and Rong, S and Ma, H and Zou, L},
title = {Synergistic integration of CRISPR/Cas and nanozymes in next-generation biosensors for ultrasensitive bacterial detection.},
journal = {Mikrochimica acta},
volume = {193},
number = {4},
pages = {},
pmid = {41876645},
issn = {1436-5073},
support = {82574099//National Natural Science Foundation of China/ ; LH2023H054//Natural Science Foundation of Heilongjiang Province/ ; 2022-MYHJ-014//Foundation for Huoju Plan Research of Mudanjiang Medical University/ ; YJSZX2022137//Foundation for Special Program of Supervisor Scientific Research of Mudanjiang Medical University/ ; 2024-KYYWFMY-0455//The Fundamental Research Funds for the Universities of Heilongjiang Province/ ; CYQN24028//Youth the Science and Technology Talents Team Project of Chunyan Plan of Heilongjiang Province/ ; },
}
@article {pmid41876887,
year = {2026},
author = {Xu, R and Cong, T and Yuan, J and Chen, X and Li, Y and Lan, X and Zhu, M},
title = {Tracking-seq: a universal off-target detection approach for CRISPR-Cas genome editing.},
journal = {Nature protocols},
volume = {},
number = {},
pages = {},
pmid = {41876887},
issn = {1750-2799},
abstract = {Tracking-seq is a highly sensitive method for genome-wide detection of off-target effects in cells edited with diverse genome editing modalities, including Cas9, cytosine base editors, adenine base editors and prime editors. Since most genome editors induce DNA repair pathways and generate single-stranded DNA (ssDNA) intermediates, Tracking-seq leverages this process by tracking replication protein A-a key protein that binds and protects ssDNA-to identify on-target and off-target events. Here we provide a detailed protocol for Tracking-seq, covering genome editing of cells, extraction of replication protein A-bound ssDNA, sequencing library construction and data analysis using our custom computational tool Offtracker. Tracking-seq is applicable to various genome editing scenarios with low cell input, delivering high-performance results. The entire workflow, from genome editing to data analysis, can be completed within 1-2 weeks, making it a rapid solution for assessing genome-wide off-target activity.},
}
@article {pmid41877594,
year = {2026},
author = {Cucuy, A and Ben-Tov, D and Melamed-Bessudo, C and Honig, A and Cohen, BA and Levy, AA},
title = {Features affecting Cas9-induced editing efficiency and patterns in tomato: evidence from a large CRISPR dataset.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {6},
pages = {e70809},
pmid = {41877594},
issn = {1365-313X},
support = {//Israel Innovation Authority/ ; },
mesh = {*Solanum lycopersicum/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; Promoter Regions, Genetic/genetics ; DNA Breaks, Double-Stranded ; CRISPR-Associated Protein 9 ; DNA Repair ; Introns/genetics ; Exons/genetics ; },
abstract = {CRISPR/Cas9 is a cornerstone of plant genome editing, yet the determinants of editing efficiency for a given single-guide RNAs (sgRNAs) and DNA double-strand break (DSB) repair outcomes remain poorly understood, particularly in plants. Here, we generated a large experimental dataset comprising 420 sgRNAs targeting promoters, exons, and introns of 137 genes in tomato protoplasts, and quantified editing efficiency and repair footprints together with chromatin accessibility and transcriptional state in the same cellular context. Editing efficiency was consistently higher at targets in accessible chromatin and modestly higher in promoters and introns than in exons, whereas transcriptional activity had no detectable effect. Editing efficiencies were more similar among sgRNAs targeting the same gene than among different genes, revealing a local genomic influence on Cas9 activity. A distinct subset of sgRNAs achieved near-complete editing and produced characteristic repair footprints dominated by long deletions with extended microhomology tracts, indicative of microhomology-mediated end joining (MMEJ), resembling patterns associated with high-efficiency guides in human cells, and suggesting conserved sequence-driven repair biases across species. In contrast, widely used human-trained prediction models failed to accurately rank sgRNA performance in plants, highlighting the limits of cross-species predictability. Together, this dataset provides a resource for improving guide design and mechanistic understanding of plant DNA repair.},
}
@article {pmid41877893,
year = {2025},
author = {Srinivasa, MA and Escobar, M},
title = {CRISPR-based Transcriptional Regulation: Technologies, Applications, and Future Directions.},
journal = {DNA},
volume = {5},
number = {4},
pages = {57},
pmid = {41877893},
issn = {2673-8856},
support = {25TPA1463933/AHA/American Heart Association-American Stroke Association/United States ; },
abstract = {CRISPR-based transcriptional regulation technologies, including CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi), offer precise and programmable control over gene expression, representing a major advance in gene and epigenetic therapy. CRISPRa uses nuclease-inactive Cas proteins fused to transcriptional activators to upregulate target genes, while CRISPRi employs repressor domains for gene silencing. Preclinical studies have demonstrated the efficacy of CRISPRa/i in models of metabolic, neurological, muscular, and oncological diseases. Notably, CRISPRi-based therapies have entered clinical trials for conditions like hepatitis B and muscular dystrophy, showing encouraging safety and efficacy profiles. Despite ongoing challenges related to delivery efficiency, immunogenicity, and off-target activity, innovations in protein engineering and guide RNA design are rapidly enhancing the precision and safety of these technologies. Overall, CRISPRa and CRISPRi are poised to transform the treatment of genetic and epigenetic disorders, with continued optimization expected to accelerate their clinical adoption and broaden their therapeutic impact.},
}
@article {pmid41879319,
year = {2026},
author = {Cheng, W and Li, J and Lei, L and Zhu, Y and Luo, S and Wang, X and Zhang, Q and Cao, M and Zheng, Y and Peng, W},
title = {Unlocking genome engineering in Alcaligenes faecalis by exploiting its native type I-F CRISPR-Cas.},
journal = {Microbiology spectrum},
volume = {},
number = {},
pages = {e0278625},
doi = {10.1128/spectrum.02786-25},
pmid = {41879319},
issn = {2165-0497},
abstract = {Alcaligenes faecalis is an environmentally significant bacterium for pollutant biodegradation and aerobic denitrification, yet its genetic engineering has been hindered by a lack of high-throughput tools. Conventional methods like homologous recombination are time-consuming and cannot achieve large genomic deletions, while technologies based on heterologous CRISPR-Cas systems failed due to cytotoxicity. This study resolves these limitations by developing a genome editing toolkit based on the endogenous type I-F CRISPR-Cas of A. faecalis J481. The toolkit enables efficient single-gene knockout and accomplishes the previously unattainable precise deletion of large genomic fragments. By engineering a PheS-mutant counterselection marker, we achieved rapid plasmid curing, allowing two rounds of large-fragment removal (~47 kb total) within 5 days. This breakthrough provides the first CRISPR-based platform for complex genome engineering in A. faecalis, overcoming intrinsic constraints of heterologous systems. The work establishes a scalable genetic toolbox to enhance A. faecalis' capabilities in bioremediation and eutrophication control. Moreover, the strategy of harnessing endogenous CRISPR-Cas systems offers a blueprint for developing advanced genome editing tools in other prokaryotes.IMPORTANCEThis study breaks through the longstanding genetic engineering bottleneck in an environmentally crucial bacterium, Alcaligenes faecalis, by creating a fast, efficient, and versatile toolkit using its native CRISPR-Cas system. This enables complex edits, such as large genomic deletions previously impossible, unlocking new potential for bioremediation and eutrophication control, providing a blueprint for other prokaryotes, and setting a precedent for genetic tool development in other hard-to-engineer microbes.},
}
@article {pmid41880574,
year = {2026},
author = {Katsumura, T and Sato, S and Yamashita, K and Oda, S and Gakuhari, T and Tanaka, S and Fujitani, K and Nishimaki, T and Imai, T and Yoshiura, Y and Takeshima, H and Hashiguchi, Y and Sekita, Y and Mitani, H and Ogawa, M and Takeuchi, H and Oota, H},
title = {DNA methylation site loss for plasticity-led novel trait genetic fixation.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {13},
pages = {e2534817123},
pmid = {41880574},
issn = {1091-6490},
support = {JP16K21352//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP19K16201//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP19H05737//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP24K02078//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP17H01453//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP17H03738//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP16J07227//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; },
mesh = {Animals ; *Oryzias/genetics ; *DNA Methylation/genetics ; Epigenesis, Genetic ; CpG Islands/genetics ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {Phenotypic plasticity allows organisms to adapt traits in response to environmental changes, yet the molecular basis by which such plastic traits become genetically fixed remains unclear. Here, we investigated gut-length plasticity in medaka fish (Oryzias latipes) through genome-wide methylation profiling, CRISPR/Cas9-mediated deletion, and population genomic analyses. We found that seasonal methylation of CpG sites upstream of the Plxnb3 is correlated with gut-length plasticity, and deletion of this region abolishes plasticity. Additionally, standing variation in Ppp3r1 is associated with genetically fixed longer gut length in populations lacking plasticity. These results suggest that loss of epigenetic regulation via CpG site reduction triggers the genetic fixation of novel traits. Our findings provide molecular evidence linking epigenetic plasticity and genetic assimilation, advancing understanding of plasticity-led evolution in natural populations.},
}
@article {pmid41881766,
year = {2026},
author = {Li, S and Tang, S and Xu, W and Zhou, J and Li, X and Wang, J},
title = {SIMPLE-CRISPR: A Sample-to-Result Platform for Point-of-Care Detection of Nucleic Acids via a Functionalized Magnetic-Bead-Based CRISPR Assay.},
journal = {Analytical chemistry},
volume = {98},
number = {13},
pages = {9964-9971},
doi = {10.1021/acs.analchem.5c08178},
pmid = {41881766},
issn = {1520-6882},
mesh = {*Point-of-Care Systems ; *CRISPR-Cas Systems/genetics ; Humans ; *Nucleic Acids/analysis/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Smartphone ; *Point-of-Care Testing ; Magnetic Phenomena ; },
abstract = {Clustered regularly interspaced short palindromic repeat (CRISPR) systems show great promise for next-generation molecular diagnostics due to their programmability and specificity. Amplification-free CRISPR detection has a strong potential for point-of-care (POC) testing, and the digital format of the assay naturally improves the sensitivity of amplification-free CRISPR detection. However, integrating sample preparation, reaction implementation, and signal readout into a streamlined and user-friendly POC workflow remains a major technical challenge. We address this by developing SIMPLE-CRISPR (Sample-to-Result Integrated platform with Magnetic-bead extraction, Polydisperse droplets, Low-complexity operation, and Engineered smartphone readout), an amplification-free CRISPR-Cas12a system for POC diagnostics. Our innovation features functionalized magnetic-bead extraction for the efficient enrichment of low-abundance nucleic acid targets from large-volume samples with minimal loss, vortex-driven polydisperse emulsification for facile digital droplet generation, and smartphone-integrated fluorescence imaging for on-site portable signal detection. This integrated approach significantly improves sensitivity compared to bulk CRISPR, removes the need for amplification and complex microfluidics, and provides sample-to-result functionality for POC environments. Clinical validation for human papillomavirus type 18 (HPV18) detection confirmed that the assay achieved diagnostic performance consistent with quantitative real-time polymerase chain reaction (qRT-PCR), demonstrating its great potential for accessible POC nucleic acid diagnostics.},
}
@article {pmid41882914,
year = {2026},
author = {Kambakam, S and Thomas, J and Robbe-Austerman, S and Shanmuganatham, K and Palinski, R},
title = {Rapid identification of African swine fever virus in diagnostic samples using CRISPR-Cas.},
journal = {Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc},
volume = {},
number = {},
pages = {10406387261432985},
pmid = {41882914},
issn = {1943-4936},
abstract = {African swine fever virus (ASFV) is a highly transmissible pathogen affecting swine, causing a devastating disease with high mortality rates in naive populations. Given the likelihood of significant economic impacts associated with an ASF outbreak, considerable resources have been allocated in the United States to safeguard the swine industry against this threat. Ongoing outbreaks of ASF in the Dominican Republic and Haiti further threaten the U.S. swine industry, given their proximity and involvement in movement to and from North America. Although surveillance programs are ongoing, limited point-of-care (POC) tests are available during outbreaks with the sensitivity and specificity standards of laboratory testing (e.g., real-time PCR [rtPCR]). However, the recently developed CRISPR-Cas-based testing systems may offer comparable high-quality results. We sought to develop a low-cost visual detection method for ASFV by employing a recombinase polymerase amplification (RPA)-dependent CRISPR-Cas12a technique that can be utilized in the field as a POC assay. Our CRISPR-Cas12a assay had comparable sensitivity and specificity to rtPCR, both visually and when quantified using a fluorescence reader. In whole blood samples from ASFV-suspect or ASFV-negative cases, our CRISPR assay achieved a sensitivity of 98.3% (10[2] DNA copies) and a specificity of 100%. Test results of our RPA-CRISPR assay can be visualized in as few as 7 min, with peak fluorescence at 40 min (RPA and CRISPR steps). Our results lay the groundwork for a large-scale POC assay assessment for ASFV detection and offer a robust workflow that works with commonly submitted diagnostic samples.},
}
@article {pmid41883584,
year = {2026},
author = {Kobel, L and Van de Venn, L and Schröder, M and Bechter, LV and Huang, D and Abdolazimi, Y and Pertel, T and Gopalakrishnan, S and Corn, JE and Kontarakis, Z},
title = {DisTAL-Seq: A TALEN-specific adaptation of DISCOVER-Seq for off-target profiling.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {2},
pages = {102883},
pmid = {41883584},
issn = {2162-2531},
abstract = {Programmable guided nucleases have revolutionized genome editing and biomedical research, with transformative potential for gene and cell therapy. Although the widespread adoption of the CRISPR-Cas system has provided deep insights into target recognition and specificity, the behavior of clinically relevant tools like transcription activator-like effector nucleases (TALENs) remains poorly characterized in human cells. To address this gap, we implemented DisTAL-Seq, a TALEN-specific adaptation of the DISCOVER-Seq pipeline, which detects MRE11 recruitment to double-strand breaks (DSBs). Based on the DISCOVER-Seq principle, DisTAL-Seq incorporates alignment logic tailored to TALEN-binding properties, including variable RVD specificity, cleavage offset, and dimerization behavior. Using DisTAL-Seq, we identified and validated on- and off-target sites across diverse TALENs and T cell donors. This unbiased approach revealed key features of TALEN activity in human cells, including number of tolerated mismatches to a target site and relative location of the induced DSB. DisTAL-Seq thus extends DISCOVER-Seq to the TALEN family and provides a robust platform for assessing modifications in enzyme architecture and application contexts on a genome-wide scale, supporting the development of safer and more effective genome editing tools.},
}
@article {pmid41885168,
year = {2026},
author = {Hanzelova, P and Baird, C and Keshinro, B and Kadhom, R and Lalonde, RL and Akimenko, MA},
title = {An essential role for actinotrichia in zebrafish fin patterning and courtship behavior.},
journal = {Development (Cambridge, England)},
volume = {153},
number = {16},
pages = {},
doi = {10.1242/dev.204990},
pmid = {41885168},
issn = {1477-9129},
support = {RGPIN-2024-06801//Natural Sciences and Engineering Research Council of Canada/ ; PJT 166139/CAPMC/CIHR/Canada ; PJT 166139/CAPMC/CIHR/Canada ; //University of Ottawa/ ; },
mesh = {Animals ; *Zebrafish/embryology/genetics/physiology ; *Animal Fins/embryology/metabolism ; Male ; *Zebrafish Proteins/genetics/metabolism ; *Body Patterning/genetics ; Female ; *Courtship ; CRISPR-Cas Systems ; *Microfilament Proteins/genetics/metabolism ; Gene Expression Regulation, Developmental ; Cell Movement ; *Sexual Behavior, Animal/physiology ; },
abstract = {A key difference between tetrapod limb buds and teleost fin buds is the presence of rigid actinotrichia fibers that guide the migrating cells contributing to ray formation. Major structural components of actinotrichia are encoded by fish-specific actinodin (And) genes, which were lost in tetrapods. To investigate the consequences of this loss during the fin-to-limb transition, we generated deletions in zebrafish and1 and and2 using CRISPR/Cas9 mutagenesis. Double mutants (and1-/-and2-/-) lack actinotrichia. Embryos and larvae have reduced fin fold size, with disorganized cell migration. In adults, all fin fold-derived skeletal structures are disrupted, including the rays of all fins, as well as the caudal fin endoskeleton. Surprisingly, double mutant males fail to breed, despite being fertile. Video analysis revealed that defects in the fins of males impair their ability to stimulate egg release. Our findings highlight the role of actinotrichia in both fin patterning and zebrafish courtship. We propose that actinodin gene maintenance is under strong selection in fish with similar courtship. We speculate that the loss of actinodin genes and a shift in courtship strategy may have coincided during tetrapod evolution.},
}
@article {pmid41885207,
year = {2026},
author = {Yin, W and Jin, Z and Jiang, Q and Jin, S and Wang, X and He, R and Qiao, B and Qiao, J and Zhang, X and Liu, Y},
title = {Elimination of cis-cleavage in CRISPR diagnostics for one-pot rapid nucleic acid detection.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41885207},
issn = {1362-4962},
support = {2022YFC2304304//National Key Research and Development Program of China/ ; 2023DJC136//Science and Technology Innovation Talent Plan of Hubei Province/ ; 2025AFB825//Natural Science Foundation of Hubei Province/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Associated Proteins/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; *Endodeoxyribonucleases/genetics/metabolism ; COVID-19/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Bacterial Proteins/genetics/metabolism ; Tumor Suppressor Protein p53/genetics ; *COVID-19 Nucleic Acid Testing/methods ; },
abstract = {Current one-pot clustered regularly interspaced short palindromic repeats diagnostics are limited by the cis-cleavage activity of Cas nucleases, which leads to amplicon degradation during amplification. Here, we report a streamlined strategy that overcomes this limitation. By integrating a bipartite split-crRNA into Cas12a (SCas12a), we separate target recognition from PAM dependency and completely eliminate cis-cleavage while preserving robust trans-cleavage. This strategy is broadly applicable for one-pot testing, compatible with recombinase polymerase amplification, RT-RPA, and loop-mediated isothermal amplification, as well as multiple Cas12a orthologs, including As, Lb, and Ct Cas12a. Moreover, the SCas12a accelerates one-pot testing with 100-1000-fold improved sensitivity and achieves >10-fold reduction in time-to-signal, enabling detection of targets at attomolar levels within 30 min. Additionally, it provides single-base resolution with up to 91-fold selectivity. The system has been successfully applied to detect HPV16, SARS-CoV-2, and TP53 SNPs in clinical samples. Together, we have developed a PAM-independent and cis-cleavage-free one-pot Cas12a assay, which holds strong potential for point-of-care diagnostics.},
}
@article {pmid41885929,
year = {2026},
author = {Idrees, J and Shabbir, AQ and Alvi, IA and Sana, S and Rehman, SU and Asif, M},
title = {CRISPR-anti-CRISPR dynamics: evolutionary, ecological and biotechnological perspectives.},
journal = {Archives of microbiology},
volume = {208},
number = {6},
pages = {},
pmid = {41885929},
issn = {1432-072X},
mesh = {*CRISPR-Cas Systems ; *Bacteriophages/genetics/physiology ; *Bacteria/genetics/virology ; Biotechnology ; Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Ecosystem ; Biological Evolution ; Viral Proteins/metabolism/genetics ; Evolution, Molecular ; },
abstract = {Phages and bacteria are engaged in an evolutionary arms race. CRISPR-Cas systems allow bacteria to resist phage predation, contributing to competitive advantages for certain bacterial strains. However, phages generate a diverse anti-CRISPR (Acr) proteins to effectively neutralise the CRISPR-Cas system. Acr proteins restore phage infectivity, which may contribute in limiting the dominance of CRISPR-armed bacterial strains, potentially influencing the microbial diversity in certain environments. Acrs can influence microbial community dynamics which may indirectly affect ecosystem functions such as nutrient cycling in certain marine and soil ecosystems. The potential of these Acrs proteins in controlled and reversible genome editing, highlights their potential as regulatory components for genome editing systems. This review discusses the molecular mechanism of CRISPR and anti-CRISPRs, highlights the diversity and limitations of known inhibitory mechanisms, ecological role of anti-CRISPRs and highlights their expanding application in microbial evolution and biotechnology.},
}
@article {pmid41886492,
year = {2026},
author = {Tu, KJ and Roy, SK and Kingsbury, TJ and Shukla, HD},
title = {HIF1α mediates resistance to radiation and to KRAS inhibitors in pancreatic adenocarcinoma.},
journal = {PloS one},
volume = {21},
number = {3},
pages = {e0341912},
pmid = {41886492},
issn = {1932-6203},
mesh = {*Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; Humans ; *Pancreatic Neoplasms/metabolism/genetics/radiotherapy/pathology/drug therapy ; *Proto-Oncogene Proteins p21(ras)/antagonists &amp; inhibitors/genetics/metabolism ; Cell Line, Tumor ; Animals ; *Radiation Tolerance/genetics/drug effects ; Mice ; *Drug Resistance, Neoplasm ; *Carcinoma, Pancreatic Ductal/genetics/radiotherapy/metabolism/pathology/drug therapy ; Tumor Suppressor Protein p53/metabolism ; Cell Proliferation/drug effects ; Apoptosis/drug effects/radiation effects ; *Adenocarcinoma/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is highly treatment resistant and characterized by a hypoxic microenvironment. Here, we investigated the role of hypoxia-inducible factor 1α (HIF1α) in regulating resistance to radiation and KRAS-inhibitor. We employed CRISPR/Cas9 to knock out (KO) HIF1α from the murine KRASG12D/+; p53R172H/+ KPC and the KRASG12D/+; p53R273H; CDK2NA-/- Panc-1 human pancreatic cell lines. Compared to WT, the HIF1α KO cell lines demonstrated a shift toward an epithelial phenotype and had decreased proliferation and migration under hypoxia. HIF1α KO cell lines were less likely to survive after radiotherapy, and neutral comet assays demonstrated DNA damage four hours after treatment, suggesting that HIF1α promotes radioresistance through non-homologous end joining. When treated with a KRASG12D inhibitor, HIF1α KO cells exhibited significantly increased apoptosis due to decreased p53 degradation, likely mediated through Mdm2. Confirming this, enrichment of hypoxic signaling was associated with KRAS inhibitor resistance in a cohort of 31 KRASG12D cell lines. Our results thus suggest that inhibiting HIF1α may sensitize PDAC to radiation and KRAS inhibitors. To explore this, we conducted a drug repurposing screen and identified three HIF1α inhibitors (bakuchiol, BAY-87-2243, 2-methoxyestradiol) whose sensitivities were correlated with sensitivity to Deltarasin, a KRAS inhibitor. Our findings suggest that HIF1α inhibitors could be used to sensitize PDAC to radiotherapy and KRAS inhibitors.},
}
@article {pmid41886504,
year = {2026},
author = {Maestas, MM and Bradley, K and Shunkarova, M and Mukherjee, N and Ishahak, M and Lu, J and Millman, JR},
title = {Whole-genome CRISPR screening identifies genetic modifiers of stem cell-derived islet transplantation.},
journal = {Stem cells translational medicine},
volume = {15},
number = {4},
pages = {},
doi = {10.1093/stcltm/szag012},
pmid = {41886504},
issn = {2157-6580},
support = {UG3DK142188//National Institutes of Health (NIH)/ ; 2022-001//Beatson Foundation/ ; //Edward J. Mallinckrodt Foundation/ ; //Washington University School of Medicine Department of Medicine/ ; //Anita Palmer Corbin Trust/ ; T32GM139774//Cellular and Molecular Biology Training/ ; //Bill &amp; Melinda Gates Foundation through the Gates Millennium Scholars Program/ ; //Rita Levi-Montalcini Postdoctoral Fellowship in Regenerative Medicine/ ; //Rita Levi-Montalcini Postdoctoral Fellowship in Regenerative -Medicine/ ; T32DK007120/GF/NIH HHS/United States ; },
mesh = {Animals ; *Islets of Langerhans Transplantation/methods ; Humans ; Mice ; Mice, Inbred NOD ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CRISPR-Cas Systems ; Cell Differentiation ; Islets of Langerhans/metabolism/cytology ; },
abstract = {INTRODUCTION: Genetically engineering human pluripotent stem cell (hPSC)-derived islets is a promising strategy for improving transplantation for diabetes cell therapy; however, genetic perturbations that modulate transplantation outcomes have yet to be systematically explored.<br><br>METHODS: To identify potential targets, we performed an unbiased whole-genome CRISPR-activation screen in transplanted stem cell-derived islets (SC-islets). Specifically, we created a stem cell line with CRISPR-activation components (HUES8-VPR) and then transduced these stem cells with a lentiviral guide RNA library targeting the whole human genome. Following transduction, the stem cells were differentiated into SC-islets, which were subsequently transplanted into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) immunodeficient mice. After transplantation, SC-islets were extracted for next-generation sequencing.<br><br>RESULTS: The screen identified multiple candidates, including the Fc alpha/mu receptor (FCAMR). In vitro characterization revealed that FCAMR overexpression did not negatively affect SC-islet function or transcriptomic identity. Mice subcutaneously transplanted with SC-islets overexpressing FCAMR had reduced blood glucose levels and increased C-peptide compared to controls. Additionally, mice receiving FCAMR-modified grafts into the kidney capsule or hindleg muscle maintained a higher body weight compared to controls in a diabetic setting.<br><br>CONCLUSIONS: In conclusion, this study demonstrats improved glucose regulation at a subcutaneous transplant site. In addition, we show that FCAMR SC-islets could play a role in systemic metabolism when transplanted into the kidney capsule or hindleg muscle. Overall, our study establishes a functional screening approach to identify gene candidates to improve SC-islet transplantation.},
}
@article {pmid41887392,
year = {2026},
author = {Wang, H and Shen, T and Yang, S and Zhou, X and Cao, P and Yu, H and He, K and Fu, M and Yu, H and Liu, X and Zhou, T and Wang, J and Huang, M and Qian, X and Wang, X and Wang, Q and Liu, L and Fan, Z and Zhang, Y and Lin, F},
title = {Genome-wide CRISPR/Cas9 screen identified MCL1 as a senolytic target for clearing palbociclib-induced senescent and PD-L1-positive cells in colorectal cancer.},
journal = {Cancer letters},
volume = {647},
number = {},
pages = {218446},
doi = {10.1016/j.canlet.2026.218446},
pmid = {41887392},
issn = {1872-7980},
mesh = {Humans ; *Colorectal Neoplasms/drug therapy/genetics/pathology/metabolism ; *Pyridines/pharmacology ; *Piperazines/pharmacology ; Animals ; *Cellular Senescence/drug effects/genetics ; *Myeloid Cell Leukemia Sequence 1 Protein/genetics/antagonists &amp; inhibitors/metabolism ; CRISPR-Cas Systems ; Mice ; Xenograft Model Antitumor Assays ; *B7-H1 Antigen/metabolism/genetics ; Apoptosis/drug effects ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Mice, Nude ; Gene Expression Regulation, Neoplastic/drug effects ; Female ; },
abstract = {Colorectal cancer (CRC) is the most prevalent digestive system malignancy worldwide. The development of targeted therapeutics specifically effective for CRC is currently in dire need. Preclinical studies showed that CDK4/6 inhibitor palbociclib suppressed the growth of CRC, but whether this effect is durable is unclear. In this study, we aimed to evaluate the roles of palbociclib-induced senescence and find a new strategy to maximize its effectiveness in CRC treatment. Animal and cellular experiments revealed that palbociclib-induced senescence and the senescence-associated secretory phenotype (SASP) caused drug resistance, anti-apoptosis, PD-L1 upregulation and inhibition of CD8[+] T cells' function. Using CRISPR/Cas9 screening, we identified MCL1 as a senolytic target to eliminate palbociclib-induced senescent CRC cells in the presence of palbociclib. Mechanically, palbociclib-induced senescent cells upregulated ZHX2 and its transcriptional target MCL1, rendered their resistance to apoptosis and T cell-mediated cytotoxicity, whereases combining palbociclib with MCL1 inhibitor markedly induced apoptosis in senescent cells by activating both extrinsic and intrinsic apoptotic pathways. Lastly, we proposed a seno-therapy consisting of a palbociclib pre-treatment plus a combination treatment of palbociclib and MCL1 inhibitor and found it effectively inhibited tumor growth and improved the survival of CRC xenografted mice. Besides its senolytic effect, seno-therapy also reduced PD-L1-positive cells and enhancing the cytotoxic functions of CD8[+] T cells. In conclusion, co-targeting CDK4/6 and MCL1 efficiently eliminates palbociclib-induced senescent CRC cells and offers a promising CDK4/6 inhibitor-based strategy for CRC treatment, ensuring prolonged tumor suppression and reducing the risk of progression or recurrence.},
}
@article {pmid41887586,
year = {2026},
author = {Menestreau, M and Frostegård, &#xc5; and Kjos, M},
title = {Strain-specific challenges in applying CRISPR/Cas9-based genome editing in the novel genus Stutzerimonas.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107478},
doi = {10.1016/j.mimet.2026.107478},
pmid = {41887586},
issn = {1872-8359},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Plasmids/genetics ; *Genome, Bacterial ; Denitrification/genetics ; },
abstract = {Recently reclassified from the genus Pseudomonas, Stutzerimonas comprises metabolically versatile bacteria widely distributed across diverse environments and with a capacity to perform complete denitrification. Here, we evaluated the applicability of CRISPR/Cas9-based genome editing in Stutzerimonas species. Using a two-plasmid pCasPA/pACRISPR system, we achieved efficient deletion of the denitrification-associated narG and dnrE genes in Stutzerimonas decontaminans 19SMN4. On the other hand, Cas9-associated toxicity significantly limited transformation in Stutzerimonas perfectomarina ZoBell. These results highlight both the potential and the limitations of CRISPR/Cas9 editing in Stutzerimonas, emphasizing that genome editing efficiency and tolerance may vary even among closely related strains.},
}
@article {pmid41888353,
year = {2026},
author = {Wang, J and Bobrik, M and Pankaew, N and Gratacap, R and Digard, P and Bean, TP and Jin, Y and Robledo, D},
title = {Efficient genome editing in a Mozambique tilapia cell line using CAS ribonucleoprotein complexes.},
journal = {Scientific reports},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41598-026-42702-w},
pmid = {41888353},
issn = {2045-2322},
support = {STG5443//Ministry of Science and Technology of Thailand/ ; BBS/E/RL/230002A/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
abstract = {Genome editing using the CRISPR/Cas system makes it possible to rapidly characterise gene function in vitro and in vivo, and provides a powerful platform through which the genetics of farmed fish can be altered to improve traits such as resistance to important pathogens. Tilapia is one of the most important farmed fish globally; however, its farming is heavily impacted by Tilapia lake virus (TiLV). The Mozambique tilapia (Oreochromis mossambicus) brain (OmB) cell line is susceptible to TiLV, making it an ideal in vitro model for studying host-pathogen interactions and mechanisms of disease resistance. To establish OmB cells as a model for gene editing in Tilapia, it is essential to optimise genome editing protocols. In this study, we optimized a CRISPR/Cas9-based genome editing system for OmB cells using ribonucleoprotein complexes. With the optimized protocol, we successfully edited two endogenous genes with efficiencies ranging from 67% to 70%. In conclusion, we establish a highly efficient CRISPR/Cas9 RNP based gene editing workflow optimized for OmB tilapia cell lines. This optimized platform will facilitate future functional genomic studies in tilapia and support the development of TiLV-resistant tilapia.},
}
@article {pmid41889087,
year = {2026},
author = {Yang, L and Ma, W},
title = {CRISPR/Cas12a: A Comprehensive Review from Structural Foundations to Applications in Nucleic Acid Precision Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {13},
pages = {10689-10708},
doi = {10.1021/acs.jafc.5c16528},
pmid = {41889087},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acids/genetics/chemistry/metabolism ; Nucleic Acid Amplification Techniques/methods ; Biosensing Techniques/methods ; *Bacterial Proteins/genetics/chemistry/metabolism ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; Humans ; *Bacteria/genetics/isolation &amp; purification/enzymology ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a technology, characterized by its distinctive trans-cleavage activity, has evolved beyond its gene-editing function to emerge as a powerful tool for molecular detection. This review systematically delineates its structural foundation and molecular mechanism, with a focus on how the technology converts specific nucleic acid recognition into cascade signal amplification. Its applications span pathogen diagnosis, species identification, food safety, and authentication of traditional Chinese medicines. Through integration with isothermal amplification and multimodal detection platforms, Cas12a has driven molecular diagnostics toward portability, visualization, and quantification. The review further discusses challenges related to sensitivity, quantitative accuracy, crRNA design, and standardization, while outlining future directions through convergence with cutting-edge technologies such as microfluidics and artificial intelligence, offering a forward-looking perspective for the development of next-generation precision biosensing platforms.},
}
@article {pmid41889958,
year = {2026},
author = {Ocampo, RF and Orosco, C and Huang, B and West, MS and Jain, PK and Taylor, DW},
title = {Architecture of a DNA-guided Cas12a.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.03.19.712971},
pmid = {41889958},
issn = {2692-8205},
abstract = {CRISPR/Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-EM structure of Acidaminococcus sp. Cas12a (AsCas12a) bound to a pseudo-DNA (ΨDNA) guide and RNA target, revealing how Cas12a accomplishes DNA-guided RNA recognition. The ΨDNA hairpin bridges the recognition and nuclease lobes, mimicking a PAM-proximal duplex and positioning the spacer to allow formation of a canonical RNA-DNA heteroduplex along the REC lobe. This provides a structural framework for its activity and provides a blueprint for future engineering.},
}
@article {pmid41891780,
year = {2026},
author = {Gu, C and Gu, J and Li, J and Zou, D and Qi, Y and Xia, R and Zhou, Z and Li, M},
title = {Insights into the Arms Race between Prokaryotic Hosts and Their Viruses in Mangrove Ecosystem.},
journal = {Environmental science &amp; technology},
volume = {60},
number = {14},
pages = {10912-10927},
doi = {10.1021/acs.est.5c14802},
pmid = {41891780},
issn = {1520-5851},
mesh = {Wetlands ; Ecosystem ; *Viruses ; CRISPR-Cas Systems ; Bacteria/genetics ; Prokaryotic Cells ; China ; },
abstract = {The coevolutionary arms race between prokaryotes and viruses has driven the diversification of various microbial immune mechanisms including restriction-modification (RM) and clustered regularly interspaced short palindromic repeats and CRISPR-associated protein (CRISPR-Cas) systems. While recent efforts have expanded the catalog of antiviral systems, their ecological dynamics within complex microbial communities remain underexplored. Here, we analyzed prokaryotic communities, viruses, and defense systems in mangrove habitats from Futian, China, by integrating DNA and RNA sequencing of sediment cores collected across multiple depths and seasons at two sites. Prokaryotic genomes harbored 65 distinct defense system types, representing ∼43% of known systems, with transcriptional activity dominated by Pseudomonadota, Planctomycetota, and Chloroflexiota. The key systems, including abortive infection (AbiD, AbiE, AbiU), Eleos, CRISPR-Cas, MazEF, Retron, and Wadjet, exhibited high transcriptional activity across samples. Strikingly, viruses encoded highly expressed defense systems such as AbiE and RM, despite their co-occurring prokaryotic hosts lacking detectable antiviral systems. The abundance ratio of prokaryotes and viruses with defense systems shows an opposite trend as the depth variation. Heterologous validation confirmed the antiviral efficacy of the selected systems. Our findings suggest that viral-encoded defense systems may functionally augment host immunity in mangrove habitats, revealing a nuanced coevolutionary interplay within these ecosystems. This study advances our understanding of host-virus interactions at the community level in mangrove wetland microbiomes.},
}
@article {pmid41891877,
year = {2026},
author = {Wan, Y and Zhao, X and Lin, X and Wang, L and Ai, X and Jiang, J and Han, L and Huang, D and Du, H and Huang, L},
title = {Indel pattern-guided repair mapping reveals genome-wide DNA repair networks in CRISPR/Cas9 editing.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41891877},
issn = {1362-4962},
support = {2024ZDZ07//Guangdong Province Drug Administration Science and Technology Innovation/ ; 202206010073//Science and Technology Program of Guangzhou/ ; 2023A03J0542//Science and Technology Program of Guangzhou/ ; 2025A1515010459//Natural Science Foundation of Guangdong Provinc/ ; GZYZH2023001//Open Project of the China NMPA Key Laboratory for Animal Alternative Testing Technology of Cosmetics/ ; 2022A1515011733//Guangdong Basic and Applied Basic Research Foundation/ ; 2024ZDZ07//Guangdong Province Drug Administration Science and Technology Innovation Project/ ; 202206010073//Science and Technology Program of Guangzhou/ ; 2023A03J0542//Science and Technology Program of Guangzhou/ ; 2025A1515010459//Natural Science Foundation of Guangdong Provinc/ ; GZYZH2023001//Open Project of the China NMPA Key Laboratory for Animal Alternative Testing Technology of Cosmetics/ ; 2022A1515011733//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *INDEL Mutation ; DNA Breaks, Double-Stranded ; *Gene Editing/methods ; *DNA Repair/genetics ; Humans ; DNA End-Joining Repair ; Gene Regulatory Networks ; },
abstract = {CRISPR/Cas9-induced DNA double-strand breaks (DSBs) trigger diverse repair outcomes, yet the dynamic regulatory networks governing these outcomes remain incompletely understood. Here, we develop indel pattern-guided repair mapping, an integrative framework that deciphers DSB repair mechanisms by integrating repair outcome spectra, kinetic dynamics, and functional gene regulation. Our analysis categorizes Cas9-mediated repair outcomes into seven distinct patterns based on their frequency and sequence characteristics, revealing differential repair kinetics among these subtypes. Functional clustering identifies three regulatory pillars: (i) microhomology-mediated end joining (MMEJ)-driven MH deletions form a cohesive module defined by a shared regulatory network of protein-coding genes and miRNAs, rather than by the core repair enzymes themselves; (ii) non-homologous end joining coordinates 1 bp insertions and non-MH deletions, with RFC4/5 stabilizing repair templates to suppress large deletions; (iii) Atypical repair outcomes show distinct genetic signatures: large insertions are associated with polymerase-related regulators, whereas mutations are associated with a signature enriched for chromatin-associated regulators. Strikingly, S100A8 emerges as a potent MMEJ suppressor via direct interaction with PARP1, revealing unappreciated cross-talk between inflammatory signaling and DSB repair pathway choice. By linking repair outcome patterns to molecular determinants, our work provides a transformative platform to interrogate DNA repair mechanisms for precise genome editing optimization and therapeutic genome stabilization.},
}
@article {pmid41892062,
year = {2026},
author = {Tantai, W and Xu, Q and Zhang, W and Li, Y and Liu, H},
title = {A One-Pot CRISPR/Cas12a-Based Platform for Contamination-Free Nucleic Acid Amplification Detection.},
journal = {Biosensors},
volume = {16},
number = {3},
pages = {},
pmid = {41892062},
issn = {2079-6374},
support = {22074085//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; *Biosensing Techniques ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR-Cas12a enables rapid and specific detection of PCR/LAMP (loop-mediated isothermal amplification) reaction products; however, this approach often requires open-tube manipulation, rendering it prone to cross-contamination. Here, we developed a novel one-pot reaction system that eliminated carryover contamination and facilitated endpoint detection using a CRISPR/Cas12a-based system. We leveraged the dependence of the CRISPR-Cas12a cleavage system on the protospacer-adjacent motif (PAM) to design PCR/LAMP primers that incorporated the PAM site (TTT) into amplified DNA. Pre-incubation of Cas12a with crRNA1 and crRNA2 using PCR/LAMP resulted in efficient cleavage of cross-contaminating DNA, while the target gene remained intact due to the lack of PAM sites. Furthermore, a Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and the ssDNA probe) pre-stored on the lid was introduced to mix with the PCR/LAMP amplicons, which triggered the non-specific cleavage of fluorescent probes for direct visual detection under a blue LED instrument. This method effectively degraded up to 10[6] copies of carryover contaminants within one hour, demonstrating the potential of one-pot detection methods in complex samples.},
}
@article {pmid41892280,
year = {2026},
author = {Woronkowicz, M and Thomas, MN and Saram, SJ and Carr, AF and Alonso-Carriazo Fernandez, A and Butt, Z and Skopiński, P and Ramsden, CM},
title = {CRISPR and Beyond: Genome-Editing Strategies in Retinal Stem Cell Research.},
journal = {Cells},
volume = {15},
number = {6},
pages = {},
pmid = {41892280},
issn = {2073-4409},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Retina/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Stem Cell Research ; Retinal Degeneration/genetics/therapy ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Genome editing has emerged as a transformative approach for understanding and treating retinal degenerative diseases. Combining this technology with pluripotent stem cells provides an ideal platform for modeling human development and disease, and investigating emerging therapeutic strategies ultimately aimed towards in vivo correction. This approach enables both functional studies to understand retinal degeneration and the early development of targeted therapies for inherited disease. This review offers a comprehensive overview of genome-editing techniques and the ability to create new clinically relevant models to understand human disease in retinal research, focusing on the use of the CRISPR-Cas9 system in induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), as well as highlighting recent advancements in base and prime editing. Gene editing in various retinal diseases is discussed in context of studies focusing on disease modeling or developing therapeutic strategies. Continued refinement of these techniques will be essential for advancing translational applications in retinal disease treatment.},
}
@article {pmid41892424,
year = {2026},
author = {Gomes, E and Mesquita, TG and Serra, P and Araújo, D and Almeida, C and Machado, A and Oliveira, R and Castro, J},
title = {Antimicrobial Resistance in the Food Chain: Bridging Knowledge Gaps for Effective Detection and Control.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {15},
number = {3},
pages = {},
pmid = {41892424},
issn = {2079-6382},
support = {https://doi.org/10.54499/2024.13640.PEX//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e Tecnologia/ ; https://doi.org/10.54499/2022.07654.PTDC//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e Tecnologia/ ; APTA4shiga (number 14840)//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e Tecnologia/ ; },
abstract = {Antimicrobial resistance (AMR) poses a critical global public health threat, with the food chain serving as a significant transmission route connecting animals, environment, and humans. This review adopts a One Health perspective to analyze the key drivers of AMR dissemination across animal agriculture, aquaculture and food processing. We evaluate detection methodologies, contrasting the regulatory gold standard of culture-based phenotypic testing with rapid molecular advancements, including Whole Genome Sequencing (WGS), metagenomics, and emerging CRISPR-Cas diagnostics. While molecular tools offer unprecedented speed and resolution, challenges such as matrix interference, the viable but non-culturable (VBNC) state, and the genotype-phenotype disconnect remain. Finally, integrated mitigation strategies are also described, ranging from on-farm antimicrobial stewardship and innovative biofilm control to consumer hygiene practices. It is essential to bridge the technical and regulatory gaps in AMR surveillance in order to develop effective interventions and ensure a safer food system.},
}
@article {pmid41893903,
year = {2026},
author = {Baliyan, N and Upadhyay, P and Murugan, T and Srivastava, A and Singh, S and Tomar, BS and Mangal, M},
title = {Recent advances in generation of doubled haploid plants for genetic improvement in solanaceous vegetable crops.},
journal = {Planta},
volume = {263},
number = {5},
pages = {},
pmid = {41893903},
issn = {1432-2048},
support = {SR/WOS-A/LS-146/2019//Department of Science and Technology, Ministry of Science and Technology, India/ ; },
mesh = {*Haploidy ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; *Vegetables/genetics ; Genome, Plant ; },
abstract = {Genotype-specific protocols and advancements focusing on CRISPR/Cas9-based haploid induction for doubled haploid (DH) production are poised to revolutionize plant breeding for faster genetic improvement in solanaceous crops. The need for swift development of improved cultivars offering greater resilience to biotic and abiotic stresses stems from the emergence of climate change risks. While conventional methods are effective, novel methods for precise crop genome manipulation are required. The in vivo and in vitro protocols leading to fixation of homozygosity and rapid attainment of homozygous DH (doubled haploids) lines have led to a resurgence in research on haploids and DH. The efforts for haploid production have been primarily concentrated on in vitro androgenesis and gynogenesis. The success of these in vitro techniques depends on various parameters, including culture conditions, developmental stage of the microspore, pretreatment, culture medium, growth regulators, and other media additives. Breakthroughs in genome-editing technologies, such as the CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9) system, have provided a new avenue for exploring in vivo haploid embryogenesis for haploid induction by means of haploid inducer factors, such as DMP (DMP domain of unknown function 679 membrane protein), ECS (egg cell-specific), MTL (matrilineal), and BBM (Baby boom). A successful haploid induction system in vegetable crops will be possible through the development of efficient in vivo and in vitro androgenesis and gynogenesis protocols. For genome-editing to be feasible, protocols must be optimized for commercially significant solanaceous crops like pepper, potatoes, eggplant, and tomatoes. The existing protocols for genome doubling need to be further improved for solanaceous crops. These developments are pivotal for the advancement and harnessing of haploid technology to its full potential in crop breeding.},
}
@article {pmid41893914,
year = {2026},
author = {Pistone, D and Bevivino, G and Dipaola, MG and Bandi, C and Lombardo, F},
title = {Current and emerging molecular diagnostic approaches in the detection of human parasites.},
journal = {Parasitology research},
volume = {125},
number = {1},
pages = {},
pmid = {41893914},
issn = {1432-1955},
abstract = {Microscopy and morphological identification remain the gold standard for diagnosing most parasitic infections, yet their limited sensitivity in asymptomatic or low-burden cases, along with technical constraints, has accelerated the adoption of molecular diagnostics. Over the past three decades, advances in nucleic acid amplification and sequencing technologies have transformed parasite detection by improving sensitivity, specificity, and reproducibility, enabling earlier intervention and stronger surveillance. PCR remains the foundation of molecular diagnostics, with real-time PCR and digital PCR improving analytical performance and quantification. Multiplex qPCR supports simultaneous detection of multiple pathogens, while dPCR enables absolute quantification and rare variant detection, although broader implementation is limited by instrument cost. Isothermal amplification methods such as tHDA, NASBA, LAMP, and RPA offer rapid, low-cost amplification at constant temperature and are well suited for field diagnostics in resource-limited settings. Next-Generation Sequencing has advanced genotyping and epidemiological surveillance by resolving cryptic species, resistance mutations, and mixed infections through targeted panels, whole-genome sequencing, and metagenomics. CRISPR/Cas-based assays provide rapid and sensitive nucleic acid detection with strong potential for point-of-care deployment due to their simplicity and adaptability. Emerging biomarkers, including circulating cell-free DNA, non-coding RNAs, and microRNAs in extracellular vesicles, offer promising non-invasive diagnostic strategies, though further validation is required. This review offers a concise overview of these molecular approaches, emphasizing recent innovations such as dPCR, NGS, CRISPR/Cas systems, and biomarker-based detection. For each method, core technical principles, representative applications, and comparative strengths and limitations are presented to illustrate their diagnostic potential.},
}
@article {pmid41895443,
year = {2026},
author = {Phan, PT and Ozturk, M and Dougherty, EM and Ravishankar, J and Xue, C and Sashital, DG},
title = {Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system.},
journal = {The Journal of biological chemistry},
volume = {},
number = {},
pages = {111401},
doi = {10.1016/j.jbc.2026.111401},
pmid = {41895443},
issn = {1083-351X},
abstract = {Type I-E CRISPR-Cas systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the PAM and PAM-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated, but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four E. coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 2 and 4. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cascade conformation, and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.},
}
@article {pmid41896556,
year = {2026},
author = {Walsh, LH and Soni, V and Ancla, J and Somerville, V and Segata, N and Joyce, S and Sinderen, DV and Mahony, J and Shkoporov, AN and Kenny, JG and Cotter, PD and O'Sullivan, O},
title = {Mining of food metagenomes reveals an unexplored diversity of dsDNA bacteriophages.},
journal = {NPJ biofilms and microbiomes},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41522-026-00941-9},
pmid = {41896556},
issn = {2055-5008},
support = {DOMINO-101060218//European Union's Horizon Europe programme/ ; },
abstract = {Bacteriophages are key drivers of microbial ecology, co-existing and co-evolving with bacteria across diverse environments. Limitations in culturing, alongside advances in sequencing and bioinformatics, have driven the use of metagenomics to explore viral diversity. Viral-specific analysis of >3000 food metagenomes from cFMD produced the FVGC, comprising ~3400 metagenome-assembled viruses, most of which belong to novel Caudoviricetes lineages (n = 91), with only ~15% represented in IMG/VR v4. Together, these findings reveal extensive uncharacterized viral diversity in food systems. Beyond serving as a reference, the FVGC facilitates detailed investigation of virus-host interactions. Viral sequences were pervasive across microbial genomes, with several bacterial families exhibiting near-universal associations with viral elements. Bacterial antiviral defence systems were abundant and taxonomically diverse, dominated by restriction-modification systems, while CRISPR-Cas systems showed pronounced lineage-specific distributions; in contrast, viral anti-defence genes were detected at low frequency (<10% of MAVs). Host prediction linked MAVs to clinically relevant taxa, including expanded ESKAPE pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Enterobacter spp., highlighting the ecological connectivity between food-associated viruses and clinically important bacteria. Antimicrobial resistance signals were scarce, suggesting minimal phage-mediated AMR dissemination in food environments. This new publicly available viral database represents a valuable resource for further exploration of viral diversity.},
}
@article {pmid41897294,
year = {2026},
author = {Zhou, Z and Zhu, L},
title = {A Computational Model for Nme1Cas9 HNH Activation Driven by Dynamic Interface Engineering at Residues S593 and W596.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897294},
issn = {2218-273X},
support = {32471296//National Natural Science Foundation of China Projects/ ; },
mesh = {Molecular Dynamics Simulation ; Thermodynamics ; Protein Engineering ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; },
abstract = {Nme1Cas9 is an encouraging genome-editing tool with high fidelity and compactness, but its applications are limited by poor catalytic efficiency compared with SpyCas9. Understanding the dynamic activation mechanism of the HNH nuclease domain is the key to breaking the kinetic bottleneck. Here, we integrated Steered Molecular Dynamics (SMD) with the Traveling-Salesman-based automated Path Searching (TAPS) algorithm to reconstruct the atomic-level activation landscape of the L1-HNH module. The simulations suggest a complex "Lifting-Rearrangement-Sliding" pathway, revealing the critical role of a "Backbone Sliding" conformation; in this step, the HNH domain rotates across the R-loop surface. A thermodynamic analysis using free energy decomposition by MM/PBSA indicates that the intrinsic instability of the wild-type HNH/R-loop interface constitutes the predominant energetic barrier. Hyperactive variants (S593Q/W596K and S593Q/W596R) can overcome this barrier by substantially increasing binding affinity to the R-loop through a "Geometry-Electrostatics Synergism": S593Q improves interfacial proximity, whereas W596K/R acts as an "Electrostatic Anchor." The results of unbiased MD simulations demonstrate that strengthened interfacial interactions effectively promote spontaneous conformational drift toward the activated state. This computational study proposes a novel in silico model for "Dynamic Interface Engineering" in which reinforcing transient interfacial contacts during conformational sliding can be an effective strategy in developing high-efficiency CRISPR-Cas effectors.},
}
@article {pmid41897352,
year = {2026},
author = {Atluri, U and Cvammen, W and Kemp, MG},
title = {The Small Molecule SR8278 Inhibits Cell Proliferation Independent of the REV-ERB Nuclear Receptor Proteins in Human Keratinocytes.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897352},
issn = {2218-273X},
support = {GM103583/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *Cell Proliferation/drug effects ; *Keratinocytes/drug effects/metabolism/cytology ; *Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism/genetics/antagonists &amp; inhibitors ; CRISPR-Cas Systems ; Apoptosis/drug effects ; Gene Expression Regulation/drug effects ; *Thiophenes/pharmacology ; },
abstract = {The small molecule SR8278 was initially identified as an antagonist of the REV-ERB (reverse c-ERBAa) nuclear receptor proteins, which play important roles in metabolism and circadian rhythms. Though SR8278 has been shown to have beneficial physiological effects in a variety of different preclinical disease contexts, its impact on gene expression and cell proliferation in keratinocytes has not previously been examined. We therefore carried out an RNA-seq analysis and found that genes involved in the G1/S transition of the cell cycle were significantly impacted by SR8278 treatment, and these effects were confirmed at both the RNA and protein level by RT-qPCR and Western blotting, respectively. Cell proliferation assays showed that SR8278 slowed cell growth but did not induce genotoxic stress or apoptosis. Finally, the use of CRISPR/Cas9 genome editing and siRNA-mediated disruption of REV-ERB gene expression showed that the loss of the REV-ERB proteins did not impact the effect of SR8278 on gene expression and cell proliferation. We conclude that the anti-proliferative effects of SR8278 are not mediated by the REV-ERB proteins, and, thus, care should be taken when interpreting studies involving this compound unless complementary genetic approaches are also shown, particularly in studies involving cell proliferation.},
}
@article {pmid41897386,
year = {2026},
author = {Richter, PR and Graf, J and Haag, FWM and Scudlo, V and Wiesmeth, S and Hauslage, J and Richter, M and Geißler, D and Lebert, M},
title = {Acceleration and Light-Induced Changes in Cytosolic cAMP Concentration in Euglena gracilis.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897386},
issn = {2218-273X},
support = {50WB1128 and 50WB2218)//Deutsches Zentrum f&#xfc;r Luft- und Raumfahrt (DLR) BMWi project/ ; },
mesh = {*Cyclic AMP/metabolism ; *Euglena gracilis/metabolism/radiation effects ; *Light ; Adenylyl Cyclases/metabolism/genetics ; *Cytosol/metabolism/radiation effects ; *Acceleration ; Phototaxis ; },
abstract = {The second messenger cyclic AMP (cAMP) is very likely involved in phototactic as well as gravitactic behavior of the unicellular flagellate Euglena gracilis. A slight but significant increase in cAMP was observed when cells encountered sub-threshold acceleration (0.16 × g) force after microgravity [µg]. No differences in cAMP levels were found between cells on a clinostat and 1x-controls. This observation is consistent with the ones of earlier studies. Illumination of cells resulted in a significant increase in cellular cAMP levels. After RNAi-mediated knockdown or CRISPR-Cas9 knockout of the photoactivated adenylyl cyclases PACα and/or PACβ in the photoreceptor, light-induced changes in cAMP levels were no longer observed. In parallel, phototactic behavior was abolished, supporting the essential role of photoactivated adenylyl cyclases in phototaxis. Cells spin around their length axis during locomotion (1-2 Hz). In order to generate a signal in the light direction, the cells should be capable of synthesizing and degrading cAMP within 0.5-1 s. The rapid fixation of cells upon transition from dark to light or light to dark revealed that detectable changes in cAMP-levels (increase or decrease) occur within a 100-200 ms time window, which is sufficiently fast to account for the proposed theoretical kinetics of cAMP oscillations.},
}
@article {pmid41897402,
year = {2026},
author = {Shahannaz, DC and Sugiura, T},
title = {Next-Generation Metabolic Reprogramming in iPSC-Derived Cardiomyocytes: CRISPR-EV Synergy for Precision Cardiac Regeneration.},
journal = {Biomolecules},
volume = {16},
number = {3},
pages = {},
pmid = {41897402},
issn = {2218-273X},
mesh = {Humans ; *Myocytes, Cardiac/metabolism/cytology ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Cellular Reprogramming ; *Regeneration ; Animals ; CRISPR-Cas Systems ; Gene Editing ; Metabolic Reprogramming ; },
abstract = {Cardiovascular disease remains the leading global cause of mortality, largely due to the limited regenerative capacity of adult human myocardium. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer a scalable platform for cardiac repair and disease modeling; however, their persistent metabolic immaturity-characterized by reliance on glycolysis, reduced oxidative phosphorylation (OXPHOS), and structurally underdeveloped mitochondria-limits functional integration and long-term therapeutic efficacy. Recent advances indicate that targeted metabolic reprogramming can enhance mitochondrial biogenesis, increase ATP production, and improve stress resilience in iPSC-CMs. This review examines the complementary integration of CRISPR-based metabolic engineering and extracellular vesicle (EV)-mediated metabolic modulation as a systems-level strategy for cardiac maturation. We discuss CRISPR activation, interference, and epigenome-editing approaches targeting regulators such as PGC-1α, TFAM, and PPARs to promote stable enhancement of mitochondrial networks and respiratory capacity. In parallel, engineered EVs delivering miRNAs, metabolic enzymes, and redox modulators provide non-genomic mechanisms to optimize bioenergetic function and mitigate oxidative stress. By synthesizing mechanistic insights, quantitative bioenergetic metrics, and translational considerations, we propose CRISPR-EV synergy as a precision framework for durable metabolic maturation of iPSC-CMs, with implications for regenerative therapy, pharmacologic screening, and myocardial repair.},
}
@article {pmid41898355,
year = {2026},
author = {Ye, Y and Huang, L and Fu, H and Wang, J and Jin, Y},
title = {Advances in SRNS Gene Research: From Precision Classification to Precision Diagnosis and Treatment.},
journal = {Biomedicines},
volume = {14},
number = {3},
pages = {},
pmid = {41898355},
issn = {2227-9059},
support = {2024C03211 to Jingjing Wang//Key Research and Development Program of Zhejiang Province/ ; GZY-KJS-ZJ-2026-071//Joint TCM Science &amp;Technology Projects of National Demonstration Zones for Comprehensive TCM Reform/ ; LTGD24H050002//Natural Science Foundation of Zhejiang Province/ ; },
abstract = {To clarify the genetic classification, diagnostic strategies, and precision treatment pathways of steroid-resistant nephrotic syndrome (SRNS), this review systematically reviews the genetic stratification system of SRNS by integrating recent advances in genetic testing technologies and pathogenesis research. It contains the pathogenic mechanisms, diagnostic protocols, and therapeutic correlations of different genetic subtypes, while summarizing current progress and clinical challenges in gene therapy. Results indicate SRNS can be categorized into genetic (38-58%) and non-genetic/immune-mediated (40-60%). A stepwise diagnostic system comprising core proteinuria gene panel testing, whole-genome sequencing (WGS), whole-exome sequencing (WES), and supplementary multi-omics/long-range sequencing is proposed, suited for populations with "typical phenotypes and moderate genetic risk", "atypical phenotypes and high genetic suspicion", and "complex structural/non-coding region variants" respectively. Pathogenic mechanisms directly determine therapeutic strategies: COQ2/PDSS2 mutations respond to coenzyme Q10 suplementation, while NPHS1 mutations necessitate early renal transplantation. Adeno-associated virus (AAV)-mediated gene therapy and CRISPR-Cas editing show preclinical promise but face challenges including incomplete detection coverage and clinical translation difficulties. Genetic technologies are driving SRNS management transformation from "empirical treatment" to "mechanism-oriented precision diagnosis and therapy". Future efforts should focus on overcoming genetic testing limitations and gene therapy translation bottlenecks to enhance diagnostic and therapeutic efficacy.},
}
@article {pmid41898529,
year = {2026},
author = {Thu, KL and Jafari, S and Silvester, J and Cruickshank, J and Soria-Bretones, I and Hodgson, K and Tobin, C and Haight, J and Lau, APY and Bray, T and Wakeham, D and Bray, MR and Mak, TW and Cescon, DW},
title = {Genome-Wide CRISPR Screens Identify ABCG2-Mediated Drug Resistance to the Threonine Tyrosine Kinase (TTK) Inhibitor CFI-402257 in Breast Cancer.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898529},
issn = {1422-0067},
mesh = {Humans ; *ATP Binding Cassette Transporter, Subfamily G, Member 2/genetics/metabolism ; *Drug Resistance, Neoplasm/genetics ; Animals ; Female ; Cell Line, Tumor ; Mice ; *Triple Negative Breast Neoplasms/genetics/drug therapy/pathology/metabolism ; *Neoplasm Proteins/genetics/metabolism ; Xenograft Model Antitumor Assays ; *Protein-Tyrosine Kinases ; CRISPR-Cas Systems ; *Protein Kinase Inhibitors/pharmacology ; *Cell Cycle Proteins/antagonists &amp; inhibitors ; *Indoles/pharmacology ; Antineoplastic Agents/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Apoptosis/drug effects ; Protein Serine-Threonine Kinases ; },
abstract = {CRISPR screens are a powerful functional genomics approach for identifying genes that confer sensitivity and resistance to anti-cancer therapies. CFI-402257 (luvixasertib, 2257) is a small molecule inhibitor of threonine tyrosine kinase (TTK), a promising therapeutic target in genomically unstable cancers due to its critical role in establishing the spindle assembly checkpoint (SAC) during mitosis. To inform its ongoing development and evaluation in clinical trials, we sought to use CRISPR activation (i.e., gain of function) screens to identify cellular mechanisms of resistance to 2257 in models of triple-negative breast cancer (TNBC). In vitro screens conducted in two TNBC cell lines nominated ABCG2 as the top resistance-conferring gene in both models. Validation studies assessing clonogenic survival and apoptosis confirmed that ABCG2 overexpression enhanced TNBC resistance to 2257 in vitro, while knockdown enhanced sensitivity. These findings suggest that 2257 is a substrate of ABCG2's drug efflux activity. However, overexpression of ABCG2 failed to confer resistance to 2257 in TNBC xenografts grown in mice and treated with a moderately active dose and schedule. Our results highlight the potential impact of drug transporters in in vitro CRISPR screens and the importance of confirming the relevance of drug response mechanisms identified in cultured cells using in vivo models that recapitulate drug pharmacokinetics and pharmacodynamics.},
}
@article {pmid41898703,
year = {2026},
author = {Seh, BA and Rafiq, K and Legradi, A and Mir, MY},
title = {Targeted Gene and Genome-Editing Strategies for Epilepsy: Experimental Advances and Translational Challenges.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898703},
issn = {1422-0067},
mesh = {Humans ; *Epilepsy/genetics/therapy ; *Gene Editing/methods ; *Genetic Therapy/methods ; Animals ; CRISPR-Cas Systems ; Translational Research, Biomedical ; },
abstract = {Epilepsy affects more than 50 million individuals worldwide, and approximately one-third of patients remain refractory to existing antiseizure medications. Advances in gene therapy and genome editing have opened new possibilities for disease-modifying interventions that directly target the molecular and circuit-level mechanisms underlying epileptogenesis. Recent progress in central nervous system tropic viral vectors, non-viral delivery systems, and programmable genome-editing technologies has enabled precise manipulation of neuronal and glial function in preclinical epilepsy models. Strategies range from restoration of haploinsufficient genes implicated in monogenic epilepsies, such as SCN1A in Dravet syndrome, to modulation of neuronal excitability through engineered ion channels, neuropeptides, and astrocyte-based approaches. In parallel, CRISPR-derived platforms, including transcriptional activation and repression systems, base editing, and prime editing, offer new avenues for regulating gene expression in post-mitotic neurons without introducing double-strand DNA breaks. Despite these advances, significant translational challenges remain, including efficient and cell-type-specific delivery, long-term safety, and the risk of network-level side effects in the epileptic brain. This review critically examines recent gene therapy and genome-editing approaches for epilepsy, highlights key technological and biological barriers to clinical translation, and discusses emerging strategies that may enable durable and targeted treatments for drug-resistant epilepsies.},
}
@article {pmid41898716,
year = {2026},
author = {Calbay, O and Hsieh, CL and Lu, C and Ghosh, S and Vijaykumar, V and Watts, I and Sweigard, H and Gandhi, J and den Hollander, AI},
title = {Mapping the Hypoxic Fitness Landscape of Retinal Pigment Epithelial Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {6},
pages = {},
pmid = {41898716},
issn = {1422-0067},
mesh = {*Retinal Pigment Epithelium/metabolism/cytology ; Humans ; Cell Hypoxia/genetics ; Gene Expression Profiling ; Cell Line ; Transcriptome ; Macular Degeneration/genetics/metabolism/pathology ; CRISPR-Cas Systems ; *Epithelial Cells/metabolism ; Mitochondria/metabolism ; *Hypoxia/genetics ; Oxygen/metabolism ; },
abstract = {Chronic hypoxia is a hallmark of aging and retinal diseases such as age-related macular degeneration (AMD), yet the molecular mechanisms that enable retinal pigment epithelium (RPE) cells to survive under sustained low-oxygen conditions remain poorly understood. To address this, we conducted transcriptomic profiling and a genome-wide CRISPR-Cas9 loss-of-function screen in ARPE-19 cells exposed to chronic hypoxia (1% and 5% O2), mimicking the retinal disease environment. The CRISPR screen identified genes whose loss compromises RPE viability or fitness under hypoxia, while transcriptomic profiling revealed oxygen-dependent shifts in key functional modules. These findings converged on pathways related to mitochondrial function, extracellular matrix remodeling, vascular signaling, and cell cycle regulation, identifying unique functional nodes specific to RPE cells. These core processes are also implicated in retinal diseases, such as AMD. Together, these complementary approaches provide an integrated view of the molecular networks driving RPE adaptation to hypoxic stress and highlight novel gene candidates that may serve as therapeutic targets in retinal disease.},
}
@article {pmid41898816,
year = {2026},
author = {Abdalla Elsayed, MEA and MacLaren, RE},
title = {Precision Is Not Enough: When Tools Outpace Translation in Ocular Gene Therapy.},
journal = {Genes},
volume = {17},
number = {3},
pages = {},
pmid = {41898816},
issn = {2073-4425},
support = {//Foundation Fighting Blindness Clinical Research Fellowship/ ; //Oxford NIHR Biomedical Research Centre, UK Department of Health/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; Gene Editing/methods ; *Eye Diseases/therapy/genetics ; CRISPR-Cas Systems ; Translational Research, Biomedical ; Animals ; },
abstract = {Advances in molecular biology have positioned the eye as a leading platform for gene therapy, owing to its surgical accessibility, relative immune privilege, and the ability of the contralateral eye to serve as an anatomical control. We trace the historical evolution of gene discovery, synthesize current gene therapy strategies for inherited and acquired ocular disorders, critically evaluating the limitations of CRISPR and related genome-editing technologies, and examine the key scientific and translational challenges that must be addressed for genetic therapies to be integrated into routine ophthalmic practice.},
}
@article {pmid41899394,
year = {2026},
author = {Aliev, T and Imatdinov, A and Prudnikova, E and Taranov, O and Emtsova, K and Imatdinov, I and Agafonov, A},
title = {The Disruption of the HIV-1 Gag Start Codon via Editing Using MmCas12m-Dual Base Editor-Loaded Virus-like Particles.},
journal = {Current issues in molecular biology},
volume = {48},
number = {3},
pages = {},
pmid = {41899394},
issn = {1467-3045},
support = {The Federal Scientific-technical programme for genetic technologies development for 2019-2030, agreement &#x2116; 075-15-2025-526//The Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m-TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m-TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools.},
}
@article {pmid41900389,
year = {2026},
author = {Shuang, W and Zeng, X and Li, T and Li, J and Sun, Q and Chen, L},
title = {Screening, Safety Assessment, and Process Optimization of Lactic Acid Bacteria from Traditional Yak Yogurt as Adjunct Cultures.},
journal = {Microorganisms},
volume = {14},
number = {3},
pages = {},
pmid = {41900389},
issn = {2076-2607},
support = {Southwest Minzu University (Grant No. ZYN2025240)//the Fundamental Research Funds for the Central Universities/ ; },
abstract = {Cheese ripening is slow and costly, driving interest in accelerating maturation. This study aimed to isolate a safe, efficient adjunct starter from traditional Sichuan yak yoghurt, a niche rich in stress-adapted lactic acid bacteria. From 295 isolates, 15 strains tolerant to high salt, low pH, and low temperature were selected. Using acidification, autolysis, proteolysis, and peptidase activity as indices, principal component analysis identified Limosilactobacillus fermentum 270 as the best candidate. Phenotypic assays showed no haemolysis, gelatin liquefaction, indole production, or amino acid decarboxylase activity. Whole-genome sequencing confirmed species identity and revealed 52 protease/peptidase genes, complete pathways for diacetyl/acetoin biosynthesis and branched-chain amino acid conversion, and no functional biogenic amine synthesis genes. Stress-related genes (F-ATPase, glycine-betaine transport, cold-shock proteins) support cheese adaptability. Antibiotic resistance gene homologs were mainly chromosomal and unlinked to mobile genetic elements; a functional CRISPR-Cas system lowers horizontal transfer risk. The strain was developed as a freeze-dried direct-vat starter (97.3% viability). Orthogonal optimisation of yak Gouda cheese-making defined best conditions: 0.018% adjunct, 45 min acidification, pH 5.8, and 30% curd washing. L. fermentum 270 thus combines proteolytic, flavour-enhancing, genetic safety, and processing traits, offering a promising adjunct for accelerated cheese ripening.},
}
@article {pmid41901428,
year = {2026},
author = {Hou, J and Li, H and Zhang, F and Yang, D and Xiong, Y and Zhu, X and Wen, M},
title = {From Gene Knockouts to Genome Remodeling: Large DNA Fragment Deletion Technologies in Plants.},
journal = {Plants (Basel, Switzerland)},
volume = {15},
number = {6},
pages = {},
pmid = {41901428},
issn = {2223-7747},
support = {2023YFA0913500//National Key Research and Development Program of China/ ; 25HHWCSS0007//Haihe Laboratory of Sustainable Chemical Transformations/ ; 32230012//the National Natural Science Foundation of China/ ; 32470363//the National Natural Science Foundation of China/ ; },
abstract = {Large DNA fragment deletion (LDFD) provides a powerful means to reconfigure plant genomes at the kilobase to megabase scale, enabling the dissection of genome function, elucidation of non-coding regulatory elements, modulation of gene dosage, reorganization of chromosomal architecture, and implementation of synthetic biology designs. In this review, we systematically compare the mechanisms, efficiencies, advantages, and limitations of the major LDFD technologies that have been applied in plants, including ZFNs, TALENs, CRISPR/Cas systems (Cas9, Cas12a, Cas3), site-specific recombinases, transposon-based systems, and prime editing-derived strategies. We highlight how plant-specific features of chromatin organization and DNA repair constrain large deletions, and discuss the current bottlenecks in achieving efficient, precise, and predictable LDFD across diverse crop genomes. Finally, we outline future directions for plant LDFD, emphasizing AI-assisted design of nucleases and recombinases, protein-directed evolution, and improved DNA- and RNP-based delivery systems. Together, these advances are expected to transform LDFD from a specialized tool into a broadly accessible platform for functional genomics, trait engineering and rational genome design in plants.},
}
@article {pmid41902223,
year = {2026},
author = {Zhang, J and Dang, TT and Lin, TY and Yu, X and Pellin, D and Tian, J and Simmons, O and Kou, E and Cornetta, K and Xiao, W},
title = {Development of a Novel Method to Detect AAV Vector Integration.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902223},
issn = {1999-4915},
support = {P01HL160472//National Institute of Health/ ; 75N92019D00018/HL/NHLBI NIH HHS/United States ; },
mesh = {*Dependovirus/genetics/physiology ; Humans ; CRISPR-Cas Systems ; *Genetic Vectors/genetics ; *Virus Integration ; HeLa Cells ; Nanopore Sequencing ; High-Throughput Nucleotide Sequencing ; },
abstract = {AAV integration has become an important safety consideration in gene therapy. However, accurately determining integration sites remains challenging due to biases introduced by library preparation methods, sequencing technologies, and bioinformatic pipelines. In this study, we developed a PCR-free amplification based on a CRISPR-Cas9 cleavage strategy for AAV DNA that overcomes the limitations of PCR amplification imposed by the ITR structure. When combined with long-read nanopore sequencing, this CRISPR-Cas9-based workflow preserves native AAV integration states and enables unbiased detection of integration junctions. We used AAV-transduced HeLa single-cell clones to evaluate the performance of this approach. To confirm integration site identification, AAV integration junctions were also detected using a probe hybridization capture strategy followed by Illumina short-read sequencing. Integration junctions identified by both methods were further confirmed by PCR. The results showed strong consistency between the two approaches in accurately identifying AAV integration sites in each clone. Overall, these findings demonstrate that the CRISPR-Cas9-enabled, PCR-free long-read sequencing workflow provides a promising tool for characterizing AAV integration events.},
}
@article {pmid41902231,
year = {2026},
author = {Qi, M and Liu, X and Wang, W and Lu, M and Zeng, Q and Li, N and Han, Y and Fan, S and Lu, C and Dai, J},
title = {Tree Shrew Genome-Wide CRISPR Screen Identifies RNF6 as a Proviral Host Factor for Zika Virus Replication in Brain Microvascular Endothelial Cells.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902231},
issn = {1999-4915},
support = {2019-1-R-24483//the Bureau of Science and Technology of Kunming/ ; 2017HC019//the Yunnan Science and Technology Talent and Platform Program/ ; D-2024006//the Training Program for High-level Health and Medical Technology Talents in Yunnan Province/ ; },
mesh = {Animals ; *Endothelial Cells/virology ; *Zika Virus/physiology/genetics ; *Virus Replication ; *Brain/virology/blood supply ; Humans ; *Zika Virus Infection/virology/metabolism ; CRISPR-Cas Systems ; *Host-Pathogen Interactions ; Viral Nonstructural Proteins/metabolism ; Blood-Brain Barrier/virology ; },
abstract = {Zika virus (ZIKV), a unique flavivirus with neurotropic and teratogenic potential, can cross the blood-brain barrier and persist in human brain microvascular endothelial cells (BMECs); however, no approved vaccines or specific antivirals exist, and its barrier-crossing and neuroinvasive mechanisms remain elusive. Innovative strategies to identify additional host factors mediating ZIKV infection could yield key insights and help address these challenges. To uncover novel host factors, we established the first tree shrew (Tupaia belangeri) genome-wide CRISPR/Cas9 knockout (GeCKO) library and performed a screen in BMECs, identifying ring finger protein 6 (RNF6) as a novel proviral factor for ZIKV. ZIKV infection in BMECs was significantly reduced following RNF6 knockout or knockdown but enhanced upon RNF6 overexpression or rescue. Mechanistically, RNF6 interacts with the ZIKV NS5 protein and acts as a potential negative regulator of the type I interferon and MAPK signaling pathways. Evolutionary and structural analyses revealed that RNF6 is highly conserved between humans and tree shrews; molecular docking further identified shared NS5-binding residues (Gln-59, Arg-140), supporting the conserved proviral role of human RNF6 in ZIKV infection. Our findings highlight tree shrew GeCKO screening as an efficient approach for identifying novel host factors and establish RNF6 as a critical proviral factor for ZIKV replication in BMECs, providing new insights into ZIKV neurotropic pathogenesis and informing potential antiviral strategies.},
}
@article {pmid41902262,
year = {2026},
author = {Jones, JE and Gunderson, CE and Wigdahl, B and Nonnemacher, MR},
title = {Breaking into HIV-1's Epigenetic Vault: Cure Strategies to Eliminate the Viral Reservoir.},
journal = {Viruses},
volume = {18},
number = {3},
pages = {},
pmid = {41902262},
issn = {1999-4915},
support = {MH110360/MH/NIMH NIH HHS/United States ; MH092177/MH/NIMH NIH HHS/United States ; MH079785/MH/NIMH NIH HHS/United States ; },
mesh = {*HIV-1/genetics/drug effects/physiology ; Humans ; *Virus Latency/drug effects ; *Epigenesis, Genetic/drug effects ; *HIV Infections/virology/drug therapy/therapy ; Proviruses/genetics/drug effects ; Chromatin ; *Anti-HIV Agents/pharmacology ; CRISPR-Cas Systems ; Animals ; },
abstract = {Human immunodeficiency virus type 1 (HIV-1) is a retrovirus that integrates into the host cell's DNA as a provirus. Transcription from the provirus is regulated in large part by cellular proteins and epigenetic factors. These may be repressive or permissive to productive infection. The host factors that regulate this balance are therefore attractive targets for HIV-1 therapeutics. Indeed, proviral chromatin is the focus of two of the current HIV-1 cure strategies. "Shock and Kill" uses latency reversal agents to open the provirus's chromatin, promoting high levels of gene expression that induce the killing of infected cells. "Block and Lock" uses latency promoting agents to induce heterochromatin, blocking transcription and forcing HIV-1 into a state of deep latency. Here, the compounds investigated in both strategies are reviewed, including their chemical structures, mechanisms of action, and clinical results. Finally, the use of CRISPR-Cas therapeutics and the impact of chromatin architecture on its efficacy are discussed.},
}
@article {pmid41903969,
year = {2026},
author = {Chen, F and Huang, M and Zeng, S and Wan, Y and Qin, A and Yang, X and Fu, D and Negahdary, M and Zhang, C},
title = {A CRISPR/dCas9 mediated electrochemical impedimetric biosensor for sensitive mtDNA detection.},
journal = {Analytica chimica acta},
volume = {1401},
number = {},
pages = {345323},
doi = {10.1016/j.aca.2026.345323},
pmid = {41903969},
issn = {1873-4324},
mesh = {*DNA, Mitochondrial/genetics/analysis ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; Metal Nanoparticles/chemistry ; Limit of Detection ; Electrodes ; Polymorphism, Single Nucleotide ; Silver/chemistry ; },
abstract = {BACKGROUND: The accumulation of mitochondrial DNA (mtDNA) mutations in cells is closely linked to various human diseases. Detection of single-nucleotide variation (SNV) in mtDNA plays a crucial role in understanding the heteroplasmy of mtDNAs that contain pathogenic changes. While conventional nucleic acid sequencing-based methods are instrumental and complex, which hampered their capability in revealing the extensive diversity of mtDNA. In order to realize trace DNA analysis, recent CRISPR/Cas-based detection methods facilitated with target pre-amplification, while raised the risks of non-specific amplification and cross-carryover contamination. Thus, it is imperative to develop new methods for sensitive and precise SNV detection in mtDNA.<br><br>RESULTS: This study developed a CRISPR/dCas9 (deactivated Cas9) mediated electrochemical impedimetric biosensor without target pre-amplification for sensitive and specific detection of SNVs in mtDNA. dCas9/sgRNA complexes were immobilized on the surface of indium tin oxide (ITO) electrode to specifically recognize target mtDNA sequences and initiate hybridization chain reaction (HCR) for signal amplification. Subsequently, positively charged polyethylenimine-coated silver nanoparticles (PEI-Ag NPs) were electrostatically deposited onto the HCR-generated long double-stranded DNA (dsDNA) products, leading to a marked decrease in electrochemical impedance due to the high conductivity of the nanoparticles. The concentration of mtDNA was thus quantified by monitoring the impedance change via electrochemical impedance spectroscopy (EIS). The method could distinguish single- and multi-base mismatches with a low detection limit of 67&#xa0;fM without pre-amplification. It exhibits excellent anti-interference ability and excellent recovery rates of 90.0% to 108.0% in complex matrices (10% human plasma), and enables accurate mtDNA detection in cell lysates.<br><br>SIGNIFICANCE: This free of pre-amplification strategy offers a highly sensitive analytical tool that enables the successfully detection of mtDNA mutation in diverse cells types, and exhibited excellent anti-interference ability in complicated biological specimen. The work presents a viable and promising strategy for the electrochemical detection of cancer-related biomarkers, indicating substantial potential in early clinical diagnosis.},
}
@article {pmid41905104,
year = {2026},
author = {Koutala, E and Cantarini, C and Raymond, K and Lebrin, F},
title = {Generation of ALK1 p.Gly48Glu mutant LUMCi029-A-3 for modeling Hereditary hemorrhagic telangiectasia type 2.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103974},
doi = {10.1016/j.scr.2026.103974},
pmid = {41905104},
issn = {1876-7753},
mesh = {Humans ; *Telangiectasia, Hereditary Hemorrhagic/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Activin Receptors, Type II/genetics/metabolism ; *Mutation/genetics ; CRISPR-Cas Systems ; Cell Line ; Gene Editing ; },
abstract = {Hereditary hemorrhagic telangiectasia type 2 (HHT2) is an autosomal dominant vascular disorder caused by pathogenic variants in ACVRL1, which encodes activin receptor-like kinase 1 (ALK1). Here, we report the generation and characterization of an isogenic human induced pluripotent stem cell (hiPSC) line carrying a heterozygous ACVRL1 c.143G > A (p.Gly48Glu) mutation. The mutation was introduced using CRISPR/Cas9-mediated genome editing and confirmed by PCR and Sanger sequencing. The edited line retained normal karyotype, pluripotency, and trilineage differentiation capacity. This hiPSC line represents a relevant in vitro model for HHT2 disease modelling and drug testing.},
}
@article {pmid41905455,
year = {2026},
author = {Meng, Y and Zhang, Z and Jia, K and Wang, W and Wang, H and Hu, Z and Liu, J},
title = {Membrane protein-focused CRISPR screen identifies ATP2A2 as a druggable transcriptional co-regulator of CCND1 (cyclin D1) in lung adenocarcinoma.},
journal = {Life sciences},
volume = {394},
number = {},
pages = {124358},
doi = {10.1016/j.lfs.2026.124358},
pmid = {41905455},
issn = {1879-0631},
mesh = {Humans ; *Lung Neoplasms/genetics/pathology/metabolism/drug therapy ; *Adenocarcinoma of Lung/genetics/metabolism/pathology/drug therapy ; Animals ; *Cyclin D1/genetics/metabolism ; Mice ; Cell Proliferation ; A549 Cells ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Apoptosis ; Cell Line, Tumor ; Mice, Nude ; Carcinoma, Non-Small-Cell Lung/genetics/pathology/metabolism ; Cell Movement ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; },
abstract = {INTRODUCTION: Lung adenocarcinoma (LUAD) is one of the most commonly seen non-small cell lung cancer (NSCLC). Recent progress has highlighted cyclin-dependent kinase (CDK) inhibitors for treating diverse cancers including NSCLC. However, acquired resistance of CDK inhibitors has prompted the exploration of druggable regulatory or compensatory pathways in cell cycle as alternative or combination therapies of CDK inhibitors.<br><br>AIM: This study aims to discover the upstream regulatory proteins in LUAD using a membrane protein-focused CRISPR screen in A549 cells and dissect their functions.<br><br>MATERIALS AND METHODS: The top hit ATP2A2, an endoplasmic reticulum (ER)-localized protein, was analyzed for its function in LUAD using cell proliferation assay, migration and invasion assays and apoptosis assay. The mechanistic studies of ATP2A2 were conducted using RT-qPCR, Western blotting, RNA-Seq, cell cycle assay, protein immunoprecipitation, mass spectrometry, immunofluorescence microscopy and others.<br><br>KEY FINDINGS: The correlation of ATP2A2 expression with NSCLCs was confirmed with public database and collected clinical samples. ATP2A2 promoted LUAD development by involving in cell cycle regulation. ATP2A2 interacted with another ER protein HACD3 and co-regulated the transcription of CCND1 gene (cyclin D1), which forms complex with CDK4/6. ATP2A2 and HACD3 promoted CCND1 expression by activating NF-&#x3ba;B signaling and the nuclear translocation of p65 protein, which is a known transcription factor of CCND1. Curcumin, an ATP2A2-targeting natural product, could inhibit LUAD both in vitro and in xenografted mouse model.<br><br>SIGNIFICANCE: This study revealed ATP2A2-HACD3-NF-&#x3ba;B as a regulatory axis of cyclin D1 expression and shed light on developing ATP2A2-targeted medications for LUAD treatment.},
}
@article {pmid41905499,
year = {2026},
author = {Fu, T and Wang, F and Ren, J and Chen, S and Peng, N and Zhang, L and Pu, K and Li, Q},
title = {An extraction-free HLPCR-Cas12a assay for ultra-sensitive and rapid detection of Acinetobacter baumannii and Klebsiella pneumoniae following craniotomy.},
journal = {Journal of microbiological methods},
volume = {244},
number = {},
pages = {107480},
doi = {10.1016/j.mimet.2026.107480},
pmid = {41905499},
issn = {1872-8359},
mesh = {*Acinetobacter baumannii/isolation &amp; purification/genetics ; Humans ; *Klebsiella pneumoniae/isolation &amp; purification/genetics ; *Craniotomy/adverse effects ; Sensitivity and Specificity ; *Acinetobacter Infections/diagnosis/microbiology ; *Polymerase Chain Reaction/methods ; *Klebsiella Infections/diagnosis/microbiology ; CRISPR-Cas Systems ; Postoperative Complications/microbiology/diagnosis ; },
abstract = {Postoperative intracranial infection represents a significant complication in neurosurgical procedures, with Acinetobacter baumannii (Ab) and Klebsiella pneumoniae (Kp) frequently identified as common drug-resistant pathogens. In response to this challenge, we have developed an efficient, extraction-free detection method that facilitates the rapid and highly sensitive identification of Ab and Kp. This method employs a single-tube format that integrates heat lysis with PCR, allowing for the concurrent bacterial lysis and target gene amplification. Detection of the amplified products is achieved through the Cas12a/crRNA complex, which, upon activation, exhibits trans-cleavage activity that cleaves reporters to produce a signal detectable by microplate readers or lateral flow test strips (LFTS). The Heat Lysis-PCR-CRISPR/Cas12a (HLPCR-CRISPR/Cas12a) detection platform unifies lysis and amplification in one step, streamlining the process. It exhibits high specificity (no cross-reactivity) and achieves detection limits of 10[2] CFU/μL (fluorescence) and 10[3] CFU/μL (LFTS). When validated on clinical CSF samples, it delivered results in 90 min with perfect concordance with quantitative PCR (qPCR). Combining speed, high sensitivity, specificity, and operational simplicity, this platform provides a powerful diagnostic tool for early detection of Ab and Kp in postoperative intracranial infections.},
}
@article {pmid41905621,
year = {2026},
author = {Singh, RP and Maity, P and Jaiswal, C},
title = {Genome editing in Parkinson's disease: Unlocking therapeutic avenues through CRISPR-Cas systems.},
journal = {Neurochemistry international},
volume = {196},
number = {},
pages = {106149},
doi = {10.1016/j.neuint.2026.106149},
pmid = {41905621},
issn = {1872-9754},
abstract = {Parkinson's disease (PD) is an illness that causes both motor and non-motor symptoms in the patient which occurs as a result of a progressive loss of dopamine-producing neurons in the substantia nigra. Even though the success of symptomatic treatments is promising, at the same time there is currently no effective therapy that can halt or reverse disease progression. Key genes such as SNCA, LRRK2, and PINK1 are considered as the main hopefuls aspect for the treatment of Parkinson's because mutations of these genes are the reason for the appearance of the familial and sporadic kinds of the disease, respectively. The CRISPR-Cas system, a breakthrough genome-editing technology which enables precise and targeted genetic modifications, renders the possibilities of both PD research and therapy. Examining the mechanics of prime editing, base editing, and CRISPR-Cas9 highlights how effective and precise these methods are for modifying genes. An overview of recent developments in the use of CRISPR to create PD models is also included in the current review, with a focus on the roles these models play in clarifying disease pathways and locating new treatment targets. These models include isogenic cell lines, transgenic animals, and induced pluripotent stem cells (iPSCs). This review highlights the potential of CRISPR-based strategies to correct PD-associated mutations, modulate pathogenic gene expression, and develop neuroprotective interventions targeting key processes such as mitochondrial dysfunction. Furthermore, it critically evaluates the role of CRISPR-based technologies as transformative tools in PD research and therapy while highlighting key challenges for their clinical translation.},
}
@article {pmid41908308,
year = {2026},
author = {Xu, G and Li, S and Li, H and Ren, X and Ding, Y and Pang, X and Liu, X and Tang, Q and Tu, T and Wang, Y and Luo, H and Yao, B and Tian, J and Chen, R and Guan, F},
title = {CasMiner: a deep-learning tool for high-throughput mining and rational design of efficient Cas9.},
journal = {National science review},
volume = {13},
number = {6},
pages = {nwag090},
pmid = {41908308},
issn = {2053-714X},
abstract = {Since its inception, the CRISPR-Cas system, particularly Cas9, has demonstrated immense potential for life science applications, but expansion of the Cas9 toolkit is constrained by sequence-alignment-based strategies for mining and optimization. Here, we developed CasMiner-a deep-learning model for discovering and engineering novel Cas9 proteins. CasMiner achieved 99.63% accuracy in predicting Cas9s and identified VpCas9 from public databases. Experimental validation showed that VpCas9 exhibits robust double-strand cleavage activity. Combining CasMiner and evolutionary analysis, we engineered three mutants with markedly increased structural rigidity and positive charge. In vivo cleavage assays revealed that the mutant VPM2-3 achieved a higher average editing efficiency in rice callus and maize protoplasts than the wild-type VpCas9, the editing efficiency of which rivals that of SpCas9. This study thus establishes a comprehensive platform for mining and engineering Cas9 proteins, and provides VpCas9 and derivative nucleases as powerful tools that greatly broaden the horizon for genome-editing applications.},
}
@article {pmid41909946,
year = {2026},
author = {Zhong, H and Zhou, J and Qin, F and Zhang, XE and Chen, M},
title = {Programmable, target-induced fluorogenic CRISPR-tDeg platform for live-cell RNA visualization.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909946},
issn = {1362-4962},
support = {2023YFA0915603//National Key R&amp;D Program of China/ ; 32101210//National Natural Science Foundation of China/ ; //Young Elite Scientist Sponsorship Program by CAST/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *RNA, Viral/genetics/metabolism ; Fluorescent Dyes/chemistry ; COVID-19/virology ; *RNA ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {RNA molecules display remarkable heterogeneity in structure, dynamics, and function, yet methods for their precise visualization in living cells remain limited. While CRISPR-based RNA imaging holds great potential, existing systems often suffer from high background fluorescence due to constitutive signal emission or non-specific binding. To overcome these challenges, we developed CtDeg (CRISPR-dCas13-tDeg), a modular RNA imaging platform that links fluorescence activation directly to target RNA recognition while leveraging degron-mediated degradation to suppress background signals. By engineering the crRNA scaffold to embed the Pepper RNA motif, CtDeg ensures that fluorescence is present only upon binding to the native RNA target. We systematically optimized C-terminal tDeg variants to maximize the signal-to-noise ratio and demonstrated that CtDeg achieves substantially lower background and higher specificity than conventional fluorescent protein-CRISPR-based RNA imaging approaches. Using CtDeg, we captured real-time paraspeckle assembly dynamics and visualized early-stage SARS-CoV-2 genomic RNA transport. Remarkably, CtDeg provided the first direct imaging evidence of virus-induced NEAT1_2 lncRNA accumulation, revealing a host-virus regulatory interaction. Beyond these applications, CtDeg is compatible with multiple Cas13 orthologs and fluorescent proteins, establishing a versatile, target-induced platform for probing RNA localization, dynamics, and function in living cells, with broad applications in synthetic biology and RNA biology.},
}
@article {pmid41909947,
year = {2026},
author = {Hille, F and Wang, C and Finstermeier, K and Beerens, D and Schmidt, K and Skibbe, M and Charpentier, E},
title = {Insights into spacer acquisition of the type V-A CRISPR-Cas system of Francisella novicida U112.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909947},
issn = {1362-4962},
support = {//Alexander von Humboldt Foundation/ ; //German Research Foundation/ ; //Max Planck Society/ ; },
mesh = {*Francisella/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Escherichia coli/genetics ; DNA, Single-Stranded/metabolism/genetics ; Plasmids/genetics ; Bacterial Proteins/metabolism/genetics ; Integrases/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Prophages/genetics ; },
abstract = {CRISPR-Cas systems immunize prokaryotic cells through a CRISPR adaptation process, in which short DNA fragments from foreign elements are acquired and integrated into the CRISPR array. Here, we investigated the spacer acquisition mechanism of the type V-A CRISPR-Cas system from Francisella novicida U112. We characterized the Cas1-Cas2 integrase in vitro and elucidated the sequence requirements of the pre-spacer and the CRISPR array for optimal spacer incorporation. We demonstrated that Cas2 coordinates metals at its active site to facilitate full-site spacer integration. Furthermore, we introduced this spacer acquisition system into Escherichia coli cells and observed that, in vivo, all Cas proteins are required for efficient type V-A adaptation, with Cas12 significantly improving adaptation efficiency. We showed that spacers were acquired preferentially from plasmids encoding cas genes, and from genomic regions of prophages and origins of replication. In addition, we found that Cas4 possesses a 3'-5' exonuclease activity against single-stranded DNA and an ATP-independent unwinding activity towards double-stranded DNA. Cas4 interacts with the Cas1-Cas2 complex and processes pre-spacers in a PAM-dependent manner. The presence of Cas4 in vivo ensures that new spacers are derived from DNA immediately downstream of a 5'-TTTN-3' PAM, which is critical for targeting invaders.},
}
@article {pmid41909950,
year = {2026},
author = {Deng, J and Zhou, J and Xiang, H and Li, X and Han, X and Weng, Z and Jia, J and Shao, Y and Sima, Y and Niu, M and Li, D and Ouyang, H and Xu, B and Pang, D and Yang, L and Yuan, H},
title = {Regulated transformation system (RTS): sddi-mediated programmable shut-off and mode switching of base editors.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41909950},
issn = {1362-4962},
support = {20260102218JC//Jilin Provincial Science and Technology Development Plan Project/ ; 2025YFF1000800//National Key Research and Development Program of China/ ; 32202754//National Natural Science Foundation of China/ ; 32372962//National Natural Science Foundation of China/ ; 20230205117RC//Jilin Province science and technology development plan project/ ; },
mesh = {Humans ; *Gene Editing/methods ; Animals ; CRISPR-Cas Systems ; CRISPR-Associated Protein 9/genetics/metabolism ; Cytosine/metabolism ; HEK293 Cells ; Adenine/metabolism ; Cell Line ; DNA, Single-Stranded/metabolism/genetics ; },
abstract = {Orthogonal and externally controllable base editors are critical for safe multiplexed single-nucleotide manipulation in vivo. Here, we identify ~140-aa miniature deaminase inhibitors (Sddis) that bind cognate single-stranded DNA deaminases (Sdds) with high affinity and specificity, occluding their DNA-binding surfaces to completely inhibit C-to-T activity. Based on these inhibitors, we engineer an adenine and cytosine base editing-regulated transformation system (ACBE-RTS). This platform features two inactive dSdds fused to nCas9 as docking arms, with effector modules provided by doxycycline-inducible SviSddi-SflSdd (CBE) and cumate-inducible Air1Sddi-ABE8e (ABE) fusions. Small-molecule regulation enables switching among four modes (OFF, CBE, ABE, ACBE), achieving up to 43.4% C-to-T or 42.9% A-to-G editing at four endogenous human sites. Using a 4000-member sgRNA library in MARC-145 cells stably expressing ACBE-RTS, a three-round screening identified four key amino acids in monkey CD163 that reduced replication of highly pathogenic PRRSV by >100-fold and eliminated detectable viral-antigen staining. Compact and multi-mode switchable on a single Cas9 scaffold, ACBE-RTS establishes a versatile framework for precision therapeutics and genetic interrogation. Its modular Sddi-Sdd interface could in principle be readily extended to other base editors, such as thymine and guanine base editors (TBE and GBE).},
}
@article {pmid41911023,
year = {2026},
author = {Ye, J and Tang, X and Chen, Y and Guo, R and Zhang, W and Wang, M and Yang, X and Deng, W and Zhang, Y and Feng, X and Hu, N and Zhang, D},
title = {Conformationally Regulated CRISPR/Cas12a Activation Enabled by a Programmable DNA Dumbbell for Electrochemical SNP Genotyping.},
journal = {Analytical chemistry},
volume = {98},
number = {14},
pages = {10814-10823},
doi = {10.1021/acs.analchem.6c00333},
pmid = {41911023},
issn = {1520-6882},
mesh = {*Polymorphism, Single Nucleotide ; *CRISPR-Cas Systems/genetics ; *Electrochemical Techniques/methods ; *DNA/chemistry/genetics ; *Genotyping Techniques/methods ; *CRISPR-Associated Proteins/metabolism/genetics ; *Biosensing Techniques/methods ; Genotype ; *Endodeoxyribonucleases/metabolism/genetics ; Bacterial Proteins ; },
abstract = {Single-nucleotide polymorphism (SNP) genotyping is crucial for genetic research and precision medicine, yet reliable discrimination of single-base variants in complex genomic backgrounds remains analytically challenging. Although CRISPR/Cas12a-based biosensing offers high sequence specificity, its intrinsic mismatch tolerance often leads to nonspecific activation by wild-type sequences, thereby compromising SNP fidelity. Herein, we report a highly sensitive electrochemical sensing platform based on a programmable DNA dumbbell (Dum) probe that functions as a conformational energy-barrier regulator of CRISPR/Cas12a activation. The closed-loop dumbbell architecture sterically shields the crRNA-activating sequence, establishing a high activation threshold that suppresses nonspecific Cas12a triggering. Only precise SNP hybridization induces a thermodynamically favorable conformational rearrangement, releasing the mediator and transitioning the CRISPR system from an inactive to an active state. To further enhance analytical sensitivity and reliability, nucleic acid-functionalized FeCo nanozymes were incorporated as catalytic signal transducers, enabling a self-validating dual-mode signal electrochemical readout through intrinsic metal redox and H2O2 electrocatalysis. The resulting platform achieved reliably discriminates mutation abundances down to 0.1%. Validation using soybean genomic DNA samples demonstrates the robustness and practical applicability of the proposed strategy. This work establishes a conformational energy-barrier-regulated CRISPR activation paradigm, providing a generalizable analytical framework for high-fidelity SNP genotyping in molecular breeding and clinical diagnostics.},
}
@article {pmid41911152,
year = {2026},
author = {Wu, Y and Qi, Y and Chen, Y and Liu, D and Liu, Q and Shang, X},
title = {CrisprPr: a hybrid-driven framework for CRISPR/Cas9 off-target prediction with analysis of prior-information updates.},
journal = {Briefings in bioinformatics},
volume = {27},
number = {2},
pages = {},
pmid = {41911152},
issn = {1477-4054},
support = {62433016//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; Deep Learning ; Computational Biology/methods ; Algorithms ; },
abstract = {CRISPR/Cas9 specificity is critically affected by off-target effects. However, the complex patterns of mismatches and their combinations at off-target sites remain difficult to capture, and existing approaches show limited capacity to identify informative features. Here, we present CrisprPr, a hybrid-driven off-target prediction framework that integrates both prior information and data-driven modeling to improve the characterization of off-target activity. CrisprPr employs a synchronous updating strategy that jointly optimizes prior-knowledge and deep-learning modules, together with multi-source integration, to deliver accurate and stable off-target predictions. Evaluations on independent test sets indicate that CrisprPr achieves competitive predictive performance and generalization compared with existing deep learning methods, with statistically significant improvements observed on several datasets. Beyond predictive performance, its analysis module examines the patterns of prior embedding-space updates to reveal distinctive target-site features supported by literature evidence. Overall, CrisprPr proposes a novel framework that demonstrates competitive predictive performance while offering new insights into the characteristics of off-target effects.},
}
@article {pmid41911342,
year = {2026},
author = {Chi, H and McMahon, SA and Graham, S and White, MF},
title = {The CRISPR ring nuclease Csx15 oligomerises on cyclic nucleotide binding to regulate antiviral defence.},
journal = {The Biochemical journal},
volume = {483},
number = {5},
pages = {699-712},
doi = {10.1042/BCJ20260019},
pmid = {41911342},
issn = {1470-8728},
support = {101018608//EC | European Research Council (ERC)/ ; },
mesh = {Protein Multimerization ; *Nucleotides, Cyclic/metabolism/chemistry ; Protein Binding ; *Adenine Nucleotides/metabolism/chemistry ; Binding Sites ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; CRISPR-Cas Systems ; Oligoribonucleotides ; },
abstract = {Prokaryotic type III CRISPR systems signal infection by generating cyclic oligoadenylate (cOA) second messengers, which activate defence proteins allosterically, providing immunity. cOA molecules are typically degraded by extrinsic, stand-alone ring nuclease (RN) enzymes with phosphodiesterase activity or by the intrinsic RN activity of the effectors themselves. Viruses and plasmids also encode RNs, which can function as anti-CRISPRs. Eight different families of extrinsic RNs are currently known. Here, we report the structural and biochemical analysis of one of these families: Csx15. We show that Csx15 is a dimeric protein of the CRISPR-associated Rossmann fold (CARF) superfamily with the ability to bind cyclic tetra-adenylate (cA4) molecules in a shared binding site formed by the head-to-tail stacking of dimers in a filament conformation. Some family members are non-enzymatic, relying on the sequestration (sponging) of cA4 to regulate the host immune response, while others act as canonical RNs, slowly degrading cA4.},
}
@article {pmid41911457,
year = {2026},
author = {Waild, NC and Ciesla, JH and Schafer, XL and Munger, J},
title = {Cellular antibody affinity-based CRISPR screening identifies JUNB as a broadly acting antiviral factor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {14},
pages = {e2534154123},
pmid = {41911457},
issn = {1091-6490},
support = {AI150698//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; AI118689//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R01 AI184380/AI/NIAID NIH HHS/United States ; GM068411//HHS | NIH | National Institute of General Medical Sciences (NIGMS)/ ; R01 AI181865/AI/NIAID NIH HHS/United States ; AI181865//HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; },
mesh = {Humans ; *Cytomegalovirus/genetics/immunology ; *Cytomegalovirus Infections/genetics/immunology/virology ; *CRISPR-Cas Systems ; *Antibody Affinity ; *Transcription Factors/genetics/metabolism/immunology ; Viral Proteins/genetics/metabolism ; Virus Replication ; *Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; },
abstract = {CRISPR screening is a powerful approach to identify genetic perturbations that impact viral infection. However, most virus-focused CRISPR screens utilize selection strategies that limit the ability to identify genes important for infection. Here, we developed a CRISPR screening pipeline to identify cellular determinants of human cytomegalovirus (HCMV) infection based on virally induced remodeling of cellular antibody affinity (VIRCAA), which is scalable for large libraries and can identify cellular genes that impact HCMV infection at different life cycle stages. We utilized this pipeline to interrogate proteomic and transcriptomic datasets associated with the HCMV UL26 protein, which blocks antiviral signaling during infection. We find that JUNB drives antiviral gene expression, induces protein ISGylation, and suppresses diverse viral infections. Further, UL26 interacts with JUNB and suppresses JUNB-mediated condensation of viral DNA replication compartments. These results highlight the VIRCAA pipeline's utility for identifying important determinants of viral infection.},
}
@article {pmid41911458,
year = {2026},
author = {Richards, L and Lee, D and Wiktor, J and Truedson, A and Cederblad, J and Jones, D},
title = {Molecular kinetics dictate population dynamics in CRISPR-based plasmid defense.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {14},
pages = {e2525424123},
pmid = {41911458},
issn = {1091-6490},
support = {2020-05137//Swedish Research Council/ ; CTS 21:1334//Carl Tryggers Foundation/ ; },
mesh = {*Plasmids/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Kinetics ; *Escherichia coli/genetics ; Conjugation, Genetic ; Single-Cell Analysis ; },
abstract = {Understanding and manipulating the spread of mobile genetic elements represents a great challenge with potential benefits across synthetic biology, agriculture, and medicine. A key part of this challenge is the multiple scales in play, from the molecular kinetics of defense systems such as CRISPR-Cas, to single-cell variability in immunity levels, to spatial structure in bacterial populations. In this work, we use a time-lapse, imaging-based approach to characterize conjugative plasmid dynamics at the molecular, single-cell, and population levels. By fluorescently tagging the conjugative plasmid RP4 and CRISPR-Cascade complexes, we quantify population dynamics as a function of spacer target number, Cascade expression level, and the presence of plasmid addiction modules. Using single-cell tracking, we report conjugation rate per neighboring donor cell, estimate the latent period between plasmid uptake and subsequent onward transmission, and quantify the effect of Cascade expression variability on plasmid clearance kinetics. Finally, using a spatially resolved, agent-based model, we show that plasmid population dynamics can be successfully predicted using these single-cell biophysical parameters as inputs. This synthesis of population and single-cell measurements suggests that plasmids are the subject of a dynamic tug-of-war between defense expression, spacer distribution, neighboring cell identity, and plasmid cost-benefit tradeoffs. The imaging and analysis techniques used here will facilitate the disentanglement of how these factors coordinate to realize community-wide plasmid dynamics in diverse contexts.},
}
@article {pmid41912937,
year = {2026},
author = {Zhang, D and Wang, H},
title = {Liquid Biopsy in Uterine Leiomyosarcoma: Current Biomarkers, Emerging Technologies, and Future Perspectives.},
journal = {Current oncology reports},
volume = {28},
number = {1},
pages = {},
pmid = {41912937},
issn = {1534-6269},
mesh = {Humans ; *Leiomyosarcoma/diagnosis/genetics/pathology/blood ; Liquid Biopsy/methods ; *Uterine Neoplasms/diagnosis/genetics/pathology/blood ; Female ; *Biomarkers, Tumor/genetics/blood ; Neoplastic Cells, Circulating/pathology ; Circulating Tumor DNA/blood ; },
abstract = {PURPOSE OF REVIEW: Uterine leiomyosarcoma (uLMS) is a rare but aggressive malignant mesenchymal tumor, accounting for 2-5% of uterine malignancies. Because its symptoms and imaging features often resemble those of benign uterine leiomyoma (LM), accurate preoperative diagnosis remain difficult. This review summarizes recent advances in liquid biopsy for uLMS and explores its potential for early detection, molecular characterization, and treatment monitoring.<br><br>RECENT FINDINGS: Liquid biopsy enables minimally invasive detection of tumor-derived components such as circulating tumor DNA (ctDNA), circulating tumor cells (CTCs), non-coding RNAs, and extracellular vesicles (EVs). Recurrent mutations in TP53, RB1, and ATRX have been identified through ctDNA analysis, while CTCs, ncRNAs, and EVs provide complementary information for monitoring tumor dynamics and therapeutic response. Emerging technologies including CRISPR-Cas systems, nanotechnology, electrochemical biosensors, and multi-omics integration enhance detection sensitivity and specificity. Liquid biopsy holds promise for improving uLMS diagnosis and management. However, standardization and biomarker validation remain essential to achieve reliable clinical translation and enable earlier, more precise treatment strategies.},
}
@article {pmid41914300,
year = {2026},
author = {Wang, W and Li, L and Li, X and Zhang, Q and Liu, Y},
title = {Aptamer- and Ribozyme-Engineered sgRNAs for Conditional Control of CRISPR/Cas9 Function.},
journal = {Frontiers in bioscience (Landmark edition)},
volume = {31},
number = {3},
pages = {47300},
doi = {10.31083/FBL47300},
pmid = {41914300},
issn = {2768-6698},
support = {2024B02//Fund for Creative Research of The Second People's Hospital of Foshan/ ; 4SG24185G//Postdoctoral Initial Foundation of Guangdong Medical University/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Catalytic/genetics/metabolism ; *Aptamers, Nucleotide/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Synthetic Biology/methods ; Genetic Engineering/methods ; Animals ; },
abstract = {The clustered regularly interspaced short palindromic repeats CRISPR-associated protein 9 (CRISPR/Cas9) system has emerged as a versatile platform for genome editing, transcriptional regulation, and chromosomal imaging. Recent advances in synthetic biology have enabled the engineering of single guide RNA (sgRNA) to confer conditional responsiveness on the CRISPR/Cas9 system. By integrating functional nucleic acid elements, such as aptamers, ribozymes, and aptazymes, into specific structural regions of the sgRNA, researchers have developed systems that respond to a variety of molecular signals, including small molecules, proteins, and endogenous metabolites. These engineered sgRNAs enable spatiotemporal control of gene editing, activation, repression, and imaging in both prokaryotic and eukaryotic cells. This review summarizes the structural principles, design strategies, and applications of condition-responsive CRISPR/Cas9 systems, highlighting their potential in synthetic biology, disease modeling, and therapeutic development. Current challenges and future directions for improving the specificity, efficiency, and applicability of these systems are also discussed.},
}
@article {pmid41914499,
year = {2026},
author = {Davidge, B and Carnes, J and Lewis, I and Rodshagen, T and Tracy, M and McDermott, SM and Stuart, KD},
title = {KREPA6 functions in RNA editing catalytic complex structural organization and gRNA utilization in Trypanosoma brucei.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41914499},
issn = {1362-4962},
support = {R01 AI014102/AI/NIAID NIH HHS/United States ; AI014102/GF/NIH HHS/United States ; //Seattle Children's Research Institute/ ; 2140153//NSF/ ; },
mesh = {*Trypanosoma brucei brucei/genetics/metabolism/growth &amp; development ; *RNA Editing ; *Protozoan Proteins/genetics/metabolism/chemistry ; *RNA, Guide, Kinetoplastida/metabolism/genetics ; Amino Acid Substitution ; RNA, Protozoan/metabolism ; *RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {Functional mitochondrial mRNAs in Trypanosoma brucei are generated by the post-transcriptional guide RNA (gRNA) directed insertion and deletion of uridine residues, called RNA editing, that is catalyzed by three closely related multiprotein RNA Editing Catalytic Complexes (RECCs). These RECCs contain a common set of 12 proteins including KREPA6 which is largely comprised of an oligonucleotide binding (OB)-fold domain with a predicted intrinsically disordered region (IDR) at its C-terminus. Here we show that certain single amino acid substitutions throughout KREPA6 or deletion of the IDR inhibit the growth and viability of bloodstream form (BF) parasites. These mutations variously impact RECC structure, many alter but do not eliminate RNA editing, and some result in differential utilization of gRNAs. The results indicate that KREPA6 protein has multiple functions some of which stem from its interactions with multiple RECC proteins and perhaps with substrate RNA in each of the three different RECCs. These functions likely involve dynamic interactions of KREPA6 with key domains of other RECC proteins, other editing proteins, and with messenger RNA/gRNA substrates during the multiple catalytic and noncatalytic steps that occur during the complicated editing process.},
}
@article {pmid41914500,
year = {2026},
author = {Liu, H and Jiao, K and Hao, A and Li, C and Hou, J and Lu, X and Liu, M and Qi, Q},
title = {Enhancement of single-stranded template annealing activity by Rad52 during repair of CRISPR-induced dsDNA breaks.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41914500},
issn = {1362-4962},
support = {U23A20268//National Natural Science Foundation of China/ ; ZR2025MS440//Shandong Provincial Natural Science Foundation/ ; SKLMTFCP-2023-03//SKLMT Frontiers and Challenges Project/ ; },
mesh = {*Rad52 DNA Repair and Recombination Protein/genetics/metabolism ; *DNA Breaks, Double-Stranded ; *DNA, Single-Stranded/genetics/metabolism ; *DNA End-Joining Repair ; Gene Editing/methods ; Yarrowia/genetics ; *CRISPR-Cas Systems ; DNA Repair ; },
abstract = {Single-strand annealing protein (SSAP)-mediated recombination engineering has become a powerful tool for bacterial genome editing. However, in most eukaryotes, its efficiency is constrained by the dominant non-homologous end joining (NHEJ) repair pathway and the limited activity of exogenous SSAPs. Here, in the typically NHEJ-dominated yeast Yarrowia lipolytica, we found that 18.7% of Cas9-induced double-strand breaks (DSBs) were precisely repaired upon provision of single-stranded oligonucleotide templates, even in the absence of recombinase overexpression, indicating the presence of an endogenous eukaryotic SSAP-mediated recombination activity. Overexpression of recombination-related proteins revealed that Rad52 plays a key role in single-strand annealing. Structural truncation of Rad52(1-300) boosted genome-editing efficiency to 96.3%, comparable to that achieved by disrupting NHEJ via Ku70 deletion. Our ESTAR platform (enhancement of single-stranded template annealing activity by Rad52) enables precise and efficient genome editing, including small-fragment insertions, deletions, and replacements, as well as large-fragment deletions exceeding 20 kb. This gene-editing technology was further validated in Saccharomyces cerevisiae and other non-conventional yeast, offering new insights into the single-stranded DNA annealing step during the repair of Cas9-induced DSBs.},
}
@article {pmid41914596,
year = {2026},
author = {Ma, B and Wang, X and Cai, X and He, M and Lai, S and Zhang, P and Xu, J},
title = {Isothermal amplification and CRISPR/Cas one-pot detection systems: strategies and prospects.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {14},
pages = {2824-2837},
doi = {10.1039/d5ay01976a},
pmid = {41914596},
issn = {1759-9679},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; },
abstract = {Molecular diagnostic techniques, known for their high sensitivity and specificity, are now the gold standard for detecting pathogen nucleic acids and are crucial in precision medicine. Traditional methodologies, including polymerase chain reaction (PCR) and gene sequencing, offer exceptional analytical precision and reliability. Nevertheless, their applicability for point-of-care testing (POCT) is limited due to the high cost of equipment, prolonged detection durations, and reliance on controlled laboratory settings. The CRISPR/Cas system is considered as the next-generation of nucleic acid-based molecular diagnostic techniques, attributed to its highly specific recognition of target nucleic acids, programmability, high sensitivity, and suitability for POCT. The one-pot CRISPR assay integrates the pre-amplification of DNA or RNA with CRISPR/Cas detection into a single reaction system. This consolidation streamlines experimental workflows, enhances the practicality of POCT, and minimizes the risk of aerosol contamination. Consequently, this method has received tremendous attention from researchers. However, challenges remain due to incompatibilities between isothermal amplification and the CRISPR/Cas system, including mutual interference, temperature mismatches, and buffer incompatibility. This paper reviews recent advancements in one-pot CRISPR/Cas detection strategies, highlighting potential solutions such as physical isolation, microfluidic integration, optimization of the reaction systems, modification of crRNA and PAM sequences and engineering of nucleases. Additionally, it explores the potential applications and challenges associated with these systems in the development of high-performance POCT platforms.},
}
@article {pmid41915142,
year = {2026},
author = {Gottipamula, S and Seetharam, RN and Nk, V},
title = {"Primed for Repair: Harnessing Hypoxia, Mechanobiology, and Gene Editing to Enhance MSC Potency and Clinical Translation".},
journal = {Stem cell reviews and reports},
volume = {},
number = {},
pages = {},
pmid = {41915142},
issn = {2629-3277},
}
@article {pmid41915857,
year = {2026},
author = {Ahmad, Z and Ramakrishnan, M and Varshney, RK and Shahzad, A and Rehman, S and Pant, B and Wei, Q},
title = {WUSCHEL Transcription Factor: From Stem Cell Maintenance to Crop Improvement.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {},
number = {},
pages = {e19705},
doi = {10.1002/advs.202519705},
pmid = {41915857},
issn = {2198-3844},
support = {32471977//National Natural Science Foundation of China/ ; 32071848//National Natural Science Foundation of China/ ; BK20231289//Natural Science Foundation of Jiangsu Province/ ; Y20240114//Young Foreign Talent Program/ ; QN2022014012L//Young Foreign Talent Program/ ; 163100036//Metasequoia Faculty Research Start-Up Funding/ ; 163100028//Metasequoia Faculty Research Start-Up Funding/ ; JC2019004//Natural Science Foundation For Distinguished Young Scholars of Nanjing Forestry University/ ; 202211//Project for Ground breaking Achievements of Nanjing Forestry University/ ; },
abstract = {The WUSCHEL (WUS) transcription factor, long recognized as a master regulator of stem cell maintenance in the shoot apical meristem (SAM), has expanded in significance as a multifaceted tool in plant biotechnology. With an emphasis on its new uses in crop regeneration, somatic embryogenesis (SE), stress tolerance, and developmental regulation in cereals, legumes, and other plant species, this review summarizes recent developments on WUS function outside of Arabidopsis. We emphasize how insights from WUS biology can be translated into practical strategies to improve yield, adaptability, and resilience, while also enhancing in vitro tissue culture systems. The objective of this review is to establish WUS as a crucial molecular target for future crop genetic improvement and sustainable farming methods by highlighting the current knowledge gaps and suggesting future directions.},
}
@article {pmid41916193,
year = {2026},
author = {Luo, Y and Vallone, VF and Blanc, E and Miller, DC and He, J and Stachelscheid, H and Beule, D and Scheffel, J and Siebenhaar, F},
title = {CRISPR/Cas9 engineered and whole-genome characterized KIT D816V-mutant human iPSC lines.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103975},
doi = {10.1016/j.scr.2026.103975},
pmid = {41916193},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Proto-Oncogene Proteins c-kit/genetics/metabolism ; *Mutation/genetics ; Cell Line ; Gene Editing ; Whole Genome Sequencing ; Genome, Human ; },
abstract = {We report on the generation of human induced pluripotent stem cell (iPSC) lines, BIHi005-A-86 and BIHi005-A-87, carrying the KIT D816V mutation associated with Indolent Systemic Mastocytosis (ISM). To overcome the confounding genetic backgrounds of existing leukemic models, we introduced this gain-of-function mutation into the healthy BIHi005-A line using CRISPR/Cas9 editing. The resulting clones were validated via whole-genome sequencing (WGS) to confirm specific on-target editing and lack of predicted or disease-relevant off-target effects, while maintaining genomic stability. Together with the parental line, this resource provides an isogenic controlled platform for investigating KIT D816V-driven pathogenesis.},
}
@article {pmid41917016,
year = {2026},
author = {Gong, X and Pożoga, M and Boyer, JB and Xue, Y and Meinnel, T and Bange, T and Giglione, C and Hell, R and Wirtz, M},
title = {The ribosome-associated N-terminal acetyltransferase B coordinates global proteostasis and autophagy in plants by creating Ac/N-degrons.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41917016},
issn = {2041-1723},
support = {496871662//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 544882710//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 5041140321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; ANR-17-CAPS-0001-01//Agence Nationale de la Recherche (French National Research Agency)/ ; ANR-20-CE92-0040//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {*Autophagy/genetics ; *Proteostasis ; *Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/metabolism/genetics ; Acetylation ; *Ribosomes/metabolism ; *N-Terminal Acetyltransferase B/metabolism/genetics ; Proteasome Endopeptidase Complex/metabolism ; Ubiquitin/metabolism ; Proteomics ; Proteome/metabolism ; Mutation ; CRISPR-Cas Systems ; Degrons ; },
abstract = {The N-terminal acetyltransferase B (NatB) acetylates ~20% of the eukaryotic proteome. However, the role of NatB-mediated N-terminal acetylation (NTA) for the regulation of the proteome fate remains unclear in eukaryotes. In this study, we demonstrate that CRISPR-Cas9-mediated deletion of NatB activity in plants results in significantly lowered global protein turnover due to decreased ubiquitin-proteasome system (UPS) activity and protein translation. Quantitative proteomics uncovers that NatB substrates are significantly enriched in the fraction of stabilized proteins in natb mutants. We provide direct evidence that the absent NTA of KIN11, a subunit of the autophagy-controlling energy sensor SnRK1, protects it from UPS-mediated destruction. The resulting accumulation of KIN11 is responsible for the increased resistance of natb mutants to energy limitation induced by prolonged darkness. Our findings establish NatB as a central regulator of UPS-autophagy interplay and highlight its role in maintaining proteome stability and enabling dynamic stress responses in plants.},
}
@article {pmid41917051,
year = {2026},
author = {Lu, J and Lai, J and Cheng, L and Zhan, H and Jie, K and Liu, C and Huang, L and Cen, M and Liu, S and Chen, Z and Zhang, Q and Zhang, J and Wu, J and Pan, B and Chen, S and Zhong, J and He, B and Li, H and Chen, X and Lin, T},
title = {Miniature and versatile genome regulation TnpB-ωRNA toolkits facilitate cancer immunotherapy.},
journal = {Nature communications},
volume = {},
number = {},
pages = {},
doi = {10.1038/s41467-026-71327-w},
pmid = {41917051},
issn = {2041-1723},
abstract = {CRISPR‒Cas systems represent powerful tools for genome regulation. However, the large size of Cas proteins limits their efficient delivery via an adeno-associated virus (AAV), thereby restricting their clinical translation. Here, we engineer the IS200/IS605 transposon-encoded nuclease TnpB, along with its ωRNA scaffold, to create an enhanced TnpB system, which serves as a compact toolkit for gene activation, genome editing, and base editing. The gene activator enTnpBa increases expression by 2889-fold with a minimized 93 nt ωRNA and robustly activates endogenous genes in mammalian cells. We develop a single-AAV-based regimen for immune activation (AAV-ImmunAct) that delivers enTnpBa to activate CXCL9, IL-15, and IFN-γ. AAV-ImmunAct effectively enhances T cell migration and activation, increases killing of cancer cell lines and patient-derived organoids, and synergizes with anti-PD-1 therapy in humanized mice. Here, we establish enTnpB as a compact and versatile platform for genome regulation and a promising tool for cancer immunotherapy.},
}
@article {pmid41917262,
year = {2026},
author = {Shi, R and Yang, M and Liu, Y and Gao, H and Lin, Z},
title = {Mechanistic basis for selective Csm6-2 activation by cyclic penta-adenylate in a type III CRISPR-Cas system.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {41917262},
issn = {1460-2075},
support = {32471255//MOST | National Natural Science Foundation of China (NSFC)/ ; 32271258//MOST | National Natural Science Foundation of China (NSFC)/ ; 2024J02006//| Natural Science Foundation of Fujian Province (Fujian Natural Science Foundation)/ ; },
abstract = {Type III CRISPR systems generate cyclic oligoadenylate (cOA, 3 to 6 AMPs) messengers upon detecting viral RNA, activating downstream effectors to defend against viral infection. Although cOA-activated effectors have been extensively characterized, the effectors specific to cA5-one of the most abundant cOA species produced during phage infection-have remained unexplored. Here, we report that the CRISPR ribonuclease Csm6 (Csm6-2) from Actinomyces procaprae selectively employs cA5 as its activator. Csm6-2 utilizes its HEPN domain, rather than the CARF domain, to mediate self-limiting cleavage of cOA activators. Cryo-EM structural analyses reveal that Csm6-2 functions as a homotetramer, and disruption of tetramer formation significantly reduces its ribonuclease activity. Although cA6 and cA5 bind Csm6-2 with comparable affinity, only cA5 induces CARF domain closure, stabilizes the tetramer, and remodels the active site in the HEPN domain. In contrast, the sixth AMP of cA6 imposes significant steric hindrance on CARF domain movement, preventing its closure and subsequent allosteric activation. These findings expand our understanding of the cOA signaling diversity and specific cOA recognition mechanisms in type III CRISPR immunity.},
}
@article {pmid41917354,
year = {2026},
author = {Chen, Z and Huang, X and Pi, Y and Jiang, Y},
title = {Method for Generation of adamtsl4 Knock-Out Zebrafish Lines Using CRRISPR/Cas9 System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3027},
number = {},
pages = {73-82},
pmid = {41917354},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Knockout Techniques/methods ; *ADAMTS Proteins/genetics ; Disease Models, Animal ; *Zebrafish Proteins/genetics ; Phenotype ; Gene Editing/methods ; Mutation ; Humans ; },
abstract = {Congenital ectopia lentis (CEL) is a rare pediatric ocular disorder characterized by zonular fiber defects leading to lens dislocation and is genetically heterogeneous. Among known causes, biallelic mutations in ADAMTSL-4 represent the second most common genetic contributor, frequently associated with ectopia pupillae (EP)-a distinct and clinically significant feature. However, the mechanisms by which ADAMTSL-4 mutations lead to these ocular abnormalities remain poorly understood, partly due to the lack of effective animal models. In this study, we generated adamtsl-4 knock-out zebrafish lines using the CRISPR/Cas9 system. Through microinjection of sgRNA/Cas9 complexes and multigenerational screening, we established stable homozygous mutant lines. These mutants exhibited consistent phenotypes, including lens dislocation into the vitreous body and marked pupillary displacement, faithfully recapitulating human ADAMTSL-4-related EL and EP. This method provides a practical and scalable strategy for generating loss-of-function zebrafish models, with demonstrated utility in recapitulating phenotypes associated with ADAMTSL-4 mutations. Our approach offers a valuable tool for investigating the molecular mechanisms underlying CEL and EP and may support drug screening and therapeutic discovery in the future.},
}
@article {pmid41917356,
year = {2026},
author = {Zheng, L and Wu, Z and Zheng, XL},
title = {Generating adamts13[-/-] Zebrafish via CRISPR/Cas9 Gene Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3027},
number = {},
pages = {93-111},
pmid = {41917356},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *ADAMTS13 Protein/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques/methods ; Animals, Genetically Modified ; Disease Models, Animal ; *Zebrafish Proteins/genetics ; },
abstract = {The zebrafish (Danio rerio) is a powerful vertebrate model for studying hematologic and thrombotic diseases due to its genetic tractability and conservation of hemostatic pathways with humans. In this chapter, we describe a detailed workflow for generating adamts13 knockout zebrafish using CRISPR/Cas9-mediated genome editing. Methods include the design and preparation of guide RNAs, Cas9 mRNA synthesis, and embryo microinjection at the one-cell stage. Alternative strategies for gRNA generation, including synthetic crRNA:tracrRNA duplexes, are also outlined. We provide protocols for screening founder fish, genotyping, and establishing stable mutant lines through outcrossing, thereby minimizing potential off-target effects. Practical notes on embryo handling, RNA stability, and contamination prevention are highlighted to ensure reproducibility. Together, these procedures establish a robust framework for creating zebrafish loss-of-function models, enabling mechanistic studies of ADAMTS13 function in vivo and advancing the exploration of thrombotic disease pathophysiology. This approach can be readily adapted to knock out other genes or introduce specific mutations in zebrafish simply by altering the gRNA sequence.},
}
@article {pmid41918363,
year = {2026},
author = {Yamada, HY and Rao, CV},
title = {Genome-Wide CRISPR Analysis Uncovers Metformin and NSAID Combination as a Chemopreventive Approach in Smoking-Associated Oral Cancer.},
journal = {Cancer prevention research (Philadelphia, Pa.)},
volume = {19},
number = {4},
pages = {183-185},
doi = {10.1158/1940-6207.CAPR-26-0004},
pmid = {41918363},
issn = {1940-6215},
mesh = {*Metformin/therapeutic use/pharmacology ; Humans ; *Mouth Neoplasms/prevention &amp; control/etiology/genetics ; *Anti-Inflammatory Agents, Non-Steroidal/therapeutic use/pharmacology ; *Squamous Cell Carcinoma of Head and Neck/prevention &amp; control/genetics/etiology ; *Antineoplastic Combined Chemotherapy Protocols/therapeutic use ; CRISPR-Cas Systems ; *Smoking/adverse effects ; Chemoprevention/methods ; Drug Resistance, Neoplasm/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Head and neck squamous cell carcinoma (HNSCC) is one of the most common cancers worldwide and carries substantial morbidity. Metformin, a widely used antidiabetic agent, shows promise for HNSCC prevention, but resistance arises in a subset of tumors. In a recent issue of Cancer Prevention Research, Hoang and colleagues use CRISPR screening to identify key mediators of metformin resistance, including AMPK and protein kinase A (PKA), and demonstrate that the cyclooxygenase 2-prostaglandin E2 axis acts upstream of PKA. Because this pathway is readily inhibited by common nonsteroidal anti-inflammatory drugs (NSAID), the findings support clinical evaluation of combined metformin and NSAID therapy to improve HNSCC chemoprevention. See related article by Hoang et al., p. 79 .},
}
@article {pmid41919328,
year = {2026},
author = {Singh, P and Sharma, K and Tamrakar, VK and Khare, R and Bhargava, A and Negi, SS},
title = {CRISPR-Cas system: recent advancements in prompt diagnosis of high-risk HPV genotypes in cervical cancer.},
journal = {Expert review of molecular diagnostics},
volume = {},
number = {},
pages = {1-16},
doi = {10.1080/14737159.2026.2654503},
pmid = {41919328},
issn = {1744-8352},
abstract = {INTRODUCTION: The CRISPR/Cas system has emerged as a highly versatile platform for diagnosing infectious diseases, particularly viral pathogens. Human papillomavirus (HPV) comprises of more than 200 types, with persistent infection by 14 high-risk genotypes recognized as the primary cause of cervical cancer worldwide. Early and accurate detection of these High-Risk HPV (HR-HPV) types is essential for effective clinical management and prevention of disease progression.<br><br>AREAS COVERED: This narrative review was based on literature searches in PubMed, Scopus, and Google Scholar covering studies published between 2015 and 2024. This review summarizes recent advances in CRISPR/Cas based diagnostics for HR-HPV, including both pre-amplification and amplification-free strategies. Integration of CRISPR systems with diverse readout modalities such as colorimetric, fluorescent, electrochemical, and lateral-flow biosensors has enabled rapid, sensitive, and user-friendly detection suitable for point-of-care testing (POCT), particularly in low-resource settings.<br><br>EXPERT OPINION: CRISPR/Cas assays demonstrate high sensitivity, specificity, and speed, offering a promising alternative to conventional molecular techniques for HR-HPV detection and genotyping. The convergence of CRISPR diagnostics with artificial intelligence, microfluidics, and affordable biosensors holds significant potential to transform community-level HPV screening. With continued innovation and regulatory support, CRISPR/Cas systems are poised to become indispensable tools for early HR-HPV detection and cervical cancer prevention.},
}
@article {pmid41921842,
year = {2026},
author = {Liao, YJ and Li, YG and Li, YL},
title = {CRISPR/Cas9-mediated lgp2 knockout and cross-species rescue reveal the immunoregulatory role of LGP2 in zebrafish.},
journal = {Fish &amp; shellfish immunology},
volume = {173},
number = {},
pages = {111308},
doi = {10.1016/j.fsi.2026.111308},
pmid = {41921842},
issn = {1095-9947},
mesh = {Animals ; *Zebrafish/genetics/immunology ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics/immunology ; Gene Knockout Techniques/veterinary ; *Immunity, Innate/genetics ; *Fish Diseases/immunology ; Gene Expression Profiling/veterinary ; *Gene Expression Regulation/immunology ; Species Specificity ; },
abstract = {LGP2 is a key regulator within the RIG-I-like receptor (RLR) pathway with dual roles in antiviral immunity, but its function under basal conditions and its evolutionary variation among fish species require further investigation. In this study, we constructed a zebrafish lgp2 knockout model using CRISPR/Cas9. Knockout lgp2 led to upregulation of key RLR signaling components (mda5, mavs), type I interferons (ifnphi1, ifnphi3, ifnphi4), and the interferon-stimulated gene mxa. Transcriptomic profiling of 3 dpf embryos identified 3186 differentially expressed genes (DEGs), including significantly elevated expression of five claudin-family genes. Protein-protein interaction (PPI) analysis of DEGs indicated that irf7 and claudin b exhibit an interaction at the protein level. Gene ontology analysis showed that LGP2 was enriched in terms such as helicase activity and ATPase activity, and was also significantly enriched in extracellular structure terms. Comparative sequence analysis revealed conserved DEXDc, HELICc and CTD domains in LGP2 from zebrafish, grass carp and barbel chub, with notable species-specific sequence variations. Functional rescue experiments demonstrated that all three LGP2 orthologs could restore lgp2 expression and modulate downstream interferon responses in lgp2[-/-] embryos; however, induction of the transcription factor irf7 was specifically mediated only by zebrafish-derived LGP2. These results provide insights into the functional diversity of LGP2 in teleost immunity and inform strategies for selective breeding aimed at disease resistance.},
}
@article {pmid41923586,
year = {2026},
author = {Oh, Y and Lee, H and Jang, S},
title = {Emerging synthetic biology-assisted technologies for overcoming antibiotic resistance: CRISPR-Cas, bacteriophage, microbiome, and metabolic engineering-based solutions.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {64},
number = {3},
pages = {e2512002},
doi = {10.71150/jm.2512002},
pmid = {41923586},
issn = {1976-3794},
support = {//National Research Foundation of Korea/ ; RS-2025-02214910//Ministry of Science and ICT/ ; //Incheon National University/ ; },
mesh = {*CRISPR-Cas Systems ; *Synthetic Biology/methods ; *Bacteriophages/genetics ; *Metabolic Engineering/methods ; *Anti-Bacterial Agents/pharmacology ; *Microbiota/genetics ; *Bacteria/drug effects/genetics ; Humans ; *Drug Resistance, Bacterial/genetics ; *Drug Resistance, Microbial/genetics ; },
abstract = {Antibiotic resistance has become a critical global health challenge due to the decreased efficacy of existing antibiotics and the emergence of multidrug-resistant pathogens. In particular, the rapid horizontal transfer of resistance genes and the diverse mechanisms by which bacteria acquire resistance have significantly undermined the effectiveness of conventional therapeutic strategies, revealing fundamental limitations in current infectious disease management. In this context, synthetic biology provides a promising framework to overcome the limitations of conventional antibiotics by integrating engineering principles with bioengineering approaches, thereby enabling precise and programmable control of biological processes. These synthetic biology-based approaches offer substantial potential for developing sustainable and highly specific antimicrobial strategies. This review comprehensively examines recent advances in synthetic biology-assisted antimicrobial strategies, including CRISPR-Cas systems, bacteriophage engineering, microbiome engineering, and metabolic engineering-driven antibiotic discovery. Collectively, these approaches represent a precision antimicrobial paradigm that enables selective targeting of resistant bacteria while preserving microbiome homeostasis. These strategies also provide new directions for limiting resistance dissemination and guiding the development of next-generation therapeutics.},
}
@article {pmid41925118,
year = {2026},
author = {El-Sokkary, MMA and Ali, IEH},
title = {Association of CRISPR/Cas system with integrons and antibiotic resistance in Kliebsiella pneumoniae.},
journal = {Future microbiology},
volume = {21},
number = {3},
pages = {299-306},
pmid = {41925118},
issn = {1746-0921},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; *Anti-Bacterial Agents/pharmacology ; *CRISPR-Cas Systems/genetics ; *Integrons/genetics ; Microbial Sensitivity Tests ; *Drug Resistance, Multiple, Bacterial/genetics ; Humans ; Klebsiella Infections/microbiology ; Bacterial Proteins/genetics ; *Drug Resistance, Bacterial/genetics ; },
abstract = {AIMS: The interaction between the CRISPR/Cas system and drug resistance was investigated in this study.<br><br>METHODS: In this study, 24 isolates, identified as Klebsiella pneumoniae, were subjected to antimicrobial sensitivity screening and detecting the presence of cas, integrons, and antibiotic resistance genes.<br><br>RESULTS: The highest resistance level could be detected in ceftriaxone and amoxicillin by 79% and 70.8%, respectively, while the lowest was 16.7% for chloramphenicol. Additionally, 50% of all isolates exhibited multidrug resistance (MDR); however, extensive drug resistance (XDR) was present in 12.5%. Class 1 integrons with different sizes could be identified. sul1 and sul2 were the most resistance genes identified, followed by aacA4 and aac(3)-II and qnrS, with 58%, 50%, 41.7%, 33%, and 33%, respectively. Concerning CRISPR genes, cas3 could be identified in nine different isolates; however, cas1 could be detected in 11 different isolated strains. In case of CRISPR1, it was found in seven isolates (29.3%), while CRISPR2 could be identified in three different isolates (12.5%).<br><br>CONCLUSIONS: Antibiotic resistance genes were mostly correlated with CRISPR genes. Interestingly, BOX-PCR group A, with a lower number of CRISPR positive isolates (p&#x2009;=&#x2009;0.046) had a limited number of detected genes, compared with other groups indicating a significant correlation between antibiotic resistance genes and cas genes (p&#x2009;=&#x2009;0.0023).},
}
@article {pmid41925232,
year = {2026},
author = {Farruggio, AP and Jiang, L and Duong, K and Nguyen, C and Kaddoura, R and Tsai, R},
title = {S-SELeCT: a human-evolved serine integrase system for efficient large-cargo genome integration.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41925232},
issn = {1362-4962},
support = {1R44GM136045-01/NH/NIH HHS/United States ; 4R44GM136045-02/NH/NIH HHS/United States ; 5R44GM136045-03/NH/NIH HHS/United States ; //Applied StemCell's Research and Development funds/ ; },
mesh = {Humans ; *Integrases/genetics/metabolism ; *Gene Editing/methods ; HEK293 Cells ; Gene Knock-In Techniques/methods ; CRISPR-Cas Systems ; Plasmids/genetics ; Genome, Human ; },
abstract = {As a consequence of their sizes, many loss-of-function genetic mutations fall within large genes. A major gene-therapy tool that could be used to solve large swaths of the genetic diseases that result from these inherited mutations is large-fragment knock-in. I.e. instead of attempting to create separate treatments for each and every location that these mutations occur in, large groups of patients could be aided via a single safe-harbor integration of the full-length coding sequence. Toward this goal, we have created a set of early stage gene-editing enzymes that can help mediate large cargo integration at a safe harbor locus in human cells. When expressed in stable lines, our S-SELeCT (Site-Specific Large Cargo Targeting) integrase fusions can facilitate integration of a 10 kb plasmid at frequencies up to 32%, and when delivered transiently via plasmid transfection, we were able to achieve up to 13% knock-in. These are the first serine integrase enzymes that have been evolved fully in human cells and the first to recognize an endogenous symmetric non-pseudosite-the first true human serine integrase attachment site.},
}
@article {pmid41925899,
year = {2026},
author = {Wang, B and Liu, M and Wang, H and Wang, C and Zhang, W and Yan, J},
title = {Research progress on nucleic acid amplification-based detection technologies for phytopathogenic fungi.},
journal = {Applied microbiology and biotechnology},
volume = {110},
number = {1},
pages = {},
pmid = {41925899},
issn = {1432-0614},
support = {CARS-29//China Agriculture Research System of MOF and MARA/ ; JKTD2025002//the Outstanding Scientist Project of Beijing Academy of Agriculture and Forestry Sciences/ ; },
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Plant Diseases/microbiology ; *Fungi/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; CRISPR-Cas Systems ; },
abstract = {Phytopathogenic fungi are highly diverse and globally distributed, posing a major threat to agricultural production worldwide. The annual losses caused by plant diseases can reach up to 30% of global crop yields, with over 80% of infections caused by fungal pathogens. The accurate identification of pathogenic fungal species is crucial for effective disease prevention and control. Thus, establishing accurate and rapid detection technologies for phytopathogenic fungi is crucial for implementing targeted control strategies and reducing agricultural losses. Molecular detection technologies based on nucleic acid amplification have recently become indispensable tools for pathogen detection. This review examines the principles and advancements of nucleic acid-based detection techniques, including thermal cycling-based methods (e.g., conventional PCR, real-time quantitative PCR, and droplet digital PCR) and isothermal amplification platforms (e.g., loop-mediated isothermal amplification and recombinase polymerase amplification), as well as CRISPR/Cas-assisted assays coupled with isothermal amplification (e.g., RPA-CRISPR and LAMP-CRISPR), with the aim of evaluating their strengths, limitations, and practical applicability in the rapid diagnosis and precision management of phytopathogenic fungal diseases. KEY POINTS: Nucleic acid amplification technologies enable rapid and sensitive detection of phytopathogenic fungi. Isothermal amplification and CRISPR/Cas-assisted platforms facilitate field-deployable and low-instrumentation diagnostics. Integrated workflows support early diagnosis and precision management of phytopathogenic fungal diseases.},
}
@article {pmid41926379,
year = {2026},
author = {Boy, RL and Zampieri, RA and Aoki, JI and Coelho, AC and Floeter-Winter, LM and Laranjeira-Silva, MF},
title = {Glycosomal Aquaglyceroporin 1 dual role in iron homeostasis and antimony susceptibility in Leishmania amazonensis.},
journal = {PLoS neglected tropical diseases},
volume = {20},
number = {4},
pages = {e0014141},
pmid = {41926379},
issn = {1935-2735},
mesh = {Animals ; *Antimony/pharmacology ; Mice ; Homeostasis ; *Iron/metabolism ; Macrophages/parasitology ; *Leishmania mexicana/drug effects/metabolism/genetics ; *Antiprotozoal Agents/pharmacology ; *Protozoan Proteins/genetics/metabolism ; Female ; *Aquaporin 1/genetics/metabolism ; Leishmaniasis/parasitology ; Mice, Inbred BALB C ; *Leishmania/drug effects/metabolism/genetics ; CRISPR-Cas Systems ; },
abstract = {Leishmania parasites cause a spectrum of diseases known as leishmaniases and must acquire nutrients like iron while surviving host defenses. Aquaglyceroporin 1 (AQP1) is a membrane channel that, in L. major, localizes to the flagellum and mediates antimony uptake and cell-volume regulation. Here, we show that in L. amazonensis AQP1 is instead targeted to glycosomes and that its expression is modulated by iron availability. A CRISPR-Cas9-mediated knockout of AQP1 in L. amazonensis revealed its multifunctional importance. AQP1-null promastigotes displayed a significant growth defect, particularly under iron-depleted conditions, and were impaired in regulating cell volume under osmotic stress. The mutant parasites contained approximately 50% less intracellular iron than wild-type cells and showed an increase in total superoxide dismutase activity, underscoring a role for AQP1 in iron homeostasis and oxidative stress management. AQP1 deletion also markedly reduced virulence in murine macrophages and in infected mice. Strikingly, loss of AQP1 increased resistance to trivalent antimony (SbIII), a first-line antileishmanial drug. AQP1-knockout promastigotes showed a 70% increase in SbIII IC50 and accumulated more Sb intracellularly than wild-type, suggesting an altered antimony handling. Altogether, L. amazonensis AQP1 is a glycosomal protein that links iron metabolism, osmoregulation, and antimony susceptibility. Its glycosomal targeting and multifaceted roles differ from those of AQP1 orthologs in other Leishmania species. These findings suggest the existence of additional antimony uptake mechanisms beyond AQP1, with implications for understanding drug resistance.},
}
@article {pmid41927937,
year = {2026},
author = {Zhuravlev, IY and Lyakhovets, AA and Matveenko, AG and Lebedeva, MA and Zhernakov, AI and Simonova, VY and Sulima, AS and Tikhonovich, IA and Zhukov, VA},
title = {CRISPR/Cas9-mediated knockout of PsLykX gene of pea (Pisum sativum L.) leads to loss of symbiotic nodules.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {41927937},
issn = {1573-9368},
mesh = {*Pisum sativum/genetics/microbiology/growth &amp; development ; *Symbiosis/genetics ; *CRISPR-Cas Systems ; *Root Nodules, Plant/genetics/microbiology/growth &amp; development ; *Plant Proteins/genetics ; Plants, Genetically Modified/genetics/growth &amp; development/microbiology ; Gene Knockout Techniques ; Plant Roots/genetics/microbiology/growth &amp; development ; Nitrogen Fixation/genetics ; Rhizobium/genetics ; },
abstract = {Pea (Pisum sativum L.) symbiosis with nodule bacteria supplying plants with additional nitrogen is a very specific plant-microbial interaction. Mutual recognition of the partners occurs through perception of bacterial signal molecules (Nod factors) by plant receptors, enabling bacterial entry via root hairs and formation of nitrogen-fixing nodules. The pea gene Sym2, described but not yet cloned, exists in different allelic forms defining the symbiotic specificity, and is therefore thought to encode a Nod factor receptor. The PsLykX gene is a strong candidate for the Sym2, since its alleles coincide with high or low symbiotic specificity; however, to date, no genetic evidence has been obtained for a role of PsLykX in symbiosis. Here, we knocked-out the PsLykX in European pea cultivar Caméor using Agrobacterium-mediated hairy root transformation and CRISPR-Cas9 editing. The roots with editing events confirmed by sequencing lost the ability to form nodules, providing direct functional evidence that PsLykX is essential, at least, for the symbiosis between pea cultivar Caméor and Rhizobium ruizarguesonis RCAM1026.},
}
@article {pmid41928005,
year = {2026},
author = {Migur, A and Feussner, M and Liao, C and Alkhnbashi, OS and Chauvier, A and Walter, NG and Backofen, R and Weinberg, Z and Beisel, CL},
title = {A leader-repeat hairpin blocks extraneous CRISPR RNA production in diverse CRISPR-Cas13 systems.},
journal = {The EMBO journal},
volume = {},
number = {},
pages = {},
pmid = {41928005},
issn = {1460-2075},
support = {468749960//Deutsche Forschungsgemeinschaft (DFG)/ ; 468749960//Deutsche Forschungsgemeinschaft (DFG)/ ; BA 2168/23-2//Deutsche Forschungsgemeinschaft (DFG)/ ; 865973//EC | Horizon Europe | Excellent Science | HORIZON EUROPE European Research Council (ERC)/ ; MCB 2140320//National Science Foundation (NSF)/ ; },
abstract = {CRISPR RNAs (crRNAs) guide recognition and targeting of intracellular invaders as part of adaptive immunity by CRISPR-Cas systems. crRNAs are transcribed from CRISPR arrays of conserved repeats interlaced with invader-derived spacers. While crRNA production is essential for immunity, its optimization for defense remains poorly understood. Here, we show that, in diverse RNA-targeting type VI CRISPR-Cas systems, the leader RNA encoded upstream of the CRISPR array prevents formation of an invader-independent extraneous crRNA (ecrRNA) by blocking processing of the first repeat. Using the VI-B2 system from Porphyromonas gingivalis as a model, we demonstrate that the leader RNA and first repeat form a conserved inhibitory hairpin that precludes binding and processing by the system's Cas13b nuclease. Disrupting this hairpin enables ecrRNA production, which in turn can deplete invader-derived crRNAs and reduce Cas13b-mediated phage defense. Structure prediction indicates that these leader-repeat hairpins are widespread across diverse type VI subtypes, highlighting a conserved regulatory mechanism. Our findings reveal how a prevalent branch of CRISPR-Cas systems suppresses ecrRNA formation to promote RNA-guided immunity.},
}
@article {pmid41930610,
year = {2026},
author = {Xing, B and Zhang, X and Shen, L and Zhang, X},
title = {CRISPR-mediated MLH1 disruption suppresses endometrial cancer growth via genomic instability induction and Wnt/β-catenin pathway inhibition.},
journal = {Folia histochemica et cytobiologica},
volume = {64},
number = {1},
pages = {75-87},
doi = {10.5603/fhc.111186},
pmid = {41930610},
issn = {1897-5631},
mesh = {Humans ; *Endometrial Neoplasms/genetics/pathology/metabolism ; *MutL Protein Homolog 1/genetics/metabolism ; *Wnt Signaling Pathway/genetics ; Female ; Cell Proliferation/genetics ; Cell Line, Tumor ; Animals ; *Genomic Instability ; Mice ; *CRISPR-Cas Systems ; Apoptosis ; Mice, Nude ; Cell Movement ; Gene Expression Regulation, Neoplastic ; },
abstract = {INTRODUCTION: MutL homolog 1 (MLH1) loss is a defining molecular feature of endometrial cancer (EC) and a principal driver of microsatellite instability (MSI). Ishikawa cells harbor intrinsic MLH1 promoter hypermethylation, resulting in reduced but not abolished MLH1 expression and placing these cells in a vulnerable, partially compromised mismatch repair state. This study explores the effects of MLH1 knockdown (MLH1-KD) on MSI, cellular functions, signaling pathways, and tumor growth in Ishikawa EC cells.<br><br>MATERIAL AND METHODS: Using CRISPR/Cas9, we created an MLH1-KD Ishikawa EC cell line, validated through Sanger sequencing, qRT-PCR, western blotting, comet assays, and &#x3b3;-H2AX analysis. Functional assays assessed proliferation, migration, and cell cycle progression and apoptosis. RNA sequencing identified global transcriptomic changes, and Wnt/&#x3b2;-catenin pathway activity was measured by a dual-luciferase reporter assay. A xenograft model evaluated tumor growth in vivo.<br><br>RESULTS: MLH1-KD cells showed MSI-H characteristics, increased DNA damage, and downregulation of key EC-related genes. Functionally, MLH1-KD led to significant reductions in cell proliferation and migration, which was accompanied by cell cycle arrest and a marked increase in apoptosis. RNA sequencing revealed profound alterations in the Wnt signaling pathway. Crucially, this was confirmed by a dual-luciferase reporter assay, which showed a significant inhibition of Wnt/&#x3b2;-catenin signaling activity. In vivo, MLH1-KD significantly decreased tumor weight and size in nude mice.<br><br>CONCLUSIONS: In EC cells with pre-existing MLH1 promoter methylation, MLH1-KD leads to MSI-H, enhances genomic instability, disrupts Wnt signaling, impairs cellular functions, and inhibits tumor growth, highlighting Wnt signaling and MSI-H as potential therapeutic targets in EC.},
}
@article {pmid41930845,
year = {2026},
author = {Qin, Y and Zhang, S and Zhang, H and Jiang, D and Wen, P and Li, S and Wang, G and Qu, F and Zhang, N and Wang, L and He, Y and Shao, Q and Zhou, X and Zeng, X},
title = {Dual-lock gated DNAzyme-CRISPR cascade for amplification-free ultrasensitive profiling of FTO demethylase activity.},
journal = {Talanta},
volume = {306},
number = {},
pages = {129714},
doi = {10.1016/j.talanta.2026.129714},
pmid = {41930845},
issn = {1873-3573},
mesh = {*Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism/genetics/analysis ; Humans ; *DNA, Catalytic/metabolism/chemistry/genetics ; *CRISPR-Cas Systems ; Breast Neoplasms/genetics ; Female ; Limit of Detection ; Adenosine/analogs &amp; derivatives ; },
abstract = {N6-methyladenosine, the most prevalent internal mRNA modification in eukaryotes, dynamically regulates transcriptomic homeostasis via FTO-mediated demethylation. CRISPR-based FTO detection technologies represent a significant research direction. However, most current strategies depend critically on nucleic acid pre-amplification steps, which introduce substantial risks of amplification leakage, thereby compromising their ability to achieve stable, high-sensitivity FTO detection. To address this, we designed a dual-lock gated DNAzyme-CRISPR cascading platform that integrates a double-locked DNAzyme with a redundant structure regulated CRISPR-Cas12a trans-cleavage signal amplification system. This architecture achieves an unprecedented limit of detection of 0.083 pM, which is 12 times more sensitive than commercial ELISA kits. Clinical validation using 20 breast cancer patient tissues demonstrated robust cancer/normal discrimination (AUC = 0.9853) and significant FTO upregulation in triple-negative subtypes (p < 0.001). The modular design establishes a universal framework for epigenetic enzyme detection while enabling dynamic monitoring of tumor epigenetic remodeling.},
}
@article {pmid41931047,
year = {2026},
author = {Hanna, R and Frangoul, H and Pineiro, L and McKinney, C and Mapara, M and Dalal, J and Rangarajan, HG and Atkins, H and Sharma, A and Chang, KH and Jaskolka, MC and Kim, K and Yu, Q and Mei, B and Afonja, O and Walters, MC and , },
title = {CRISPR-Cas12a Gene Editing of HBG1 and HBG2 Promoters to Treat Sickle Cell Disease.},
journal = {The New England journal of medicine},
volume = {394},
number = {13},
pages = {1281-1291},
doi = {10.1056/NEJMoa2415550},
pmid = {41931047},
issn = {1533-4406},
mesh = {Adolescent ; Adult ; Female ; Humans ; Male ; Young Adult ; *Anemia, Sickle Cell/blood/diagnosis/genetics/therapy ; *CRISPR-Cas Systems/genetics ; *Fetal Hemoglobin/analysis/genetics ; *gamma-Globins/genetics ; Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; *Hematopoietic Stem Cell Transplantation/adverse effects/methods ; Promoter Regions, Genetic/genetics ; Severity of Illness Index ; Follow-Up Studies ; Transplantation, Autologous/adverse effects/methods ; Transplantation Conditioning/adverse effects/methods ; Repressor Proteins/metabolism ; Busulfan/administration &amp; dosage/adverse effects ; Myeloablative Agonists/administration &amp; dosage/adverse effects ; *Vaso-Occlusive Crises/epidemiology/genetics/prevention &amp; control/therapy ; Treatment Outcome ; },
abstract = {BACKGROUND: Renizgamglogene autogedtemcel (reni-cel) is an investigational clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a gene-edited autologous hematopoietic stem-cell therapy. The therapy was designed to disrupt the BCL11A binding sites in the HBG1 and HBG2 promoters to reactivate fetal hemoglobin production for the treatment of sickle cell disease.<br><br>METHODS: We conducted a phase 1-2, multicenter, open-label, single-group study involving patients with severe sickle cell disease who were 12 to 50 years of age and had had at least two severe vaso-occlusive events per year in the previous 2 years. The patients received a single infusion of reni-cel after myeloablative conditioning with busulfan. The patients were monitored for engraftment, hemoglobin-related measures, allelic editing levels, vaso-occlusive events, and adverse events over a 24-month period. The study was terminated early on the basis of the sponsor's reassessment of clinical development priorities. Results of an analysis that was not prespecified are reported.<br><br>RESULTS: As of October 29, 2024, a total of 28 patients with severe sickle cell disease had been treated with reni-cel. The median duration of follow-up was 9.5 months (range, 0.7 to 25.2). Among 27 patients who had neutrophil and platelet engraftment by the data-cutoff date, neutrophil engraftment occurred after a median of 23 days (range, 14 to 29), and platelet engraftment occurred after a median of 25 days (range, 17 to 51). At month 6, among 18 patients with at least 6 months of available data, the mean (&#xb1;SD) total hemoglobin level (9.8&#xb1;1.7 g per deciliter at baseline) had increased to 13.8&#xb1;1.9 g per deciliter, and the mean percentage of fetal hemoglobin (2.5&#xb1;2.5% at baseline) had increased to 48.1&#xb1;3.2%; both measures were maintained at or above these values thereafter. One patient had two severe vaso-occlusive events after infusion. Adverse events were consistent with those that occur after myeloablative busulfan-based conditioning and autologous hematopoietic stem-cell transplantation.<br><br>CONCLUSIONS: Treatment with reni-cel led to normalization of the total hemoglobin level and an increase in the percentage of fetal hemoglobin, with no vaso-occlusive events occurring in 27 of 28 patients after infusion. These results support further investigation of this gene-editing approach in the treatment of severe sickle cell disease. (Funded by Editas Medicine; RUBY ClinicalTrials.gov number, NCT04853576.).},
}
@article {pmid41931048,
year = {2026},
author = {Frangoul, H and Hanna, R and Walters, MC and Kao, RL and Carroll, C and McManus, M and Chang, KH and Jaskolka, MC and Kim, K and Yu, Q and Badamosi, N and Mei, B and Afonja, O and Thompson, A and , },
title = {CRISPR-Cas12a Gene Editing of HBG1 and HBG2 Promoters to Treat β-Thalassemia.},
journal = {The New England journal of medicine},
volume = {394},
number = {13},
pages = {1292-1301},
doi = {10.1056/NEJMoa2501277},
pmid = {41931048},
issn = {1533-4406},
mesh = {Adolescent ; Adult ; Female ; Humans ; Male ; Young Adult ; *beta-Thalassemia/therapy/genetics/blood ; *CRISPR-Cas Systems/genetics ; *Fetal Hemoglobin/analysis/genetics ; *gamma-Globins/genetics ; Gene Editing/methods ; *Genetic Therapy/adverse effects/methods ; *Hematopoietic Stem Cell Transplantation/adverse effects/methods ; Neutrophils ; Promoter Regions, Genetic ; Repressor Proteins/metabolism ; Busulfan/administration &amp; dosage/adverse effects ; Myeloablative Agonists/administration &amp; dosage/adverse effects ; Transplantation Conditioning/adverse effects/methods ; *Erythrocyte Transfusion/statistics &amp; numerical data ; Treatment Outcome ; },
abstract = {BACKGROUND: Renizgamglogene autogedtemcel (reni-cel) is an investigational clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a gene-edited autologous hematopoietic stem-cell therapy. The therapy was designed to disrupt the BCL11A binding sites in the HBG1 and HBG2 promoters to reactivate fetal hemoglobin production for the treatment of transfusion-dependent β-thalassemia.<br><br>METHODS: We conducted a phase 1-2, multicenter, open-label, single-group study of reni-cel in participants 18 to 35 years of age with transfusion-dependent &#x3b2;-thalassemia. The participants received myeloablative conditioning with busulfan before reni-cel infusion. The primary end points were neutrophil engraftment by 42 days after infusion and frequency and severity of adverse events. Participants were monitored for hemoglobin-related measures and transfusion independence. The study was terminated early on the basis of the sponsor's reassessment of clinical development priorities. Results of an analysis that was not prespecified are reported.<br><br>RESULTS: Nine participants with transfusion-dependent &#x3b2;-thalassemia (four &#x3b2;[0]/&#x3b2;[0] or &#x3b2;[0]/&#x3b2;[0]-like and five non-&#x3b2;[0]/&#x3b2;[0] genotypes) received reni-cel and were included in the analysis. The median duration of postinfusion follow-up was 17.5 months (range, 3.8 to 23.4), and six participants could be evaluated for transfusion independence at 12 months or more. All the participants had neutrophil and platelet engraftment by 42 days after infusion. Rapid increases in total and fetal hemoglobin levels resulted in each of the nine participants being transfusion-free at their last follow-up visit. The six participants who could be evaluated at 12 months or later were transfusion-independent. The mean total and fetal hemoglobin levels were greater than 12 g per deciliter and greater than 11 g per deciliter, respectively, between months 6 and 18. A total of 69 grade 3 or 4 adverse events with onset or worsening during or after reni-cel infusion were reported in the nine participants. Six serious adverse events (infections, pyrexia, or pneumonitis) were reported in four participants. Adverse events were generally consistent with myeloablative conditioning. One patient had decreased lymphocyte counts attributed to reni-cel.<br><br>CONCLUSIONS: Treatment with reni-cel resulted in rapid neutrophil engraftment, an increase in fetal hemoglobin expression, and transfusion independence. These data support further investigation of Cas12a gene editing of the promoters of HBG1 and HBG2 in the treatment of transfusion-dependent &#x3b2;-thalassemia. (Funded by Editas Medicine; EdiThal ClinicalTrials.gov number, NCT05444894.).},
}
@article {pmid41934615,
year = {2026},
author = {Wang, Z and Tamura, Y and Hashimoto, M and Nakazato, I and Yamaguchi-Nishimura, A and Arimura, SI},
title = {Using ngTALEN to improve genome editing efficiency on targets containing 5-methylcytosines.},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {1},
pages = {e70826},
pmid = {41934615},
issn = {1365-313X},
support = {24H02271//Japan Society for the Promotion of Science/ ; JPJSCCA20230008//Japan Society for the Promotion of Science/ ; },
mesh = {*Gene Editing/methods ; *Arabidopsis/genetics/metabolism ; DNA Methylation/genetics ; *5-Methylcytosine/metabolism ; Arabidopsis Proteins/genetics/metabolism ; *Transcription Activator-Like Effector Nucleases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic/genetics ; Genome, Plant/genetics ; },
abstract = {We recently discovered distinct methylation patterns between the mitochondrial genome and the nuclear-encoded mitochondrial DNA sequences (NUMTs), with the mitochondrial genome being hypomethylated and NUMTs being hypermethylated. Given that genome editing using mitochondrial targeted transcription activator-like effector nucleases (TALEN) is highly efficient, while editing at NUMT is difficult, we hypothesized that the methylation status might affect editing outcomes. To test this, we attempted to use ngTALEN [employing RVD-NG to recognize 5-methylcytosine (5mC)] to target the Flowering Wageningen (FWA) locus of Arabidopsis thaliana, specifically the promoter and gene body regions with varying levels of cytosine methylation. Comparative analysis using the active epimutant allele fwa-d and wild-type Columbia-0 (Col-0) carrying a naturally silenced allele of FWA revealed that editing was impeded by 5mC at both the promoter and gene body of FWA for both CRISPR/Cas9 and TALEN. Overall, TALEN editing is robust and comparable to that of CRISPR/Cas9 at multiple sites, while ngTALEN showed improved editing at the CG-hypermethylated promoter of FWA compared with TALEN. Additionally, when targeting multiple genomic loci with identical sequences that differ in methylation levels and chromatin states, ngTALEN was less effective to induce edits. Therefore, this study represents the first systematic comparison of editing efficiency between CRISPR/Cas9 and TALEN in dealing with methylated or unmethylated DNA in plants. Furthermore, we have developed ngTALEN as a specific and robust tool for enhancing editing at sites with various levels of CG methylation.},
}
@article {pmid41935002,
year = {2026},
author = {Zhang, H and Zhu, H and Gao, H and Zhou, Y and Que, L and Rong, S and Ma, H and Chang, D and Pan, H},
title = {A colorimetric/fluorescent/electrochemical tri-modal biosensor based on dual CRISPR/Cas12a system for detection of microRNA.},
journal = {Analytica chimica acta},
volume = {1402},
number = {},
pages = {345393},
doi = {10.1016/j.aca.2026.345393},
pmid = {41935002},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *Electrochemical Techniques/methods ; *Colorimetry/methods ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The precise detection of tumor markers is crucial for early cancer diagnosis and monitoring. Existing unimodal detection methods are susceptible to interference in complex biological samples, making it difficult to simultaneously achieve high sensitivity and reliability. MicroRNAs (miRNAs), as a key class of cancer-related biomarkers, necessitate novel detection methods capable of multi-layered verification. This study aims to develop an biosensing platform with multi-signal outputs to address the critical challenge of balancing sensitivity, specificity, and result credibility in current tumor marker detection.<br><br>RESULTS: We successfully constructed a tri-modal biosensor based on a dual CRISPR/Cas12a system for the highly sensitive and specific detection of miRNA let-7a. The sensor generates a trigger strand via an exponential amplification reaction (EXPAR), which concurrently regulates three independent signaling pathways: &#x2460; It initiates the first CRISPR/Cas12a to suppress G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme) formation, turning off the ABTS colorimetric signal. &#x2461; It also activates duplex-specific nuclease (DSN) to inhibit the hybridization chain reaction (HCR), thereby blocking FAM fluorescence coupling to streptavidin-coated magnetic nanoparticles (SMBs) and turning off the fluorescence signal. &#x2462; The absence of HCR products on the SMBs inhibits the second CRISPR/Cas12a system, thereby preserving the electrode's P1 probe for binding with P2-3D-CdCo-ONSs@AuNPs and maintaining a high "turn-on" electrochemical signal from the nanocomposite. Thus, the concentration of miRNA let-7a, ranging from 50&#xa0;fM to 1 pM, can be precisely quantified and validated through colorimetric, fluorescent, and electrochemical signals.<br><br>SIGNIFICANCE: This study integrates a dual CRISPR/Cas12a system with a tri-modal output strategy encompassing colorimetric, fluorescent, and electrochemical detection, thereby constructing a detection platform featuring a cross-verification mechanism. This design not only significantly enhances detection accuracy and anti-interference capability but also lays a solid foundation for developing next-generation, highly reliable molecular diagnostic tools. It holds considerable application potential in the fields of early cancer screening and precision medicine.},
}
@article {pmid41935011,
year = {2026},
author = {Zhang, Y and Liu, W and Guo, R and Qi, Y and Bai, B and Zhang, J and Hu, N and Gu, Y and Yang, Y and Wang, S},
title = {CRISPR/Cas12a-mediated electrochemiluminescent biosensor integrating Ag modified Co-doped metal-organic frameworks for dual detection of malathion and phorate.},
journal = {Analytica chimica acta},
volume = {1402},
number = {},
pages = {345402},
doi = {10.1016/j.aca.2026.345402},
pmid = {41935011},
issn = {1873-4324},
mesh = {*Malathion/analysis ; *Biosensing Techniques/methods ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems ; *Electrochemical Techniques/methods ; Silver/chemistry ; *Luminescent Measurements/methods ; Limit of Detection ; Food Contamination/analysis ; Cobalt/chemistry ; Metal Nanoparticles/chemistry ; CRISPR-Associated Proteins ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {BACKGROUND: With growing concerns about food safety, the detection of pesticide residues in food has become increasingly important. The mixed organophosphorus pesticides (OPs) formulations are commonly employed to maximize crop production; however, the excessive application of OPs has posed severe threats to food safety and human health. Therefore, there is an urgent need to develop rapid and sensitive analytical methods that could simultaneously detect the multiple OPs in food samples. Currently, studies on high-performance electrochemiluminescence (ECL) sensors for two or more target sensing have been rarely reported.<br><br>RESULTS: This work reports a CRISPR/Cas12a-mediated ECL biosensor for the dual detection of two organophosphorus pesticides, i.e., malathion and phorate. The composite of metal-organic framework material (Co-PTC) loaded with silver nanoparticles (AgNPs@Co-PTC) serves as the single-signal probe, in which Co-PTC was the ECL emitter and AgNPs function as co-reaction accelerators to amplify ECL signals effectively. The method obtains an off-ECL signal by incubating the biosensor with malathion and DNA labelled with black hole quencher 1 (BHQ1-DNA); subsequently, after converting phorate to activator DNA, the sideloading activity of CRISPR/Cas12a was activated, which resulted in an on-ECL signal. By integrating spherical nucleic acid switching strategy with CRISPR/Cas12a for signal amplification, the method achieved the detection limits of 0.108 pM for malathion and 1.01 pM for phorate (S/N&#xa0;=&#xa0;3), with satisfactory recovery rates of 95.7%-106.4% in food samples (cabbage and lettuce).<br><br>SIGNIFICANCE: The established dual-detection mode eliminates the need for multiple signal reporters, simplifying the detection procedure and effectively avoiding cross-interference. Accordingly, the CRISPR/Cas12a-based ECL biosensors featured high selectivity and stability and offered a novel analytical strategy for food safety monitoring.},
}
@article {pmid41935495,
year = {2026},
author = {Zhang, J and Zhang, W and Yan, J and Zhang, Y and Zheng, J and Liu, Y and Gu, N and Han, K},
title = {A proximity induced strand displacement amplification and CRISPR/Cas12a-based SERS assay for ultrasensitive detection of dengue virus.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118651},
doi = {10.1016/j.bios.2026.118651},
pmid = {41935495},
issn = {1873-4235},
mesh = {*Dengue Virus/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; *Spectrum Analysis, Raman/methods ; *Dengue/virology/diagnosis ; *CRISPR-Cas Systems/genetics ; Humans ; Limit of Detection ; Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Dengue virus (DENV) is a mosquito-borne single-stranded RNA virus that causes dengue fever and can progress to severe disease, posing a significant public-health threat. Rapid and highly sensitive detection is crucial for the early diagnosis of DENV, which, however, suffers from difficult discrimination from similar symptoms of other virus infection, such as Chikungunya or Zika virus, as well as insufficient specificity and time-consuming procedures. Here, we present a surface-enhanced Raman scattering (SERS) biosensor that integrates strand displacement amplification (SDA) with CRISPR/Cas12a for ultrasensitive detection of DENV. In the absence of DENV, SDA and Cas12a stay inactive and the SERS probes retain an aggregation state due to the crosslinking of single-stranded Linker, exhibiting strong SERS signals. In the presence of target sequences, SDA generates abundant single-stranded Trigger-strands, activating Cas12a through crRNA recognition, cleaving the Linker required to enable the crosslinking and leaving the SERS nanoprobes in a monodispersed state, demonstrating weak SERS signals. This method shows exceptional sensitivity for Dengue virus type 1 (DENV-1), with a limit of detection as low as 4.68 fM. It also provides excellent specificity by accurately distinguishing DENV-1 from other pathogens, while maintaining reliable performance. The modular design of this strategy endows the system capability to be adapted for different targets, making it not only a promising tool for the highly sensitive diagnosis of DENV-1, but also a versatile platform for the precise recognition of other pathogens.},
}
@article {pmid41935496,
year = {2026},
author = {Wu, Y and Lv, B and Zhou, R and Zhang, H and Li, D},
title = {Highly sensitive closed-tube detection of alkaline phosphatase based on phosphate-mediated CRISPR/Cas12a regulation.},
journal = {Biosensors &amp; bioelectronics},
volume = {304},
number = {},
pages = {118659},
doi = {10.1016/j.bios.2026.118659},
pmid = {41935496},
issn = {1873-4235},
mesh = {*Alkaline Phosphatase/blood/isolation &amp; purification ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Phosphates/chemistry/metabolism ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; *Endodeoxyribonucleases/chemistry/genetics/metabolism ; },
abstract = {Recently, we reported that a 5'-terminal phosphate at the junction of split activators inhibits the trans-cleavage activity of Cas12a. In this work, we discovered that phosphate-mediated Cas12a inhibition has two key features: strong position dependence and notable dual-phosphate enhancement. AlphaFold 3 (AF3) structural predictions indicate that the inhibition is mediated by interactions between the phosphate and specific local residues on Cas12a. On the basis of these findings, we developed a highly sensitive alkaline phosphatase (ALP) biosensor by harnessing the principle that ALP controls Cas12a activation through dephosphorylating activators. Without the need for upstream signal amplification, the biosensor achieves a high sensitivity of 6.07 × 10[-6] U/L for ALP across a broad dynamic range of 7.5 × 10[-6] to 2.5 × 10[-2] U/L. Furthermore, the assay is readily extendable to the evaluation of ALP inhibitors. Notably, a closed-tube assay system with lyospheres (CASL) was developed. Our biosensor equipped with the CASL enables closed-tube ALP detection in human serum, effectively preventing environmental contamination and interference. Owing to its simple closed-tube workflow and excellent performance, this biosensor holds great potential for point-of-care clinical diagnostics. Beyond its application, the phosphate-mediated CRISPR/Cas12a regulation strategy elucidated herein offers deeper mechanistic insight and a versatile blueprint for future biosensor design.},
}
@article {pmid41937163,
year = {2026},
author = {Zhou, HR and Doan, DTH and Hartwig, T and Turck, F},
title = {Cis-regulatory architecture downstream of FLOWERING LOCUS T underlies quantitative control of flowering in Arabidopsis thaliana.},
journal = {Genome biology},
volume = {27},
number = {1},
pages = {},
pmid = {41937163},
issn = {1474-760X},
support = {EXC 2048/1 Project ID: 390686111//Cluster of Excellence on Plant Sciences/ ; },
mesh = {*Arabidopsis/genetics/growth &amp; development ; *Flowers/genetics/growth &amp; development ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; Enhancer Elements, Genetic ; Gene Editing ; Chromatin/metabolism ; Regulatory Sequences, Nucleic Acid ; },
abstract = {BACKGROUND: The FLOWERING LOCUS T (FT) gene is a central integrator of floral induction in Arabidopsis thaliana, with its precise expression controlled by complex transcriptional networks. While upstream regulatory regions are well-studied, the role of downstream cis-regulatory elements in modulating FT expression remains poorly characterized.<br><br>RESULTS: Systematic dissection of the FT downstream region in its native chromosomal context using CRISPR/Cas9-mediated genome editing provides genetic evidence that a 2.3-kb sequence, encompassing the Block E enhancer immediately adjacent to the FT coding sequence, is essential for proper FT expression and timely flowering. Fine-scale deletions within Block E reveal that a 63-bp sequence containing one CCAAT-box and one G-box, both closely spaced, forms a core functional module, whereas other conserved motifs contribute modestly in a context-dependent manner. Strikingly, a cryptic CCAAT-box module downstream of Block E that becomes active when repositioned. This coincides with increased transcription factor occupancy and local chromatin accessibility.<br><br>CONCLUSIONS: Our work reveals that quantitative FT expression and flowering time are governed by the spatial organization and chromatin context of downstream cis-regulatory elements. The positional sensitivity and modular logic of these elements provide framework for understanding and engineering quantitative gene regulation through targeted cis-regulatory design, a concept broadly applicable across diverse developmental systems.},
}
@article {pmid41937789,
year = {2026},
author = {Lv, J and He, X and Wu, Q and Sun, Y and Pu, W and Dai, C},
title = {Research progress and applications of gene activation editing technology in crops.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1787461},
pmid = {41937789},
issn = {1664-462X},
abstract = {In recent years, CRISPR/Cas gene editing technology has become a fundamental method in biological breeding. As a vital tool for overcoming technological obstacles, it is currently widely used in functional gene research and genetic enhancement across a variety of organisms. Currently, CRISPR activation (CRISPRa) technology based on dCas9 fusion transcription activation domains has emerged as a powerful tool for expanding the application of CRISPR/Cas systems in improving traits in plants, animals, and microorganisms. This overview starts by going over the underlying principles and components of gene activation editing technology, as well as the phases of development of its three generations. It summarises the present difficulties and potential directions in this field while concentrating on the use of gene activation editing in important crop traits including growth and development regulation, stress resistance, and quality regulation. The objective is to offer valuable insights for the research and development of crop breeding.},
}
@article {pmid41938062,
year = {2026},
author = {Wang, S and Thach, T and De Vita, A},
title = {Editorial: The role of nano-therapeutics in precision cancer medicine.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {14},
number = {},
pages = {1818253},
pmid = {41938062},
issn = {2296-4185},
}
@article {pmid41938130,
year = {2026},
author = {Yin, X and Fan, Z and Tong, Z},
title = {CRISPR-based electrochemiluminescence biosensors: Principles, optimization strategies, and translational challenges - A review of recent progress.},
journal = {Food chemistry. Molecular sciences},
volume = {12},
number = {},
pages = {100392},
pmid = {41938130},
issn = {2666-5662},
abstract = {The integration of CRISPR/Cas systems with electrochemiluminescence (ECL) has emerged as a promising strategy for constructing high-performance biosensing platforms. CRISPR systems, particularly Cas12a and Cas13a, offer programmable recognition of nucleic acid targets and activatable trans-cleavage activity. ECL provides sensitive signal readout with low background and wide dynamic range. As a narrative review, this article provides a comprehensive overview of recent advances in CRISPR-ECL biosensors, with an emphasis on optimization strategies and practical applications. We first discuss the working principles of Cas12a and Cas13a relevant to biosensing, highlighting their distinct kinetic properties, crRNA design considerations, and reaction condition requirements. We then examine optimization approaches at three interconnected levels: nucleic acid probe design (signal-on, signal-off, and auxiliary probes), sensing interface engineering (probe structures, luminophores, electrode materials, and magnetic nanomaterials), and cascade signal amplification (PCR, CHA, RCA, SDA, EDA, and RPA). Through cross-study comparison, we evaluate the strengths and limitations of different approaches and identify critical knowledge gaps. Their applications in detecting disease biomarkers, pathogen nucleic acids, environmental contaminants, and enzyme activities are summarized. Despite remarkable sensitivity achieved, challenges remain in assay time, reproducibility in complex matrices, and clinical validation. From industrialization and global health perspectives, regulatory approval, manufacturing scalability, cost control, and deployment in low-resource settings are also discussed. Finally, future directions toward simplified workflows, enhanced matrix robustness, standardized validation, multiplexed detection, and point-of-care compatible platforms are proposed. This review provides a structured reference and critical perspective for researchers working on CRISPR-ECL biosensing and related fields.},
}
@article {pmid41938355,
year = {2026},
author = {Ma, G and Xu, H and Zhang, S and Li, X and Liu, J and Xie, J and Yan, F and Zhou, H},
title = {Enhancing the efficiency of nuclease-based prime editing in rice with the Tf1 reverse transcriptase.},
journal = {aBIOTECH},
volume = {7},
number = {2},
pages = {100026},
pmid = {41938355},
issn = {2662-1738},
abstract = {The development of efficient and precise genome-editing tools is crucial for advancing functional genomics and improving crops. Our previously established nuclease-mediated prime editing (NM-PE) system, which combines the SpCas9 nuclease with prime editing based on microhomology-mediated end joining, enables the seamless insertion of small DNA fragments into plant genomes to add tags to genes of interest. However, the efficiency of 3 × FLAG sequence insertion via NM-PE requires further improvement. Here, we report a significant optimization of this system by replacing the M-MLV reverse transcriptase (RT) with evolved variants of the retrotransposon RT Tf1 derived from the mammalian PE6 system. Through codon optimization, we generated the evoTf1M4 variant, which substantially enhanced the efficiency of NM-PE. The optimized construct rPE20aV3 achieved up to 18.75% precise insertion of a 66-bp 3 × FLAG sequence at endogenous loci, representing a three-fold improvement over the original NM-PE system. Our results demonstrate that Tf1-aided optimization of NM-PE serves as an efficient platform for seamless insertion of a 3 × FLAG sequence in rice, offering broad potential for advanced genome engineering in plants.},
}
@article {pmid41939725,
year = {2026},
author = {Xu, H and Liu, R and Zhou, H and Kong, B and Shen, K and Zhao, T and Du, X and Zhang, H and Song, H and Guo, D and Gu, X and Wang, Q and Lee, CW and Yin, G and Zhang, Y and Chen, W},
title = {Engineered small extracellular vesicles as bioactive materials: Integrating engineering strategies for cargo loading and targeted delivery systems.},
journal = {Bioactive materials},
volume = {59},
number = {},
pages = {96-134},
pmid = {41939725},
issn = {2452-199X},
abstract = {Small extracellular vesicles (sEVs) are increasingly regarded as a unique class of bioactive materials whose intrinsic membrane composition and nanoscale architecture provide a versatile platform for therapeutic engineering. Rather than passive carriers, sEVs can be actively programmed through diverse strategies to achieve efficient loading, precise targeting, and functional integration with synthetic systems. Endogenous modulation of donor cells-via genetic editing, priming with bioactive glass, cytokine stimulation, or hypoxic cues-enables selective packaging of nucleic acids, proteins, and metabolites into secreted vesicles. Exogenous techniques, including electroporation, sonication, and extrusion, allow controlled incorporation of therapeutic drugs or genome-editing complexes such as CRISPR/Cas. In parallel, surface modifications based on Lamp2b-fusion scaffolds, aptamers, antibodies, and click chemistry confer tissue tropism and extend circulation time. Integration with nanomaterials, scaffolds, and microfluidic platforms further enhances stability, scalability, and reproducibility, positioning sEVs at the intersection of biology and materials science. This review highlights recent advances in engineering sEVs as programmable bioactive materials and discusses their potential to transform regenerative medicine, oncology, and precision therapeutics.},
}
@article {pmid41940952,
year = {2026},
author = {Wang, N and Tang, J and Hou, X and Quan, C and Mai, J and He, C and Chen, R and Tao, B and Gu, Y and Jia, D and Fu, T and Zou, J and Zhao, L and Wen, J and Shen, J},
title = {BnaCIPK9 homoeologs mediate the dosage-dependent regulation of seed oil in allotetraploid Brassica napus L.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {139},
number = {4},
pages = {},
pmid = {41940952},
issn = {1432-2242},
support = {2022ZD04008//Biological Breeding-National Science and Technology Major Project/ ; 2022YFD1200804//National Key Research and Development Program of China/ ; 2021HSZD004//Hubei Hongshan Laboratory research funding/ ; 2662023PY004//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Brassica napus/genetics/metabolism ; *Seeds/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; *Plant Oils/metabolism ; Phylogeny ; CRISPR-Cas Systems ; Tetraploidy ; *Gene Dosage ; Haplotypes ; Arabidopsis/genetics ; Genes, Plant ; },
abstract = {CRISPR/Cas9 mutagenesis and overexpression lines analyses revealed CIPK9 homoeologs function in oil regulation of allotetraploid Brassica napus, cooperative multicopy interactions, and a superior haplotype on chromosome A10. Rapeseed (Brassica napus, B. napus), a globally significant allopolyploid oilseed crop, fulfills substantial annual vegetable oil demand. Evolutionary adaptation in this species is underpinned by gene duplication and homoeolog retention, enhancing plasticity under dynamic environmental stresses. This study focuses on BnaCIPK9, a regulator of seed oil content. Phylogenomic and structural analyses demonstrate that BnaCIPK9 homoeologs underwent duplication followed by consistent evolutionary retention within the Brassica lineage, exhibiting remarkable sequence and structural conservation. Expression profiling revealed tissue-partitioned functional specialization among homoeologs, with BnaA10.CIPK9 and BnaC05.CIPK9 showing seed-preferential expression. CRISPR/Cas9 knockout in B. napus and heterologous overexpression in Arabidopsis demonstrate these homoeologs act as dosage-dependent regulators of oil accumulation, dependent on their distinct expression patterns. They further exhibit expression-driven functional diversification in abiotic stress responses during seedling development. Population genomics reveal differential evolutionary trajectories among duplicates, with intensified selection on chromosome A10 driving adaptive divergence. Crucially, haplotype-trait association identifies hap.qCIPK9.A10.0 as a major haplotype linked to elevated oil content. This work elucidates how homoeolog subfunctionalization fine-tunes critical agronomic traits, oil biosynthesis, and stress resilience, in polyploid crops, establishing haplotype-assisted breeding as imperative for developing crop cultivars. Favorable haplotypes, exemplified by hap.qCIPK9.A10.0, offer precise targets for high-oil crop breeding improvement.},
}
@article {pmid41942919,
year = {2026},
author = {Behera, L and Samal, KC and C, P and Agrawal, PK and Achary, VMM and Dash, M and Mishra, A and Rani, M and Masika, FB and Goud, GSD and Kesawat, MS and Samantaray, S},
title = {An improved Agrobacterium-mediated transformation method for genome editing using CRISPR/Cas9 in elite indica rice (Oryza sativa L.).},
journal = {BMC genomics},
volume = {},
number = {},
pages = {},
doi = {10.1186/s12864-026-12800-0},
pmid = {41942919},
issn = {1471-2164},
}
@article {pmid41943368,
year = {2026},
author = {Sangeetha, VJ and Pawase, PA and Vasanthkumar, SS and Patrimath, SS and Bashir, O},
title = {Precision-engineered starch: Integrating metabolic engineering and cell-free synthetic biology for sustainable bioplastics and functional foods.},
journal = {Carbohydrate polymers},
volume = {381},
number = {},
pages = {125146},
doi = {10.1016/j.carbpol.2026.125146},
pmid = {41943368},
issn = {1879-1344},
mesh = {*Metabolic Engineering/methods ; *Starch/chemistry/biosynthesis/genetics/metabolism ; *Synthetic Biology/methods ; *Functional Food ; Amylose/chemistry/metabolism ; Gene Editing ; },
abstract = {This review consolidates recent advances that reposition starch development from downstream modification towards deliberate biosynthetic engineering. It critically examines the starch biosynthetic pathway as a foundation for rational design, highlighting the coordinated roles of granule-bound starch synthase, soluble starch synthases, starch branching enzymes, and debranching enzymes in defining granule structure. Clear structure-function-nutrition relationships are delineated, demonstrating how amylose-amylopectin ratio, chain-length distribution, and phosphate esterification govern physicochemical behaviour, digestibility, glycemic response, and resistant starch formation. Insights derived from naturally occurring starch mutants are integrated to elucidate genotype-phenotype-function linkages. It further evaluates the recent progress in planta metabolic engineering, with particular emphasis on CRISPR-Cas-based genome editing as a precise strategy for generating food-relevant starch phenotypes without foreign DNA insertion. Representative applications include high-amylose, low-glycemic staple crops and starches with engineered branching patterns to enhance processing performance. In parallel, emerging cell-free synthetic biology platforms are presented as complementary systems for producing structurally defined glucans with high purity and reproducibility. Collectively, these developments establish engineered starch as a next-generation bio-based material aligned with nutritional quality, technological functionality and sustainability objectives.},
}
@article {pmid41943836,
year = {2026},
author = {Xu, J and Cheng, L and Ma, S and Gan, C and Chai, J and Zheng, X and Hu, L and Ling, M and Zhang, M and Zhao, B and Cheng, H},
title = {In vivo CRISPR/Cas9 Screening Reveals that UBE2L3 Modulates Autophagic Flux through TSC2 Ubiquitination and Potentiates PD-1 Blockade in Triple-Negative Breast Cancer.},
journal = {International journal of biological sciences},
volume = {22},
number = {6},
pages = {2950-2969},
pmid = {41943836},
issn = {1449-2288},
mesh = {*Triple Negative Breast Neoplasms/metabolism/genetics ; *Ubiquitin-Conjugating Enzymes/metabolism/genetics ; Humans ; *Autophagy/genetics/physiology ; *Tuberous Sclerosis Complex 2 Protein/metabolism/genetics ; Ubiquitination ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Female ; Animals ; Mice ; *Programmed Cell Death 1 Receptor/metabolism/antagonists &amp; inhibitors ; Mice, Nude ; },
abstract = {Triple-negative breast cancer (TNBC), a distinct breast cancer subtype, poses significant challenges to conventional therapeutic approaches, and effective targeted therapies are limited. CRISPR/Cas9 library screening has demonstrated unprecedented efficiency and revolutionary potential in the identification of therapeutic targets. In this study, we performed In vivo CRISPR/Cas9 library screening and identified the E2 ubiquitin-conjugating enzyme UBE2L3 as a critical regulatory factor in the progression of TNBC. Loss of UBE2L3 restricted tumor cell growth by modulating autophagy in TNBC cells. Mechanistically, UBE2L3 downregulation led to increased tuberous sclerosis complex 2 (TSC2) expression, suppressing mTOR activity and altering autophagic processes in tumor cells. This regulation was mediated through the interaction between UBE2L3 and the E3 ubiquitin ligase SMURF2, which together control TSC2 protein ubiquitination and degradation. Autophagy and the tumor microenvironment are closely associated, and we observed that UBE2L3 knockdown in TNBC tumors significantly increased CD8+ T lymphocyte infiltration and enhanced tumor sensitivity to anti-PD-1 therapy. Collectively, our findings provide a theoretical foundation for considering UBE2L3 as a potential therapeutic target in TNBC.},
}
@article {pmid41943869,
year = {2026},
author = {Katayama, R and Hayashi, R and Ueda, M and Sakamoto, T and Nakazawa, M},
title = {Elucidation of Enzymatic Routes Underlying Odd-Chain Fatty Acid Synthesis and Propionate Assimilation in Euglena gracilis.},
journal = {The Journal of eukaryotic microbiology},
volume = {73},
number = {3},
pages = {e70078},
doi = {10.1111/jeu.70078},
pmid = {41943869},
issn = {1550-7408},
support = {JPMJGX23B0//Japan Science and Technology Agency/ ; JP24K01897//Japan Society for the Promotion of Science/ ; //Women Researchers Support Ofice of Osaka Metropolitan University/ ; },
mesh = {*Propionates/metabolism ; *Euglena gracilis/metabolism/enzymology/genetics ; *Fatty Acids/biosynthesis ; Gene Knockout Techniques ; Acyl Coenzyme A/metabolism ; Methylmalonyl-CoA Mutase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Wax esters (WEs) produced by Euglena gracilis under anaerobic conditions contain significant proportions of odd-chain fatty acids and fatty alcohols (30%-40%). Propionyl-CoA, the primer for odd-chain fatty acid synthesis, is generally thought to be generated via the methylmalonyl-CoA pathway; however, the enzymatic basis of this pathway in E. gracilis remains unclear. Here, we generated knockout (KO) mutants of candidate enzymes in the methylmalonyl-CoA pathway-succinyl-CoA synthetase (SCS), methylmalonyl-CoA mutase (MCM), methylmalonyl-CoA epimerase (MCE), and propionyl-CoA carboxylase (PCC)-using the CRISPR/Cas9 system and examined their roles in odd-chain fatty acid synthesis and propionate assimilation. The methylmalonyl-CoA pathway exhibited direction-dependent functions. KO of MCE and PCC did not affect the proportion of odd-chain WEs, indicating that these enzymes are not required for odd-chain WE synthesis and suggesting an alternative route converting methylmalonyl-CoA to propionyl-CoA. In contrast, both enzymes were required for propionate assimilation. Functional differentiation was observed between SCSβ isozymes: SCSβ1 contributed to odd-chain WE synthesis, whereas SCSβ2 primarily functioned in propionate assimilation. Partial impairment of propionate assimilation in SCSα or SCSβ2 KO strains suggests involvement of additional routes. These findings improve our understanding of direction-dependent roles in the methylmalonyl-CoA pathway in E. gracilis and support further studies of odd-chain lipid biosynthesis.},
}
@article {pmid41945389,
year = {2026},
author = {Li, M and Gao, G and Jiao, Y},
title = {A Hierarchical Screening Strategy for Genome-Edited Events in Polyploid Species: A Case Study on Hexaploid Common Wheat.},
journal = {Current protocols},
volume = {6},
number = {4},
pages = {e70354},
doi = {10.1002/cpz1.70354},
pmid = {41945389},
issn = {2691-1299},
support = {2024YFF1000301//National Key R&amp;D Program of China/ ; tsqn202312306//Taishan Scholars Program/ ; 2025HWYQ-071//Shandong Provincial Natural Science Fund for Excellent Young Scientists Fund Program (Overseas)/ ; SYS202206//Shandong Provincial Natural Science Foundation/ ; ZR2022ZD22//Shandong Provincial Natural Science Foundation/ ; ZR2021ZD30//Shandong Provincial Natural Science Foundation/ ; },
mesh = {*Triticum/genetics ; *Gene Editing/methods ; *Polyploidy ; *Genome, Plant ; CRISPR-Cas Systems ; },
abstract = {Genome editing via CRISPR/Cas9 has been widely adopted in cereal crops. In diploid species such as rice and barley, the generation of knockout mutants is relatively straightforward for functional characterization of the genes of interest due to their single-copy nature in the genome. In contrast, common wheat (Triticum aestivum L.) is a hexaploid species comprising three subgenomes (AABBDD); consequently, most genes are present as three homoeoalleles that retained substantial function redundancy during evolution. The generation of a complete set of single, double, and triple mutants is therefore essential for elucidating homoeoallele-specific functions and dissecting their contributions to the developmental and agronomic traits. Moreover, ensuring germplasm purity through the elimination of residual T-DNA is critical for maintaining stable mutation, particularly in single- and double-mutant lines. Here, we describe a hierarchical screening strategy for efficient identification of a comprehensive series of CRISPR/Cas9-induced mutants. This approach integrates high-throughput DNA isolation, selection of T-DNA-free mutants, maintenance of a uniform genetic background via backcrossing, systematic screening of all mutant combinations, and molecular confirmation of genome edits. This screening pipeline has proven effective in hexaploid common wheat and is readily adaptable to other polyploid species that are amenable to crossing. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Wheat cultivation and leaf sample preparation Basic Protocol 2: High-throughput DNA isolation Basic Protocol 3: Genotyping using an optimized, cost-efficient T7E1 assay Alternate Protocol 1: Genotyping using the KASP assay Support Protocol 1: Screening of T-DNA-free triple mutants.},
}
@article {pmid41946009,
year = {2026},
author = {Sutaoney, P and Singh, P and Malakar, S and Arsi, L and Ghosh, P},
title = {Microbial lipases: Catalyzing sustainable solutions for industrial innovations.},
journal = {Enzyme and microbial technology},
volume = {198},
number = {},
pages = {110869},
doi = {10.1016/j.enzmictec.2026.110869},
pmid = {41946009},
issn = {1879-0909},
abstract = {Microbial lipases are multifaceted biological catalyst that have surfaced as a key driver in various industries and are both eco-friendly and cost efficient.In large scale applications, lipases produced from bacteria, fungi and yeasts function better than their equivalents generated from plants and animals due to their wide substrate specificity, catalytic efficacy and stability under physicochemical circumstances. Recent developments in microbial lipase research, including sources, screening techniques, assay procedures, production methods, purification tactics, and biochemical characterisation, are critically examined in this review.The structural and mechanistic elements that control lipase function-such as lid domains, interfacial activation, and catalytic triads-are given special attention since they all have an impact on the stability, specificity, and industrial performance of the enzyme.Large-scale screening is done to check for the production of lipase in Bacillus sp., Achromobacter sp., Alcaligenes sp., Arthrobacter sp., Pseudomonas sp., and Penicillium sp. Additionally, the combination of synthetic biology, metagenomics, CRISPR-Cas technologies, enzyme engineering, and AI-assisted modelling is emphasized as a revolutionary strategy for identifying and customizing lipases with desired characteristics, including extreme environment microbes and application-specific variants.The review also highlights the growing industrial uses of microbial lipases in the bio-fuel, food and beverage, detergent, textile, leather, pharmaceutical, and medical industries, highlighting their contribution to the development of economically feasible and ecologically safe bioprocesses. All things considered, microbial lipases are an important biotechnological tool for developing sustainable industrial innovation and green chemistry.},
}
@article {pmid41946722,
year = {2026},
author = {Malone, HA and Myers, JA and Gruss, EG and Morgan, MA and Friske, JD and McCarty, TC and Navarro, JJ and Robinson, S and Halliburton, RL and Kietlinska, SJ and De Luna Vitorino, FN and Hansen, BS and Pruett-Miller, SM and Garcia, BA and Roussel, MF and Partridge, JF and Roberts, CWM},
title = {PHIP suppresses NuRD to enable the growth of SWI/SNF-mutant cancers.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41946722},
issn = {2041-1723},
support = {R01-CA-273455//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-172152//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01-CA-113794//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; F31-CA-278355//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; P01-CA-96832//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01-HD-106051//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P01-CA-196539//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Humans ; *Neoplasms/genetics/metabolism/pathology ; *Mi-2 Nucleosome Remodeling and Deacetylase Complex/metabolism/genetics ; Cell Line, Tumor ; *Transcription Factors/genetics/metabolism ; Mutation ; *Chromosomal Proteins, Non-Histone/genetics/metabolism ; Chromatin Assembly and Disassembly/genetics ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Chromatin/metabolism ; Promoter Regions, Genetic ; Transcriptional Activation ; Ubiquitination ; },
abstract = {SWI/SNF chromatin remodeling complexes are perturbed in 20% of all cancers and in several developmental disorders, yet the mechanisms by which these mutations dysregulate transcription and drive disease are poorly understood. To both elucidate these mechanisms and identify vulnerabilities caused by these mutations, we leverage genome-wide CRISPR-Cas9 screening in hundreds of cancer cell lines and identify the chromatin reader protein PHIP as a specific dependency in cancers with broadly disrupted SWI/SNF function. Mechanistically, we reveal that PHIP cooperates with SWI/SNF to facilitate transcriptional activation by ubiquitinating and suppressing subunits of the repressive Nucleosome Remodeling and Deacetylase (NuRD) complex. We demonstrate that loss of SWI/SNF results in NuRD complexes accumulating at promoters where they would otherwise cause widespread transcriptional silencing if not antagonized by PHIP. Collectively, we identify PHIP as a regulator of the interplay between distinct chromatin regulators that function in development and disease and as a targetable vulnerability in cancers with broad SWI/SNF inactivation.},
}
@article {pmid41946842,
year = {2026},
author = {Akana, RV and Yoe, J and Laveroni, O and Sun, C and Kim, YM and Jerby, L},
title = {High-content CRISPR activation screens identify synthetically lethal RNA-based mechanisms to sensitize cancer cells to targeted T cell cytotoxicity.},
journal = {Nature genetics},
volume = {58},
number = {4},
pages = {841-853},
pmid = {41946842},
issn = {1546-1718},
support = {1019508.01//Burroughs Wellcome Fund (BWF)/ ; OPP1113682//Bill and Melinda Gates Foundation (Bill &amp; Melinda Gates Foundation)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Line, Tumor ; Receptors, Antigen, T-Cell/immunology/genetics ; *T-Lymphocytes/immunology ; *RNA/genetics ; Animals ; Mice ; *Melanoma/genetics/immunology ; *Neoplasms/genetics/immunology ; *Cytotoxicity, Immunologic/genetics ; },
abstract = {T cells recognize their target cells through the T cell receptor (TCR). Combining gain-of-function, single-cell and optical high-content screens, we identified RNA-based mechanisms that selectively sensitize target cells to TCR-specific T cell cytotoxicity. First, CRISPR activation screens in melanoma cells identify functionally diverse regulators of TCR-specific cytotoxicity, including SAFB, KHDRBS1, MYC, CD44, WNT3A, WNT1 and others. Expressing sensitizing hits in cancer and virally infected cells restores TCR-specific cytotoxicity. Next, we developed in situ Perturb-seq for optical pooled genetic screens with in situ detection of perturbations and spatial transcriptomic readouts. Perturb-seq and in vivo-in situ Perturb-seq show that the hits converge on shared cell-autonomous and intercellular mechanisms, map gene-environment interactions and reveal that Wnt ligands activate T cells. Introducing a scalable approach to decode gene function at the cell and tissue level, the study uncovered context-specific gene functions to restore targeted T cell-based elimination of dysfunctional cells via synthetically lethal, RNA-based interventions.},
}
@article {pmid41946927,
year = {2026},
author = {Fang, GQ and Deng, Y and Lyu, XY and Yin, CQ and Song, J and Zhang, Y and Zhong, J and Shen, EZ and Song, CQ},
title = {Boosting prime editing with engineered non-canonical pegRNAs.},
journal = {Nature biomedical engineering},
volume = {},
number = {},
pages = {},
pmid = {41946927},
issn = {2157-846X},
support = {32471552//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Prime editing (PE) enables precise genetic modifications using canonical prime editing guide RNA (pegRNA), with the reverse transcription template and primer binding site (RTT-PBS) attached to the 3' ends of CRISPR-Cas guide RNAs. Although PE ribonucleoprotein (RNP) delivery holds great therapeutic potential, its weak genomic editing capability limits therapeutic applications. Here we present structure-guided engineering of the PE complex using non-canonical pegRNAs (npegRNAs), with the RTT-PBS integrated within the single guide RNA loops, to improve PE efficiency. This approach demonstrates enhanced precise editing rates across various genomic sites and cell types, and improves therapeutic gene correction in a tyrosinaemia mouse model. Cas9-associated npegRNAs are more resistant to exonuclease degradation, probably enhancing the PE complex's targeting efficiency in living cells. Using PE RNP delivery, npegRNAs achieve increased average editing yields of 26.8-fold over canonical pegRNAs and 5.9-fold over engineered pegRNAs (epegRNAs). Furthermore, npegRNA-mediated RNPs increased the efficiency of installing disease-relevant mutations up to 123-fold in human cell lines, including Jurkat T cells and induced pluripotent stem cells. Collectively, our findings demonstrate a robust PE strategy and highlight the potential of npegRNAs for therapeutic PE applications.},
}
@article {pmid41949766,
year = {2026},
author = {Mostafa, K and Scarano, A and Abdulla, MF and Hacıkamiloğlu, S and Kurt, O and Santino, A and Kavas, M},
title = {A rapid Agrobacterium rhizogenes-mediated transient expression for assessing sgRNA efficiency in CRISPR-Act3.0 in tomato.},
journal = {Plant cell reports},
volume = {45},
number = {5},
pages = {},
pmid = {41949766},
issn = {1432-203X},
support = {121O463//T&#xfc;rkiye Bilimsel ve Teknolojik Ara&#x15f;t&#x131;rma Kurumu/ ; },
mesh = {*Solanum lycopersicum/genetics/metabolism ; *Agrobacterium/genetics ; Plants, Genetically Modified ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Plant ; Fatty Acids/metabolism ; Transformation, Genetic ; Promoter Regions, Genetic ; },
abstract = {CRISPR-Act3.0 is a robust tool for modulating fatty acid profiles in plants. We demonstrate that Agrobacterium rhizogenes-mediated transformation provides a rapid, cost-effective, and equipment-independent platform for validating sgRNA efficiency and metabolic outcomes within a short time. The CRISPR-Act3.0 system offers a powerful strategy for activating endogenous gene expression in plants. However, the labor-intensive and time-consuming nature of stable transformation often hinders the rapid validation of multiple sgRNAs. In this study, we optimized a rapid Agrobacterium rhizogenes-mediated transient expression system in tomato to evaluate sgRNA efficiency within the CRISPR-Act3.0 framework. As a proof-of-concept, we targeted four genes involved in fatty acid biosynthesis: SlFATA, SlFATB-01, SlFATB-02, and SlFATB-03. To ensure precise control, we utilized the root-specific pSMB promoter to drive the CRISPRa components. Our results demonstrate that this system can successfully induce significant transcriptional activation and alter fatty acid compositions specifically increasing palmitic acid levels by up to 45%-within approximately 30 days. This approach bypasses the requirement for whole-plant stable transformation during the initial screening phase and prevents potential pleiotropic effects by restricting activation to root tissues. Overall, this study provides a highly efficient diagnostic pipeline for functional genomics and metabolic engineering in tomato, offering a significant advantage for rapid trait evaluation before committing to stable transgenic line production.},
}
@article {pmid41950725,
year = {2026},
author = {Clémençon, M and Brogard, J and Rozen, M and Hourton, C and García, RM and Goureau, O and Bigou, S and Reichman, S},
title = {Generation of human P347L RHO-associated retinitis pigmentosa iPSC lines by a mutation insertion in the RHODOPSIN gene carrying the RHO c.1040C &gt; T variant using CRISPR/Cas9.},
journal = {Stem cell research},
volume = {93},
number = {},
pages = {103968},
doi = {10.1016/j.scr.2026.103968},
pmid = {41950725},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Retinitis Pigmentosa/genetics/pathology/metabolism ; *Rhodopsin/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Mutation ; Cell Line ; Cell Differentiation ; },
abstract = {The P347L RHODOPSIN-related retinal dystrophy leads an autosomal dominant Retinitis Pimentosa. Here we describe the generation of two isogenic human induced pluripotent stem cell (hiPSC) lines carrying the mutation c.1040C > T, p.Pro347Leu in the RHODOPSIN gene using CRISPR/Cas9 engineering from a control hiPSC clone. The two generated hiPSC lines can be differentiated in all the three germ layers, showed pluripotency makers expression and presented a normal karyotype. These hiPSCs will provide a new cell tool to better understand physiopathological mechanisms of retinitis pigmentosa and for the development of innovative treatment.},
}
@article {pmid41951520,
year = {2026},
author = {He, Y and Ma, Y and Wu, Y and Tang, X and Liu, S and Yin, D and Zheng, X and Qi, Y and Zhang, Y and Zhang, T},
title = {Harnessing diverse tRNAs and AI-guided mining for compact and efficient plant multiplex genome editing.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2026.02.016},
pmid = {41951520},
issn = {1879-3096},
abstract = {The widespread use of CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) in plants highlights the need for compact and efficient multiplexed genome editing systems. This study optimizes single-guide RNA (sgRNA) expression in CRISPR by leveraging endogenous tRNA processing mechanisms for efficient multiplexed genome editing. Screening in Arabidopsis thaliana and Oryza sativa identified superior tRNAs that outperformed the widely used AtGly-tRgcc. Leveraging tRNA's dual functions in sgRNA processing and their intragenic RNA polymerase III promoter activity, we established a compact multiplexed system for simultaneous editing of at least ten genomic loci in rice and soybean. Moreover, we developed plant tRNA large language models that learn sequence representations to identify both canonical and noncanonical tRNAs, uncovering thousands of tRNAs missed by traditional algorithms and expanding the repertoire for genome editing. This work provides a robust tRNA-based CRISPR platform, an artificial intelligence-guided tRNA mining framework, and a comprehensive tRNA resource for advanced plant genome engineering and germplasm innovation.},
}
@article {pmid41951915,
year = {2026},
author = {Zhu, Y and Moerner, WE and Qi, LS},
title = {CRISPR-Cas-based live cell imaging of genome dynamics.},
journal = {Nature reviews. Genetics},
volume = {},
number = {},
pages = {},
pmid = {41951915},
issn = {1471-0064},
abstract = {The 3D architecture and dynamics of the genome are crucial for regulation of genome stability, transcription and cellular function. CRISPR-based live imaging technologies have enabled real-time visualization of specific genomic loci and transcripts in living cells. These tools harness customized guide RNAs and nuclease-deactivated Cas effectors to achieve precise genomic targeting, and recent methodological advances provide the 3D spatiotemporal resolution required to decipher real-time chromatin communication. These methods are elucidating the biophysical properties of chromatin, linking dynamic enhancer-promoter interactions directly to transcription, and revealing the role of 3D genome dynamics in basic cellular processes and disease. Here, we summarize the development of CRISPR-based live-cell imaging techniques, highlight the complementary 3D microscopy and analysis methods compatible with these methods, and offer perspectives on their applications to uncover fundamental principles that govern genome dynamics and function.},
}
@article {pmid41952056,
year = {2026},
author = {Salum, YM and Chen, J and Dang, J and Qiao, Q and Chen, T and Lin, T and Wei, H and He, W},
title = {Leveraging RNAi and CRISPR/Cas9-based strategies for target gene characterization and control of western flower thrips (Frankliniella occidentalis): Advances and perspectives.},
journal = {Pest management science},
volume = {},
number = {},
pages = {},
doi = {10.1002/ps.70782},
pmid = {41952056},
issn = {1526-4998},
support = {//Major Project of Science and Technology of Fujian Province (2024NZ029029)/ ; //Fujian Provincial Science and Technology Project (2023L3021 and 2025N0081)/ ; },
abstract = {Frankliniella occidentalis, the western flower thrips, is among the most destructive agricultural pests worldwide and a major vector of orthotospoviruses. Escalating insecticide resistance has intensified efforts to develop molecularly informed control strategies. RNA interference (RNAi) and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) genome editing have emerged as principal tools for functional genomics in this species, enabling targeted analysis of genes involved in development, metabolism, and insecticide resistance. This review synthesizes recent advances in RNAi and CRISPR applications in F. occidentalis, with particular emphasis on delivery strategies, editing efficiencies, and resulting phenotypes. RNAi studies demonstrate growing technical feasibility through injection, feeding-based approaches, and plant-mediated systems, and emerging CRISPR/Cas investigations confirm the possibility of targeted gene knockouts affecting resistance traits and visible genetic markers. Despite these advances, genetic manipulation of F. occidentalis remains technically challenging, primarily due to biological and technical constraints, including inefficient delivery of ribonucleoprotein to the germline, the extremely small and fragile nature of eggs, and narrow developmental windows for manipulation. We discuss these barriers and highlight methodological innovations required to improve RNAi stability, embryo manipulation, and genome editing efficiency. Finally, we propose how these molecular and genetic tools may be incorporated into existing integrated pest management, emphasizing their potential as complementary, species-specific strategies rather than standalone control solutions toward sustainable F. occidentalis management. © 2026 Society of Chemical Industry.},
}
@article {pmid41952275,
year = {2026},
author = {Das, U and Prasad, SS and Sahoo, T and Paramanik, S and Halder, A and S, P},
title = {Unveiling the potential of banana (Musa spp.) improvement through genetic manipulation: current trends and future implications.},
journal = {Plant signaling &amp; behavior},
volume = {21},
number = {1},
pages = {2656013},
pmid = {41952275},
issn = {1559-2324},
mesh = {*Musa/genetics/microbiology ; Gene Editing ; Plants, Genetically Modified/genetics ; Genetic Engineering/methods ; Disease Resistance/genetics ; Plant Breeding ; Plant Diseases/microbiology/genetics ; },
abstract = {Banana (Musa spp.) is a globally important fruit crop and a staple food for millions of people. However, its narrow genetic diversity and clonal propagation make it highly vulnerable to pests, diseases, and abiotic stresses. Genetic improvement is limited by sterility, and triploid plants are reproduced clonally in most cultivated varieties, preventing traditional breeding based on genetic advancements. Transgenesis and gene editing are among the genetic engineering techniques used to increase yield, improve quality, and enhance resilience. Advances in Agrobacterium-mediated transformation and CRISPR/Cas tools have enabled the development of bananas with enhanced resistance to Fusarium wilt tropical race 4 (TR4), Black Sigatoka, and bacterial wilt, along with increased provitamin A content, longer shelf life, and reduced postharvest losses. DNA-free genome editing provides a promising approach to overcoming certain regulatory barriers and enhancing public acceptance. While challenges such as genotype-specific transformation efficiency, regulatory hurdles, and public perception persist, genetic manipulation holds the potential to both preserve and improve global banana production. This review synthesizes recent progress, key targets, and future prospects for the genetic improvement of banana.},
}
@article {pmid41952451,
year = {2026},
author = {Mengistu, G},
title = {CRISPR-Cas Systems in Human Disease Therapy: Advances, Clinical Applications, Limitations, and Future Directions.},
journal = {The journal of gene medicine},
volume = {28},
number = {4},
pages = {e70091},
doi = {10.1002/jgm.70091},
pmid = {41952451},
issn = {1521-2254},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods/trends ; Precision Medicine ; Animals ; },
abstract = {CRISPR-Cas systems have emerged as versatile platforms for targeted genome and transcriptome engineering, enabling precise manipulation of disease-associated genetic pathways. Continued advances in CRISPR technologies including base editing, prime editing, and epigenome modulation have expanded therapeutic possibilities beyond nuclease-mediated DNA cleavage, allowing programmable gene correction and regulation. Early clinical studies demonstrate sustained therapeutic benefit in selected monogenic disorders and highlight the feasibility of both ex vivo and in vivo editing strategies. However, clinical translation remains constrained by challenges such as off-target activity, delivery inefficiency, immune responses to Cas proteins, editing heterogeneity, and uncertainties regarding long-term safety. This review critically synthesizes recent advances in CRISPR-Cas systems for human disease therapy, integrating molecular innovations, delivery strategies, clinical progress, and ethical considerations. By evaluating both technological achievements and unresolved limitations, this article outlines key priorities for advancing CRISPR-based therapeutics toward safe, effective, and equitable precision medicine.},
}
@article {pmid41952472,
year = {2026},
author = {Martinkienė, J and Cui, T and Ciotta, G and Kupčinskas, J and Pažėraitis, D},
title = {RP-HPLC-based purification of long single-stranded DNA for CRISPR knock-in applications.},
journal = {BioTechniques},
volume = {78},
number = {1-12},
pages = {149-157},
doi = {10.1080/07366205.2026.2649857},
pmid = {41952472},
issn = {1940-9818},
mesh = {*DNA, Single-Stranded/isolation &amp; purification/genetics/chemistry ; Chromatography, High Pressure Liquid/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; *Chromatography, Reverse-Phase/methods ; },
abstract = {BackgroundLong single-stranded DNA (ssDNA; >200 nucleotides) is valuable for DNA nanotechnology, precision medicine, and as a CRISPR-Cas9 knock-in donor template, but existing preparation methods are laborious, low-yield, or difficult to scale. MethodsWe developed a workflow combining enzymatic digestion with high-temperature reversed-phase high-performance liquid chromatography (RP-HPLC) to purify kilobase-length ssDNA. The method was evaluated across analytical and semi-preparative formats. ResultsThe approach enables clean resolution of linear and circular ssDNA species ranging from 1.5 to 4.5 kb and is scalable across formats. A 1.5 kb ssDNA donor supported efficient CRISPR knock-in at the T-cell receptor alpha constant (TRAC) locus in primary human CD8[+] T cells, without adversely affecting viability or expansion. ConclusionsThis RP-HPLC workflow provides a scalable and reproducible method for generating high-purity long ssDNA suitable for genome engineering applications.},
}
@article {pmid41954204,
year = {2026},
author = {Zheng, S and Wang, C and Huang, Y and Meng, X and Zhao, D and Guan, E and Ma, F and Chen, A and Zheng, L},
title = {Dual-Mode CRISPR/Cas13a Assay for the Detection of Human Metapneumovirus in Clinical Respiratory Samples.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70921},
doi = {10.1002/jmv.70921},
pmid = {41954204},
issn = {1096-9071},
support = {2025YFC2311704//National Key R&amp;D Program of China/ ; },
mesh = {Humans ; *Metapneumovirus/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *Paramyxoviridae Infections/diagnosis/virology ; *Respiratory Tract Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *CRISPR-Cas Systems ; Reproducibility of Results ; *Nucleic Acid Amplification Techniques/methods ; Real-Time Polymerase Chain Reaction ; RNA, Viral/genetics ; },
abstract = {Human metapneumovirus (HMPV) is a significant pathogen, causing widespread acute respiratory infections (ARIs). Rapid and accurate detection is crucial for timely diagnosis and outbreak control. To address the limitations of real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based HMPV detection, we developed a novel method integrating recombinase-aided amplification (RAA) with CRISPR-Cas13a technology. Based on the alignment of 335 HMPV whole-genome sequences, a conserved region of the nucleocapsid (N) gene was selected for RAA primer and CRISPR RNA (crRNA) design. The concentrations of Cas protein, crRNA, and probe were optimized for specificity, sensitivity, and repeatability. A dual-mode platform was established, combining a fluorescence assay for quantitative detection and a lateral flow assay (LFA) for visual, on-site readout. Both methods were validated using 106 clinical samples and compared with RT-qPCR. Both methods demonstrated high specificity (no cross-reactivity with other respiratory viruses), a detection limit of 1 copy/μL, and excellent repeatability. Clinical validation showed complete concordance (κ = 1, p < 0.001) between the fluorescence assay and RT-qPCR, and high agreement for LFA (κ = 0.924, p < 0.001), with a sensitivity of 94.23%, specificity of 98.15%, positive predictive value (PPV) of 98%, and negative predictive value (NPV) of 94.6%. This study presents an RAA-CRISPR/Cas13a dual-mode platform for HMPV detection, providing a robust molecular diagnostic framework that paves the way for the future development of rapid testing assays.},
}
@article {pmid41954977,
year = {2026},
author = {Duan, X and Zhang, H and Lv, J and Fan, X and Liu, X and Wang, Q and Zhang, F and Zhang, Q and Liu, L and Liang, L and Liu, R},
title = {PE-STAR: prime editing with SOS-triggered and RecJ-augmented repair enables high-efficiency editing in Escherichia coli.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954977},
issn = {1362-4962},
support = {2023YFC3402300//National Key R&amp;D Program of China/ ; SRICSPYF-ZY2025107//Scientific Research Innovation Capability Support Project/ ; 22278058//National Natural Science Foundation of China/ ; 22578048//National Natural Science Foundation of China/ ; XLYC2203075//Xingliao Talent Plan/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; *Escherichia coli/genetics ; *Escherichia coli Proteins/genetics/metabolism ; *SOS Response, Genetics/genetics ; *DNA Repair ; CRISPR-Cas Systems ; Exodeoxyribonucleases/genetics/metabolism ; Plasmids/genetics ; },
abstract = {Prime editing enables precise genome modifications without DNA double-strand breaks, yet bacterial applications are limited by low efficiency and small edit sizes. Here, we develop PE-STAR, Prime Editing with SOS-Triggered and RecJ-Augmented Repair, to enhance prime editing in Escherichia coli. Removing three inhibitory 3'→5' exonucleases (SbcB, ExoX, and XseA) improved edited-strand retention, and extending post-transformation outgrowth increased editing efficiency. RecJ overexpression strengthened 5'-directed processing during flap resolution and gap expansion, biasing repair toward incorporation of the reverse-transcribed edited strand. To enrich edited cells, we integrated an SOS-responsive counter-selection circuit that links PE3-associated dual nicking to LexA-dependent gRNA expression targeting a plasmid encoding the toxin CcdB, thereby eliminating unedited cells. PE-STAR achieved up to 80%-90% editing efficiency for short-fragment modifications, representing up to 16-fold improvement across loci. The platform supported insertions, deletions, and replacements of up to 46 bp with high efficiency. Furthermore, installing an attB site by prime editing, followed by Bxb1 integrase recombination, enabled chromosomal integration of 3.2 and 8.0 kb cassettes with 100% recombination efficiency among screened colonies, including GFP reporter and riboflavin biosynthetic pathway. PE-STAR expands both the efficiency and functional scope of bacterial prime editing for programmable genome engineering.},
}
@article {pmid41954981,
year = {2026},
author = {Sentmanat, MF and Wang, ZT and Kouranova, E and Peters, ST and Chan, WC and Lin, J and Miao, Y and White, JM and Wallace, M and Cui, X},
title = {Efficient multi-kilobase knock-ins in mice and cell lines using CRISPR/Cas9 and rAAV donors with unbiased whole-genome characterization by LOCK-seq.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954981},
issn = {1362-4962},
support = {//Alvin J. Siteman Cancer Center/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Gene Knock-In Techniques/methods ; *Dependovirus/genetics ; *Gene Editing/methods ; Cell Line ; RNA, Guide, CRISPR-Cas Systems/genetics ; Humans ; Genome ; },
abstract = {Multi-kilobase knock-ins (KIs) are a necessary, yet challenging type of genome editing to create and characterize in cell lines and animals. The combination of rAAV donor transduction and electroporation of single-cell mouse embryos with Cas9/gRNA ribonucleoprotein complex enables highly efficient KI, but the insert size is limited by the viral packaging capacity. Here, we report the creation of up to 6.7 kb precise KI achieved in one step by using three rAAVs designed to insert one after the other. To fully characterize the edited genome with large KIs, we developed LOCK-seq (LOng-read sequencing of Captured Kilo-base targets), where relevant genomic regions are enriched via hybridization, achieving over 100-fold greater coverage compared with other long-read methods with enrichment. LOCK-seq simultaneously detects the presence of precise KI alleles, imprecision in the insert and donor concatenation, genotypes of non-KI alleles, and more importantly, uniquely identifies and localizes random integration of the full or partial donor(s). Additionally, the multi-rAAV donor approach is successfully applied to cell lines, including lines intolerant of plasmid DNA, whereas LOCK-seq reliably and efficiently screens for KI clones. Together, the two approaches significantly improve the creation and precision of knock-in models.},
}
@article {pmid41954985,
year = {2026},
author = {Zhang, S and Liu, Y and Wu, W and Liu, Z and He, Q and Wang, T and Yang, J and Yin, H and Yuan, Z and Zhang, H},
title = {Allosteric activation mechanism of the type VII CRISPR-Cas system.},
journal = {Nucleic acids research},
volume = {54},
number = {6},
pages = {},
pmid = {41954985},
issn = {1362-4962},
support = {32571682//National Natural Science Foundation of China/ ; 25JCZDJC00740//Natural Science Foundation of Tianjin Municipal Science and Technology Commission/ ; TJYXZDXK-009A//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; TJYXZDXK-3-004B//Tianjin Key Medical Discipline (Specialty) Construction Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Allosteric Regulation ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; RNA/metabolism/chemistry/genetics ; Catalytic Domain ; Models, Molecular ; Ribonucleases/chemistry/metabolism/genetics ; },
abstract = {Type VII CRISPR-Cas system, evolutionarily associated with type III systems, utilizes a Cascade complex formed by Cas5 and catalytically inactive Cas7 copies for target RNA binding, but instead incorporates a specialized Cas14 ribonuclease for target cleavage. Here, we report a high-quality cryo-EM structure at the target engagement state with a shortened crRNA and elucidate how the recruited Cas14 captures the target RNA and undergoes target-mediated activation. The signature Cas14 is homologous to eukaryotic CPSF73 and prokaryotic RNase J, comprising two conserved subdomains, MβL and β-CASP. Different from canonical type III systems, 5'-end target RNA, rather than 3'-end, is bent into the positively charged binding channel formed by the two subdomains to access the conserved catalytic pocket on Cas14. Two special structural features, α1 helix from Cas7 and α10 helix from Cas14, promote the bent target RNA docking into the catalytic pocket of Cas14 nuclease in concert. A dual-functional loop, displaced by the entering target RNA, induces a closed-to-open transition between the two subdomains for nuclease activation. More importantly, the flipped dual-functional loop also maintains the stabilization of incoming target RNA. Altogether, our work provides a more comprehensive understanding of type VII system mechanism, laying a mechanistic foundation for RNA-targeting tool development.},
}
@article {pmid41956064,
year = {2026},
author = {Wang, M and Sternberg, SH},
title = {'Insane in the membrane': 2',3'-cGAMP triggers filamentous phage defense.},
journal = {Cell host &amp; microbe},
volume = {34},
number = {4},
pages = {556-558},
doi = {10.1016/j.chom.2026.03.009},
pmid = {41956064},
issn = {1934-6069},
mesh = {*Nucleotides, Cyclic/metabolism ; *Inovirus/physiology ; Signal Transduction ; Humans ; Membrane Proteins/metabolism ; Nucleotidyltransferases/metabolism ; *Cell Membrane/metabolism/virology ; *Bacteria/metabolism/virology ; Animals ; },
abstract = {In this issue, Tak et al. report that bacteria produce 2',3'-cGAMP, a signaling molecule once considered unique to metazoans. This cyclic dinucleotide activates a SAVED-domain effector that polymerizes into membrane-disrupting filaments, inducing abortive infection and preventing phage replication, broadening the evolutionary scope of cGAS-STING-like signaling.},
}
@article {pmid41956072,
year = {2026},
author = {Decloquement, M and Macauley, MS},
title = {Revealing cancer glycome drivers using CRISPR activation screens.},
journal = {Cell genomics},
volume = {6},
number = {4},
pages = {101215},
pmid = {41956072},
issn = {2666-979X},
mesh = {Humans ; *Neoplasms/genetics/metabolism ; Glycosylation ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Glycomics/methods ; },
abstract = {In this study, Wisnovsky and colleagues[1] investigate the genetic determinants underlying cancer-associated glycome remodeling. Using genome-wide CRISPR-based approaches, they identified regulators of cell surface glycosylation. Specifically, genetic networks are discovered that upregulate cancer-related ligands of the sialic acid-binding immunomodulatory Siglecs.},
}
@article {pmid41959582,
year = {2026},
author = {Carrasco, E and Gutierrez-Marcos, J},
title = {Novel genome editing approaches to manipulate apical meristem activity for crop yield.},
journal = {Frontiers in plant science},
volume = {17},
number = {},
pages = {1743528},
pmid = {41959582},
issn = {1664-462X},
abstract = {Meristem function underlies organogenesis and yield potential in crop species, and its regulation depends on the crosstalk of genetic and hormonal networks that balance stem-cell niche maintenance and differentiation. During the shoot apical meristem (SAM) transition, developmental reprogramming shifts the meristem from a vegetative to a reproductive state, referred to as inflorescence meristem (IM). Major regulatory events in this transition include the cytokinin-gibberellin crosstalk, that regulate the expression of the CLAVATA/WUSCHEL (CLV/WUS) negative feedback loop and key transcription factor families like KNOTTED-LIKE HOMEOBOX (KNOX) and SHOOT MERISTEMLESS (STM). Despite the basic principles of apical meristem differentiation are well-described nowadays, major phenotypic bottlenecks were reached in major staple crops during the artificial selection process, known as domestication, leading to a final reduction in total crop yield. This review aims to describe the key processes and genes that play a role in this transition and how they can be artificially targeted to overcome these limitations. Major bioengineering approaches are covered, ranging from classical random mutagenesis with chemicals like ethyl methanesulfonate (EMS) to targeted genome editing using diverse Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR/Cas) systems. Finally, emerging strategies such as epibreeding are considered as promising tools to achieve precise, reversible modulation of meristem activity and to unlock new routes for crop yield enhancement.},
}
@article {pmid41961863,
year = {2026},
author = {Emond, A and Laflamme, C and Therrien, M and Liao, M and Maios, C and Labarre, A and Drapeau, P and Parker, JA},
title = {Characterization of a C9orf72 Knockout Danio rerio model for ALS and cross-species validation of potential therapeutics screened in Caenorhabditis elegans.},
journal = {PloS one},
volume = {21},
number = {4},
pages = {e0346613},
pmid = {41961863},
issn = {1932-6203},
mesh = {Animals ; *Zebrafish/genetics ; *Caenorhabditis elegans/genetics ; *C9orf72 Protein/genetics ; *Amyotrophic Lateral Sclerosis/genetics/drug therapy/pathology ; Disease Models, Animal ; Gene Knockout Techniques ; Humans ; CRISPR-Cas Systems ; Motor Neurons/pathology/metabolism ; Larva ; Animals, Genetically Modified ; Phenotype ; },
abstract = {Intronic hexanucleotide repeat expansions in the C9orf72 gene represent the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This expansion decreases C9orf72 expression in affected patients, indicating that loss of C9orf72 function (LOF) acts as a pathogenic mechanism. Several models using Danio rerio (zebrafish) for C9orf72 depletion have been developed to explore disease mechanisms and the consequences of C9orf72 LOF. However, inconsistencies exist in reported phenotypes, and many have yet to be validated in stable germline ablation models. To address this, we created a zebrafish C9orf72 knockout model using CRISPR/Cas9. The C9orf72 LOF model demonstrates, in a generally dose-dependent manner, increased larval mortality, persistent growth reduction, and motor deficits. Additionally, homozygous C9orf72 LOF larvae exhibited mild overbranching of spinal motoneurons. To identify potential therapeutic compounds, we performed a screen on an established Caenorhabditis elegans (C. elegans) C9orf72 homologue (alfa-1) LOF model, identifying 12 compounds that enhanced motility, reduced neurodegeneration, and alleviated paralysis phenotypes. Motivated by the shared motor phenotype, 2 of those compounds were tested in our zebrafish C9orf72 LOF model. Pizotifen malate was found to significantly improve motor deficits in C9orf72 LOF zebrafish larvae. We introduce a novel zebrafish C9orf72 knockout model that exhibits phenotypic differences from depletion models, providing a valuable tool for in vivo C9orf72 research and ALS therapeutic validation. Furthermore, we identify pizotifen malate as a promising compound for further preclinical evaluation.},
}
@article {pmid41964260,
year = {2026},
author = {Muthusamy, B and Nizan, S and Bar-Ziv, A and Perl-Treves, R},
title = {Functional Validation of the Melon Fom-1 Gene, Controlling Resistance to Fusarium oxysporum Races 0 and 2, by CRISPR/Cas9 Mutagenesis.},
journal = {Molecular plant pathology},
volume = {27},
number = {4},
pages = {e70258},
pmid = {41964260},
issn = {1364-3703},
support = {1137/16//Israel Science Foundation/ ; 873-0074-11//Chief Scientist grant for Biotechnology, Ministry of Agriculture, Israel/ ; },
mesh = {*Fusarium/pathogenicity/physiology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *CRISPR-Cas Systems/genetics ; Mutagenesis/genetics ; *Plant Proteins/genetics/metabolism ; *Genes, Plant ; *Cucumis melo/genetics/microbiology ; },
abstract = {Functional validation of NLR genes is critical for confirming their specific roles and developing durable disease-resistant crops. The Fom-1 gene of Cucumis melo, controlling resistance to races 0 and 2 of F. oxysporum f. sp. melonis (FOM), had been identified by map-based cloning as MELO3C022146, which encodes a TIR-NBS-LRR (TNL) protein. It resides in a head-to-head orientation adjacent to another TNL gene, Prv, controlling resistance to papaya ring spot virus (PRSV). In this study we validated the function of Fom-1 in mediating FOM resistance by applying CRISPR/Cas9 editing to the resistant cultivar, Védrantais. Two gRNAs were designed to target exons 1 and 2, respectively, and mutations were introduced at both target sites, resulting in truncated open reading frames in both alleles. Inoculation assays of T1 plants with FOM races 2 and 0 revealed a breakdown of resistance, manifested by leaf necrosis and wilting, and susceptibility was stably inherited in the T2 generation. This proved that MELO3C022146, the candidate gene for Fom-1, is responsible for resistance in melon cultivar Védrantais. Future studies will address the molecular functions of this gene as well as possible interactions between Fom-1 and its neighbour R-gene, Prv.},
}
@article {pmid41965285,
year = {2026},
author = {Yang, X and Cai, J and Wang, J and Meng, Y and Shi, Y and Cai, H},
title = {CRISPR-Cas9 knockout screens to identify drug resistance genes in acute myeloid leukemia.},
journal = {Methods in cell biology},
volume = {205},
number = {},
pages = {199-216},
doi = {10.1016/bs.mcb.2026.01.006},
pmid = {41965285},
issn = {0091-679X},
mesh = {*CRISPR-Cas Systems/genetics ; *Leukemia, Myeloid, Acute/genetics/drug therapy/pathology ; Humans ; *Drug Resistance, Neoplasm/genetics ; *Gene Knockout Techniques/methods ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology ; },
abstract = {Acute Myeloid Leukemia (AML) is a hematopoietic malignancy characterized by the uncontrolled proliferation of aberrant myeloid blasts within the bone marrow, resulting in disrupted hematopoiesis and severe clinical consequences. Drug resistance represents a major barrier in AML treatment, frequently manifesting as relapse following initial remission with conventional chemotherapeutic agents such as cytarabine and venetoclax. The underlying mechanisms of drug resistance include enhanced drug efflux, altered drug metabolism, and activation of pro-survival signaling pathways, necessitating the elucidation of specific genetic determinants to enable the development of effective therapeutic strategies. The advent of CRISPR/Cas9 system has facilitated precise genomic modifications, permitting the generation of cell libraries with targeted gene knockouts in AML cells. This approach can identify genes whose disruption alters drug sensitivity, implicating their involvement in survival and resistance to cell death. This protocol outlines a systematic strategy to uncover genes associated with drug resistance in AML cells by leveraging CRISPR/Cas9-mediated functional genomic screening. By employing this methodology, genes conferring drug susceptibility upon knockout are noted as potential drivers of drug resistance, offering valuable insights for the rational design of targeted therapies.},
}
@article {pmid41965303,
year = {2026},
author = {Zhai, X and Yang, B and Deng, F and Gulati, S and Mckinnirey, F and Wu, X and Li, Y and Goldys, EM},
title = {CbAgo-enriched Cas12a biosensor for cancer mutations screening.},
journal = {Analytica chimica acta},
volume = {1403},
number = {},
pages = {345420},
doi = {10.1016/j.aca.2026.345420},
pmid = {41965303},
issn = {1873-4324},
mesh = {Humans ; *Biosensing Techniques/methods ; Mutation ; CRISPR-Cas Systems ; *Endodeoxyribonucleases/genetics/metabolism ; *Pancreatic Neoplasms/genetics/diagnosis ; Circulating Tumor DNA/genetics/blood ; Proto-Oncogene Proteins p21(ras)/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; DNA Mutational Analysis/methods ; Bacterial Proteins ; },
abstract = {BACKGROUND: Accurate detection of low-frequency DNA mutations in body fluids is essential for cancer monitoring and treatment evaluation. However, the high abundance of wild-type DNA often masks rare mutant signals, making sensitive detection particularly challenging.<br><br>RESULTS: We developed a screening strategy termed the CbAgo-enriched Cas12a mutation screening system (CECMS). By integrating the single-nucleotide resolution of CbAgo with the trans-cleavage activity of CRISPR-Cas12a, this system selectively eliminates wild-type DNA while enriching targeted mutant alleles. CECMS achieves up to 100-fold higher sensitivity at 37&#xa0;&#xb0;C compared with conventional Cas12a biosensors, enabling reliable detection of variant allele frequencies (VAFs) as low as 0.01%. In undiluted serum spiked samples for circulating tumor DNA (ctDNA) detection, the method successfully detected pancreatic cancer-associated KRAS G12D mutations at a VAF of 0.1%.<br><br>SIGNIFICANCE: By leveraging CbAgo-mediated enrichment, the capability of exposing rare SNV for downstream detection is markedly improved. With its high efficiency and ease of use, CECMS holds strong potential as a convenient tool for clinical cancer diagnostics and monitoring.},
}
@article {pmid41965876,
year = {2026},
author = {Seibert, M and Kurrle, N and Kaleab, S and Wempe, F and von Metzler, I and Serve, H and Schnütgen, F},
title = {Endogenous protein tagging coupled with a CRISPR screening approach identifies UBE3C as a potential MYC oncogene regulator.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {},
pmid = {41965876},
issn = {2045-2322},
mesh = {Humans ; *Proto-Oncogene Proteins c-myc/genetics/metabolism ; *CRISPR-Cas Systems ; *Ubiquitin-Protein Ligases/genetics/metabolism ; Cell Line, Tumor ; *Multiple Myeloma/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The transcription factor MYC is a key regulator of cellular proliferation and metabolism and is frequently dysregulated in malignancies such as multiple myeloma (MM). Despite its clinical relevance, direct therapeutic targeting of MYC remains limited, emphasizing the need to identify upstream regulators that control endogenous MYC expression. To systematically uncover such regulators, we developed a genome-wide CRISPR-Cas9 loss-of-function screening approach, employing a custom-engineered MM reporter cell line (RPMI8226-F11), in which oncogenic MYC protein was endogenously tagged with EGFP (referred to as GFP). This fluorescent readout enabled a direct, quantitative assessment of endogenous MYC expression levels. A pooled genome-wide sgRNA library was introduced, and cells were sorted based on GFP fluorescent intensity to reflect varying MYC levels. Next-generation sequencing of sgRNA distributions across sorted populations enabled the identification of candidate MYC regulators. Validation of screen hits, including the established MYC activator IRF4 and repressor FBXW7, confirmed the reliability of our system. To further dissect regulatory networks, we performed an overrepresentation analysis of target genes, which revealed the enrichment of Mediator complex subunits among MYC activators and ubiquitin-proteasome pathway components among MYC repressors. Functional validation of prioritized hits-MED30 (Mediator complex) and UBE3C (E3 ubiquitin ligase)-demonstrated a strong impact on endogenous MYC levels. Notably, the knockout of UBE3C markedly increased MYC expression, whereas its paralogs, UBE3A and UBE3B, showed no measurable effect, suggesting a specific regulatory role for UBE3C in MM cells. Together, our study provides a comprehensive CRISPR screen-based resource for the discovery of MYC regulators and highlights UBE3C as a potential therapeutic node for modulating MYC expression in MM.},
}
@article {pmid41966744,
year = {2026},
author = {Tang, Q and Song, XH and Guo, AN and Chen, Y and Shao, JW},
title = {CRISPR-mediated regulation of apoptosis in cancer: Molecular targets, mechanisms, and translational challenges.},
journal = {Biochemical and biophysical research communications},
volume = {817},
number = {},
pages = {153719},
doi = {10.1016/j.bbrc.2026.153719},
pmid = {41966744},
issn = {1090-2104},
abstract = {The CRISPR/Cas system, owing to its high gene-editing efficiency and relatively low off-target effects, has emerged as a pivotal technological platform in cancer research. By precisely modulating oncogenic signaling pathways and apoptosis-related molecules, CRISPR provides a critical tool for elucidating and reprogramming the regulatory mechanisms of apoptosis in tumor cells. However, despite substantial progress in preclinical studies, achieving efficient and selective induction of apoptosis in tumor cells remains a major translational challenge. Increasing evidence indicates that CRISPR-based strategies can achieve more effective antitumor outcomes by reprogramming tumor cell sensitivity to apoptosis, rather than relying solely on single-gene editing. This review systematically summarizes the molecular targets and underlying mechanisms of CRISPR-mediated regulation of tumor cell apoptosis, with particular emphasis on key apoptotic signaling pathways and representative research advances. It highlights the pivotal role of targeting survival-associated genes in suppressing tumor progression and promoting apoptotic cell death. Furthermore, this review discusses the potential synergistic mechanisms of CRISPR in combination with chemotherapy or immunotherapy, as well as the value of CRISPR-based functional screening in identifying apoptosis-regulatory targets and drug resistance-associated mechanisms. Finally, we analyze the key challenges facing the clinical translation of CRISPR-mediated apoptosis regulation and propose future research directions and conceptual frameworks to optimize CRISPR-based anticancer strategies and facilitate their clinical application.},
}
@article {pmid41968241,
year = {2026},
author = {Martin-Vicente, A and Fortwendel, JR},
title = {Cas9 Ribonucleoproteins (RNPs) for Gene Deletion.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {3-12},
pmid = {41968241},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Gene Deletion ; *Aspergillus fumigatus/genetics ; *Ribonucleoproteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; Homologous Recombination ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Targeting/methods ; },
abstract = {Genetic manipulations in pathogenic microorganisms, like Aspergillus fumigatus, allow us to study the function of genes in pathogenicity or other important traits. Classic genetic engineering tools in filamentous fungi rely on DNA fragment sub-cloning or fusion PCR to build a gene deletion cassette containing extended flanking regions of homology to promote efficient recombination. However, the introduction of CRISPR technology into fungal genetics has greatly simplified and accelerated the genome editing process. Here, we describe a simple and universal, one-step CRISPR-Cas9-mediated genetic tool employing repair templates containing microhomology regions. This gene targeting system displays high homologous recombination efficiency rates and can be easily implemented across different genetic backgrounds.},
}
@article {pmid41968242,
year = {2026},
author = {Handelman, M and Werner, H and Osherov, N},
title = {Generation of Seamless Point Mutations with Cas9 RNP and pTel-hygR Plasmid in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {13-21},
pmid = {41968242},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/drug effects ; *CRISPR-Cas Systems ; Drug Resistance, Fungal/genetics ; *Plasmids/genetics ; *Point Mutation ; Antifungal Agents/pharmacology ; Fungal Proteins/genetics ; Gene Editing/methods ; Cytochrome P-450 Enzyme System/genetics ; Triazoles/pharmacology ; },
abstract = {The environmental mold Aspergillus fumigatus is a common human fungal pathogen that causes a wide range of diseases. The antifungal triazoles that inhibit the Cyp51 enzyme involved in ergosterol biosynthesis are used to treat A. fumigatus infections. However, triazole resistance is an increasing concern due to mutations in the genes cyp51A, hmg1, and others, and efflux pumps overexpression. The process of verifying mutations is time-consuming, even with CRISPR-Cas9 methods, as it still requires constructing repair templates with selectable markers. This study presents a faster and more efficient method to introduce mutations conferring triazole resistance in A. fumigatus by using in vitro assembled CRISPR-Cas9, along with a recyclable selectable marker. With this approach, we successfully introduced triazole resistance-conferring mutations in A. fumigatus genes (cyp51A, cyp51B, and hmg1), both individually and in combination. The technique has the potential to introduce mutations for resistance to other antifungals, toxic metals, and environmental stressors, thus enhancing the ability to generate dominant mutations in A. fumigatus.},
}
@article {pmid41968243,
year = {2026},
author = {Valero, C and Tindale, J and Bromley, MJ and van Rhijn, N},
title = {Marker-Free CRISPR/Cas9 RNP Transformation for Gene Disruption in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {23-30},
pmid = {41968243},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics ; *CRISPR-Cas Systems/genetics ; *Transformation, Genetic ; Gene Editing/methods ; *Ribonucleoproteins/genetics ; Genetic Engineering/methods ; },
abstract = {CRISPR-based genetic engineering has resulted in the possibility of far more efficient and challenging genetic engineering in filamentous fungal pathogens. In this respect, selection marker-free CRISPR-Cas9 transformation has been possible in fungi such as Aspergillus fumigatus, one of the most prevalent fungal pathogens of humans. Here, we describe a protocol to perform this technique, which can be widely adapted to use multiple strains and other species.},
}
@article {pmid41968244,
year = {2026},
author = {Guo, Y and Scharf, DH},
title = {CRISPR-Cas9 and Microhomology-Mediated End-Joining for Biosynthetic Pathway Reconstruction in Aspergillus fumigatus.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {31-45},
pmid = {41968244},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Biosynthetic Pathways/genetics ; Plasmids/genetics ; *DNA End-Joining Repair ; Genetic Vectors/genetics ; Gene Editing/methods ; },
abstract = {We have recently engineered Aspergillus fumigatus using an AMA1-based episomal expression CRISPR vector and a microhomology-mediated end-joining repair system. We adopted A. fumigatus as a heterologous expression host to investigate the biosynthetic pathways of epipolythiodioxopiperazine-type compounds. In the present chapter, we describe the assembly of the CRISPR-Cas9 vector and microhomology template plasmid, their transformation into A. fumigatus, and metabolite detection.},
}
@article {pmid41968255,
year = {2026},
author = {Campanella, JEM and Arentshorst, M and Ram, AFJ and van den Hondel, CAMJJ and Malavazi, I},
title = {Molecular Biosensing of the Aspergillus fumigatus Cell Wall Integrity Pathway Using the (p)agsA::luc (Luciferase) Reporter System.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3028},
number = {},
pages = {199-223},
pmid = {41968255},
issn = {1940-6029},
mesh = {*Aspergillus fumigatus/genetics/metabolism ; *Cell Wall/metabolism/genetics ; *Biosensing Techniques/methods ; *Genes, Reporter ; *Luciferases/genetics/metabolism ; CRISPR-Cas Systems ; Fungal Proteins/genetics/metabolism ; Promoter Regions, Genetic ; Luminescent Measurements ; Gene Expression Regulation, Fungal ; },
abstract = {In this protocol, we describe a robust luciferase-based biosensor assay to monitor the activity of the cell wall integrity (CWI) pathway in Aspergillus fumigatus in real time. The method relies on the stable integration of a markerless, synthetic reporter cassette (p)agsA::luc at the aft4 Safe Haven (Sh) genomic locus using CRISPR-Cas9. This cassette comprises a modified A. niger agsA promoter containing three tandem RlmA-binding sites, which drives the expression of the luciferase gene. Upon exposure to cell wall stress, the endogenous transcription factor RlmA activates the reporter, generating a luminescent signal proportional to promoter activity. The protocol includes the construction of aft4 locus-specific CRISPR-Cas9 plasmids, A. fumigatus transformation and candidate selection, as well as the setup of the luminescence bioassay in white 96-well microplates. This system enables highly sensitive, nondestructive, and time-resolved quantification of CWI pathway activation during early fungal growth or biofilm under various genetic or chemical perturbations. Moreover, it supports comparative studies across wild-type and mutant strains, offering a powerful platform for dissecting stress response signaling and identifying antifungal compounds that target the CWI pathway.},
}
@article {pmid41968752,
year = {2026},
author = {Zhang, X and Li, X and Li, X and Peng, C and Wang, W},
title = {A Pangenome Framework Reveals Conserved Pathogenicity-Associated Biosynthetic Gene Clusters in Fusarium equiseti, a Root Rot Pathogen of Panax notoginseng.},
journal = {Molecular plant pathology},
volume = {27},
number = {4},
pages = {e70260},
pmid = {41968752},
issn = {1364-3703},
support = {2025J0492//Yunnan Provincial Department of Education Science Research Fund Project/ ; 22377105//National Natural Science Foundation of China/ ; 202401AV070003//National Nature Science Foundation of Yunnan Province/ ; 202505AF350066//Yunnan provincial science and technology talent/ ; //Xingdian Talent Support Program-Yunling Scholat (W.-G. Wang)/ ; },
mesh = {*Fusarium/genetics/pathogenicity/metabolism ; *Panax notoginseng/microbiology ; *Multigene Family/genetics ; *Plant Roots/microbiology ; *Plant Diseases/microbiology ; Genome, Fungal/genetics ; Virulence/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Panax notoginseng (Chinese ginseng, sanqi) is an economically and medicinally important herb whose cultivation is increasingly threatened by destructive soilborne root rot. Although Fusarium species represent the dominant pathogens in this disease complex, the virulence determinants underlying Fusarium-P. notoginseng interactions remain largely unknown, particularly the contribution of secondary metabolite (SM) biosynthetic gene clusters (BGCs) in medicinal plants. Here, we present a pangenome-informed and experimentally validated framework linking Fusarium SM to disease development. By constructing a genus-scale BGC landscape from 54 Fusarium genomes, integrating it with infection-stage transcriptomics of Fusarium equiseti P26, and establishing an efficient CRISPR/Cas9-based genome-editing system, we functionally prioritized and interrogated conserved, infection-induced BGCs from dozens of predicted clusters. This integrative pipeline identified two broadly conserved and infection-essential BGCs that are indispensable for disease development, including an evolutionarily conserved ICS-NRPS hybrid cluster and a ferrichrome-type siderophore NRPS cluster widely distributed across Fusarium species. Notably, the pathogenic contribution of the ICS-NRPS pathway was independently corroborated in Fusarium oxysporum, supporting a conserved role for this BGC class across the genus. Together, our findings provide the first pangenome-guided gene-to-phenotype dissection of SM-associated virulence in P. notoginseng root rot, establishing conserved SM biosynthetic backbones as causal pathogenicity determinants and tractable targets for disrupting Fusarium disease development across BGC-rich fungal genomes.},
}
@article {pmid41972732,
year = {2026},
author = {Rutkowska, A and Strózik, T and Wasiak, T and Ciunowicz, D and Kapelan, N and Szczepaniak, N and Sosnowski, J and Goślińska, W and Bartkowiak, J and Budny-Lewandowska, A and Antończyk, P and Markiewicz, M and Gustaw, P and Filiks, K and Jaskólska, M and Stoczyńska-Fidelus, E},
title = {CRISPR-Cas9 Therapeutics in Early Clinical Development: Delivery and Molecular Diagnostics.},
journal = {Cells},
volume = {15},
number = {7},
pages = {},
pmid = {41972732},
issn = {2073-4409},
support = {2024/ABM/05/KPO/KPOD.07.07-IW.07-0242/24-00//LEK-AM Pharmaceutical Company Ltd/ ; 503/0-154-01/503-01-001//Medical University of Lodz/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; *Pathology, Molecular/methods ; *Genetic Therapy/methods ; *Molecular Diagnostic Techniques ; *Gene Transfer Techniques ; },
abstract = {CRISPR-Cas9 has progressed from an experimental tool to a therapeutic modality, marked by the first regulatory approvals of an ex vivo-edited autologous CD34+ hematopoietic stem cell product that induces fetal hemoglobin (CASGEVY/exa-cel). In this narrative review, we synthesize modality-specific molecular diagnostic strategies used across early CRISPR clinical translation. In parallel, early clinical experience has begun to demonstrate the feasibility of in vivo editing, including subretinal delivery for CEP290-associated inherited retinal degeneration (EDIT-101 programme) and hepatocyte-targeted lipid nanoparticles (LNPs) for liver-derived targets such as transthyretin and plasma prekallikrein (KLKB1). As translation expands across hematologic, metabolic, ocular and oncology indications, development is increasingly constrained by the predictability and safety of editing outcomes, delivery-determined biodistribution and exposure time, and immune recognition of bacterial Cas9 orthologs and delivery components. We summarize diagnostic readouts for confirming patient genotype, quantifying on-target editing and expression changes, assessing off-target and structural outcomes using orthogonal assays, and monitoring clonal dynamics and immune responses during long-term follow-up. We also discuss how these readouts interface with CMC controls and regulatory expectations for advanced therapy medicinal products (ATMPs), highlighting the need for fit-for-purpose, standardized testing frameworks in early trials.},
}
@article {pmid41972830,
year = {2026},
author = {Chokwassanasakulkit, T and Ranasinghe, V and Woods, E and Nguyen, LQ and McMillan, NAJ},
title = {What If Trojan Horse Nanoparticles Could Change the Game for HPV Gene-Targeted Therapies?.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70916},
pmid = {41972830},
issn = {1096-9071},
support = {2027649//National Health and Medical Research Council (NHMRC)/ ; 2027569//National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Nanoparticles/administration &amp; dosage ; *Papillomavirus Infections/therapy/virology ; *Genetic Therapy/methods ; Uterine Cervical Neoplasms/therapy/virology ; Female ; RNA, Small Interfering/administration &amp; dosage/genetics ; *Papillomaviridae/genetics ; Animals ; CRISPR-Cas Systems ; },
abstract = {Human papillomavirus (HPV) is a common sexually transmitted infection linked to various cancers, particularly cervical cancer, primarily driven by high-risk strains like HPV16 and HPV18. While vaccines are effective in preventing new infections, they do not address existing cases, highlighting the need for innovative therapies. Gene-targeted approaches, such as CRISPR/Cas and siRNA, show promise in inhibiting HPV oncogenes. Recent advancements in Trojan horse nanoparticles (NPs) offer a strategy for delivering these therapies directly to HPV-infected cells. These NPs improve stability and targeted delivery, enhancing the biodistribution of CRISPR/Cas systems and siRNAs while protecting them from degradation. However, challenges like immune responses and regulatory hurdles persist. Therefore, this review emphasizes the potential of Trojan horse NPs in treating HPV-related cancers, identifies critical areas for future research, and provides updates on gene-targeted therapy encapsulated NPs in preclinical and clinical trials.},
}
@article {pmid41973392,
year = {2026},
author = {Shojaei Baghini, S and Esfahani, K and Rad, N and Arezoumandi, M and Taghipour, E and Salmanian, AH},
title = {Targeted multiplex gene knockouts in Lemna minor using CRISPR/Cas9.},
journal = {Transgenic research},
volume = {35},
number = {1},
pages = {},
pmid = {41973392},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems/genetics ; *Araceae/genetics ; *Gene Editing/methods ; *Gene Knockout Techniques/methods ; *Plants, Genetically Modified/genetics/growth &amp; development ; RNA, Transfer/genetics ; },
abstract = {Lemna minor (commonly known as duckweed) is a fast-growing aquatic plant recognized as a promising green bioreactor for recombinant protein production. Its rapid proliferation, high protein yield, environmental adaptability, and edibility make it highly attractive for biotechnological applications. It is essential to develop and expand genetic tools tailored to this species to maximize these advantages and further unlock its biotechnological potential. A key strategy for achieving this goal is the implementation of advanced genome editing technologies, such as the CRISPR/Cas9 system. Although multiplex CRISPR/Cas9 gene editing has previously been successfully applied in Lemna aequinoctialis, the capability of the endogenous plant tRNA processing system for multiplex editing in L. minor using the polycistronic tRNA-sgRNA (PTG)/Cas9 system has not yet been explored. In this study, a PTG construct was engineered to include four sgRNAs designed to simultaneously target two plant-specific glycosyltransferase genes: α-1,3-fucosyltransferase (FucT) and β-1,2-xylosyltransferase (XylT). As anticipated, the PTG-Cas9 system successfully induced frameshift mutations, characterized by insertions and deletions (indels), in regenerated L. minor plants derived from transformed calli. Validation via PCR and RT-PCR analysis, followed by sequencing of the target loci, confirmed the presence of indels at the target sites. Furthermore, western blot analyses utilizing antibodies specific to XylT and FucT in two homozygous lines (lines 44 and 217) revealed truncated XylT proteins in both lines. Moreover, an in-frame FucT protein was detected in line 217, whereas FucT expression was absent in line 44. This study marked the first successful demonstration of PTG-Cas9 system for multiplex genome editing in L. minor, paving the way for advanced genetic engineering in this species.},
}
@article {pmid41973928,
year = {2026},
author = {Lian, G and Khabazeh, A and Sheen, V},
title = {A modified CRISPR/Cas9 approach in silencing the triplication in Down syndrome: A treatment path XISTs.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {16},
pages = {e2517953123},
doi = {10.1073/pnas.2517953123},
pmid = {41973928},
issn = {1091-6490},
support = {1R01HD109794-01//Foundation for the NIH (FNIH)/ ; 5R21NS115593-02//Foundation for the NIH (FNIH)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Long Noncoding/genetics ; *Down Syndrome/genetics/therapy ; Gene Silencing ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genetic Therapy/methods ; },
abstract = {Down syndrome (DS) is one of the most common developmental human genetic disorders and is due to triplication of chromosome 21 (HSA21). Although previous studies using epigenetic suppression of HSA21 by the long noncoding RNA XIST showed a potential for DS treatment, integration efficiency of XIST by conventional zinc finger nucleases is too low to allow for practical implementation. Here, we report a modified CRISPR/Cas9 approach, which enhances the efficiency of XIST gene integration. First, a codon-optimized λ-phage exonuclease (exo) was fused with Cas9 to create 5'- and 3'-end overhangs at cutting sites of donor DNA and acceptor chromosome DNA. Second, four sgRNAs, two of which selectively targeted each the acceptor or donor DNA, were assembled tandemly into one Cas9 plasmid (PX459) to increase the Cas9-cutting efficiency and promote donor DNA integration. Third, sgRNAs were designed by searching for unique single nucleotide polymorphism nucleotides distinct between the three HSA21 copies, as a protospacer adjacent motif site to specifically target one HSA21 copy. Fourth, donor DNA plasmid containing XIST was modified to disable replication and inhibit transcription function and allow for inducible expression. Our modified CRISPR method significantly enhanced the integration efficiency (20 to 40%) of long XIST gene (14 kb) into an extra chromosome 21 (HSA21), as was identified with PCR, cell cloning, immunostaining, and FISH. RNA sequencing results showed that imbalance of gene transcription across extra HSA21 can be partially corrected by XIST gene integration. The modified CRISPR method with XIST paves a road for therapeutic treatment for DS.},
}
@article {pmid41974871,
year = {2026},
author = {Vitiello, A and Boccellino, M and Zovi, A and Bassetti, M},
title = {Antimicrobial resistance and gene therapy: emerging molecular strategies for a global health threat.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {41974871},
issn = {1476-5462},
abstract = {Antimicrobial resistance (AMR) is one of the most serious and pressing health challenges facing modern medicine. Despite advances in antimicrobial stewardship, diagnostics and infection prevention, the rapid emergence and spread of resistant pathogens continues to limit treatment options and increase morbidity, mortality and healthcare costs. The discovery of new innovative antimicrobial therapies remains of paramount importance. In this context, gene therapy is gaining attention as a complementary strategy that can directly target the molecular genetic determinants of antimicrobial resistance. Recent advances in RNA-based technologies and CRISPR-Cas systems have enabled increasingly precise manipulation of microbial genomes, opening up the possibility of restoring antimicrobial susceptibility, reducing virulence and limiting the spread of resistance genes.},
}
@article {pmid41977119,
year = {2026},
author = {Abubakar, S and Abdulsalam, L and Fatty, L and Kanwal, R and Naeem, M and Ahmad, I},
title = {Emerging CRISPR Approaches for Countering Immune Evasion: Insight from Recent Studies.},
journal = {International journal of molecular sciences},
volume = {27},
number = {7},
pages = {},
pmid = {41977119},
issn = {1422-0067},
mesh = {Humans ; *CRISPR-Cas Systems ; *Neoplasms/therapy/immunology/genetics ; Gene Editing/methods ; Immunotherapy/methods ; *Tumor Escape/genetics ; Animals ; *Immune Evasion/genetics ; Immunotherapy, Adoptive/methods ; },
abstract = {Cancer immunotherapy has recently become an essential approach for treating cancer, showing considerable promise as a substitute for surgery, radiation therapy, and conventional chemotherapy. It primarily aims to boost the host's natural defense system to combat cancer malignancies by utilizing components of immune checkpoint blockades (ICBs), mainly programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), along with elements of adoptive cellular therapies (ACTs) like Chimeric Antigen Receptor (CAR) therapy, T Cell Receptor (TCR) therapy and Tumor-Infiltrating Lymphocyte (TIL) therapy. However, cancer cells tend to undermine the effectiveness of cancer immunotherapeutic strategies by employing one or more immune evasion mechanisms. This review briefly highlights how key mechanisms of cancer immune evasion confer resistance to immunotherapy and how the Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR)/Cas9 systems, as gene-editing tools, are poised to enhance cancer immunotherapy for treating challenging cancers. We emphasize that (CRISPR/Cas9) systems can be used to explore and positively alter the genes of the immune system, boosting the effectiveness of cancer immunotherapy by editing immune checkpoints, TILs, and CAR-T cells, and disrupting genes, facilitating tumors' evasion of the immune system. Furthermore, we highlight the growing interest in emerging base editor technology to engineer natural killer (NK) cells to overcome NK-cell-based immunotherapy challenges, particularly human leukocyte antigens (HLA)-mediated limitations, and to engineer CAR-T cells for improved immunotherapy outcomes.},
}
@article {pmid41977403,
year = {2026},
author = {Ma, S and Li, Y and Fei, T},
title = {Functional CRISPR Screens Define Genetic Drivers for Cancer Transformation and Progression from Non-Cancerous Cells.},
journal = {International journal of molecular sciences},
volume = {27},
number = {7},
pages = {},
pmid = {41977403},
issn = {1422-0067},
support = {2023A1515140084//Guangdong Basic and Applied Basic Research Foundation/ ; 32470673//National Natural Science Foundation of China/ ; B16009//the 111 Project/ ; 2022JH13/10200026//the Construction Project of Liaoning Provincial Key Laboratory, China/ ; },
mesh = {Humans ; Animals ; Mice ; *Cell Transformation, Neoplastic/genetics ; *CRISPR-Cas Systems ; *Liver Neoplasms/genetics/pathology ; Female ; *Carcinoma, Hepatocellular/genetics/pathology ; Disease Progression ; Gene Expression Regulation, Neoplastic ; *Breast Neoplasms/genetics/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Tumor initiation and metastatic progression are driven by context-dependent genetic alterations that disrupt tumor suppressor pathways, metabolic homeostasis, and signaling networks. However, the initial drivers that transform normal cells into malignant ones and their context dependency remain elusive. To address this, we aimed to systematically identify and characterize these drivers across cancer types, species, and microenvironments. We constructed customized clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) knockout (KO) libraries targeting high-frequency mutated and downregulated genes associated with liver hepatocellular carcinoma (LIHC) and breast carcinoma (BRCA) and conducted parallel functional screens in non-cancerous mouse and human fibroblast cell lines under two-dimensional (2D), three-dimensional (3D), and in vivo conditions. Strikingly, TP53 and NF1 emerged as pan-context drivers consistently enriched across immortalization, tumorigenesis, and metastasis in both LIHC and BRCA settings, while most other identified drivers were largely species-, tissue-, and microenvironment-specific with limited cross-model overlap. Despite this heterogeneity, all drivers converge on core pathways including epigenetic regulation, metabolic reprogramming, and growth factor signaling. Unlike prior studies on established cancer cells, this work defines the genetic barriers restricting the malignant transformation of primary normal cells, offering a new framework for early cancer evolution.},
}
@article {pmid41978257,
year = {2026},
author = {Zhang, Y and Zhou, H and Xu, X and Zhang, K and Huang, G and Fan, F and Zhao, D and Li, Q and Zhu, X and Zhang, X},
title = {A versatile CRISPR-associated transposases-mediated toolkit for transcriptional regulation library.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
pmid = {41978257},
issn = {1362-4962},
support = {32300072//National Natural Science Foundation of China/ ; 32225031//National Natural Science Foundation of China/ ; 32488301//Basic Science Center Program/ ; //Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project/ ; 32300072//National Natural Science Foundation of China/ ; },
mesh = {Escherichia coli/genetics/metabolism ; Promoter Regions, Genetic ; Gene Library ; Vibrio cholerae/genetics ; *Transposases/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Bacterial ; Lycopene/metabolism ; Metabolic Engineering/methods ; Transcription, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; },
abstract = {Genome-scale and multigene transcriptional regulation are crucial technologies in metabolic engineering. However, in Escherichia coli, a stable and universal tool for whole-genome transcriptional activation, and an in situ tool for multigene regulation remain lacking. Here, we present CAGER, a versatile clustered regularly interspaced short palindromic repeats-associated transposases (CAST)-mediated gene regulation toolkit. Through rational mutagenesis, we mitigate the intrinsic transcriptional interference in the left end of CAST system derived from Vibrio cholerae. Using promoters or terminators as cargoes, CAGER constructs the genome-wide activation (3272 genes) or termination (3339 genes) libraries, from which new activation or inhibition targets relevant to cellular acetic acid assimilation are identified. Furthermore, with the aid of M13 phage and the promoter library, CAGER facilitates rapid in situ multigene expression diversification. Applied to lycopene synthesis, a library targeting seven genomic sites is constructed within 24 h, achieving a 73.6-fold yield increase. This work highlights the modifiability of CAST elements and broadens CAST's application in transcriptional regulation.},
}
@article {pmid41978267,
year = {2026},
author = {Fang, Q and Koczor, CA and Roos, WP and McClellan, S and Andrews, JF and Sobol, RW},
title = {Ubiquitylation of DNA polymerase β via atypical K27 chains signals a DNA damage response.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
pmid = {41978267},
issn = {1362-4962},
support = {//Legoretta Cancer Center Endowment Fund/ ; R01 CA238061/CA/NCI NIH HHS/United States ; P01 ES028949/ES/NIEHS NIH HHS/United States ; ES028949/NH/NIH HHS/United States ; R01 ES014811/ES/NIEHS NIH HHS/United States ; NSF-1841811//National Science Foundation/ ; ES014811/NH/NIH HHS/United States ; CA238061/NH/NIH HHS/United States ; GRANT11998991//DOD/ ; CA148629/NH/NIH HHS/United States ; R01 CA148629/CA/NCI NIH HHS/United States ; U01 ES029518/ES/NIEHS NIH HHS/United States ; ES029518/NH/NIH HHS/United States ; },
mesh = {*Ubiquitination ; *DNA Polymerase beta/metabolism/genetics/chemistry ; *DNA Damage ; Humans ; *Lysine/metabolism ; Ubiquitin/metabolism ; X-ray Repair Cross Complementing Protein 1/metabolism ; Ubiquitin-Protein Ligases/metabolism ; DNA Repair ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {DNA polymerase β (Polβ) stability is tightly regulated by specific binding partners that prevent its degradation by the proteasome. Ubiquitin (Ub) chains, differing in their structural arrangements (topology), contribute to distinct cellular functions. To investigate Polβ ubiquitylation, we utilized CRISPR/Cas9-modified cell lines expressing endogenous, mClover-tagged Polβ. Our results reveal that Polβ is primarily modified with ubiquitin chains linked through lysine residues 27 [Ub(K27)] and 29 [Ub(K29)]. We further observed that Ub(K27) and Ub(K29) chains impact the utilization of ubiquitin chains linked through lysine residues 6 [Ub(K6)] and 48 [Ub(K48)] upon Polβ ubiquitylation. The ubiquitin ligase TRIP12 preferentially attaches Ub(K27) and Ub(K29) chains to Polβ. While Polβ ubiquitylation with Ub(K27) appears to have a non-degradative role, mixed ubiquitin chains and Ub(K48) chains trigger Polβ degradation. Following DNA damage, Polβ and XRCC1 translocate from the cytosol to the nucleus, while ubiquitylated Polβ relocates from the chromatin to the cytosol. This translocation of Polβ, modified with Ub(K27) chains, promotes the dissociation of the Polβ/XRCC1 complex within chromatin, and oxidative stress enhances their association in the cytosol. These findings demonstrate that atypical ubiquitin chain modifications play crucial roles in DNA repair and the DNA damage response, underscoring the unexpected importance of these chain topologies in maintaining genome integrity.},
}
@article {pmid41981893,
year = {2026},
author = {Jia, M and Xie, Y and Wei, C and Wang, H and Xue, L and Zou, C and Zhu, JK and Wang, M},
title = {Base editing in rice using nuclease-deactivated CRISPR/Cas-SF01.},
journal = {Journal of integrative plant biology},
volume = {},
number = {},
pages = {},
doi = {10.1111/jipb.70265},
pmid = {41981893},
issn = {1744-7909},
support = {YBXM2514//Nanfan special project of CAAS/ ; 32188102//National Natural Science Foundation of China/ ; KQTD20240729102038044//Shenzhen Science and Technology Program/ ; //Agriculture Science and Technology Major Project/ ; 2024B1111130001//Guangdong S&amp;T Program/ ; },
abstract = {Adenine and cytosine base editing using dCas-SF01 and the 35S-CmYLCV-U6 composite promoter successfully introduced targeted base substitutions at multiple loci in rice, with average editing efficiency of 33.3%. Using the protospacer adjacent motif (PAM)-relaxed SF01-IKRR variant enabled base editing using 5'-NTN PAMs in rice.},
}
@article {pmid41982105,
year = {2026},
author = {Cong, X and Zhang, X and Gu, F and Tan, N and Huang, S and Jia, P and Su, J and Sun, C and Tan, Q and Fang, L and Wang, J and Yan, J and Yu, C and Li, B and Huang, J},
title = {Development of a Reverse Transcription Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Visual and Highly Specific Identification of Zika Virus.},
journal = {Journal of medical virology},
volume = {98},
number = {4},
pages = {e70917},
pmid = {41982105},
issn = {1096-9071},
support = {2026ZD01909700//Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project/ ; 2024A1515010148//Natural Science Foundation of Guangdong Province/ ; 0720240122//Guangdong Provincial Center for Disease Control and Prevention Supports Talent Projects/ ; 2023B1212010010//Guangdong Provincial Key Laboratory of Pathogen Detection for Emerging Infectious Disease Response/ ; A2024665//Medical Scientific Research Foundation of Guangdong Province, China/ ; 2024D343//Talent Support Program of Guangdong Provincial Center for Disease Control and Prevention/ ; },
mesh = {*Zika Virus/isolation &amp; purification/genetics ; Humans ; *Zika Virus Infection/diagnosis/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Reproducibility of Results ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; RNA, Viral/genetics ; Reverse Transcription ; Recombinases/genetics ; DNA Primers/genetics ; },
abstract = {Zika virus (ZIKV), a single-stranded positive-sense RNA virus of the Flaviviridae family (Flavivirus genus), causes acute febrile illness and severe congenital anomalies. Serological cross-reactivity with Dengue virus (DENV) and Chikungunya virus (CHIKV) complicates diagnosis, underscoring the urgency of developing specific point-of-care tests for early detection, outbreak mitigation, and reduced misdiagnosis risks. This study established and optimized an RPA-CRISPR/Cas12a assay for the rapid, visual, and highly specific detection of ZIKV. Primers and crRNAs targeting the highly conserved capsid (C) gene were designed, and the assay was systematically evaluated for its specificity, sensitivity, reproducibility, and clinical applicability. The RPA-CRISPR/Cas12a assay enabled naked-eye detection under UV light within 35 min. It demonstrated single-copy sensitivity (1 copy/μL), no cross-reactivity with DENV 1-4, CHIKV, or Japanese encephalitis virus (JEV), and 100% concordance with RT-qPCR in clinical validation. Repeatability tests showed low variability (coefficient of variation (C.V.) < 15%), confirming robust reproducibility. This instrument-free platform integrates rapid visual detection, single-copy sensitivity, high specificity, and field-deployable features, making it particularly suitable for point-of-care testing (POCT) in resource-limited settings. The developed assay provides critical support for early outbreak containment and prenatal screening in ZIKV-endemic regions.},
}
@article {pmid41982408,
year = {2025},
author = {Ni, D},
title = {The Kongming defense: Host-pathogen battles take a new face.},
journal = {Engineering microbiology},
volume = {5},
number = {2},
pages = {100209},
pmid = {41982408},
issn = {2667-3703},
abstract = {Bacteria employ diverse immune systems, such as CRISPR-Cas, to fend off phage infections. A recent study uncovered the unprecedented mechanistic features of the Kongming bacterial defense system, which uniquely exploits phage-derived enzymes to synthesize deoxyinosine triphosphate (dITP), thereby triggering host immunity through NAD+ depletion. In response, some phages have evolved countermeasures to disrupt dITP synthesis, highlighting the ongoing evolutionary arms race between hosts and pathogens. This discovery not only deepens our understanding of bacterial defense strategies but also paves the way for new insights in biomedical research and synthetic biology.},
}
@article {pmid41984502,
year = {2026},
author = {Rogers, LM and Firestone, K and Chinni, R and Branco, AC and Wrobleski, K and Chou, T and Goldstein, JA and Gu, X and Mason, E and Chu, S and der Lohe, MR and Low, P and Aronoff, DM},
title = {Folate receptor beta drives NLRP3 inflammasome activation and pyroptosis in macrophages independent of folate binding.},
journal = {Journal of immunology (Baltimore, Md. : 1950)},
volume = {215},
number = {4},
pages = {},
doi = {10.1093/jimmun/vkag051},
pmid = {41984502},
issn = {1550-6606},
mesh = {Humans ; *NLR Family, Pyrin Domain-Containing 3 Protein/metabolism/immunology ; *Inflammasomes/metabolism/immunology ; *Macrophages/immunology/metabolism ; *Folic Acid/metabolism ; *Pyroptosis/immunology ; *Folate Receptor 2/metabolism/genetics/immunology ; THP-1 Cells ; Interleukin-1beta/metabolism ; DNA Methylation ; Signal Transduction ; CRISPR-Cas Systems ; Caspase 1/metabolism ; },
abstract = {Folate receptor beta (FRβ), encoded by FOLR2, is selectively expressed in monocytes and macrophages, yet its function in innate immune signaling remains poorly defined. Here, we identify FRβ as a novel regulator of NLRP3 inflammasome activation and pyroptosis in human THP-1 macrophages. Using CRISPR/Cas9-mediated gene deletion, we show that loss of FOLR2 severely impaired caspase-1 activation, gasdermin D cleavage, and IL-1β release in response to multiple NLRP3 stimuli, without altering pro-IL-1β induction. These defects were not rescued by exogenous folate and were independent of extracellular folate concentrations. Mechanistically, FOLR2/FRβ appears to potentiate potassium efflux and the expression of multiple potassium channel-encoding genes. Single-cell RNA sequencing revealed broad transcriptional repression in FRβ-deficient macrophages, including genes involved in inflammasome signaling and ion transport. Genome-wide methylation profiling showed increased CpG hypermethylation in FOLR2-deficient cells, consistent with reduced transcriptional activity. Our findings indicate that FRβ promotes NLRP3 activation in a folate-independent manner potentially by regulating DNA methylation, gene transcription, and K+ efflux in macrophages. These findings uncover a previously unrecognized immunoregulatory function for FRβ, positioning it as a potential modulator of macrophage-driven inflammation in contexts such as host defense, autoimmunity, and tissue-specific immune responses at the tumor and maternal-fetal interface.},
}
@article {pmid41985328,
year = {2026},
author = {Jigheh, MP and Ravanlo, ZZ and Shahrak, MZ and Mohabbat, A and Baghi, HB},
title = {Targeting human oncogenic viruses with CRISPR/Cas: New therapeutic opportunities and challenges.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {198},
number = {},
pages = {119284},
doi = {10.1016/j.biopha.2026.119284},
pmid = {41985328},
issn = {1950-6007},
abstract = {CRISPR/Cas systems, initially characterized as bacterial adaptive immune mechanisms, have rapidly emerged as precise and versatile genome-editing tools with significant potential for antiviral research and therapeutic development. This review highlights the role of CRISPR/Cas systems in targeting persistent and human oncogenic viruses, including HPV, HBV, HCV, EBV, KSHV, HTLV-1, and MCPyV, as well as HIV, which may indirectly contribute to cancer through immune dysregulation. Many of these viruses can integrate into the host genome or persist as chronic or latent infections, contributing to cancers for which curative options are limited. CRISPR-based strategies enable the excision of integrated proviral DNA, disruption of viral replication, targeted silencing of viral transcripts, and modulation of host tumor-suppressor pathways. Cas9 efficiently targets DNA viruses, such as HBV and HPV, whereas RNA-targeting Cas13 allows precise silencing of RNA viruses, like HCV. Editing T-cell receptors, including CCR5 and CXCR4, offers the potential for long-term resistance to HIV. CRISPR-based preclinical studies indicate the potential to disrupt HBV cccDNA, suppress EBV and KSHV latency gene expression, and inactivate HTLV-1 oncogenes, thereby potentially reducing viral persistence and oncogenic progression. Despite these advances, challenges remain regarding off-target effects, delivery efficiency, immune responses, and ethical considerations. Innovations such as high-fidelity Cas variants, base and prime editing, and non-viral delivery systems are expected to enhance both safety and therapeutic precision. This review provides an overview of viral life cycles, oncogenic pathways, and therapeutic vulnerabilities of human oncogenic viruses and CRISPR-based genome-editing approaches under investigation for viral elimination and cancer therapy.},
}
@article {pmid41986860,
year = {2026},
author = {Armenta-Medina, A and Mora-Macias, J and Massange-Sánchez, JA and López-Valenzuela, BE and Rangel-Chavez, CP},
title = {Perspectives of Gene Editing for the Conservation of Plant Genetic Resources.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {3011},
number = {},
pages = {365-381},
pmid = {41986860},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; *Conservation of Natural Resources/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Plants/genetics ; Crops, Agricultural/genetics ; Plant Diseases/genetics ; },
abstract = {The rapid development of genome editing tools, particularly CRISPR-Cas9 and virus-induced genome editing (VIGE), offers transformative opportunities for the conservation of plant genetic resources. These technologies enable precise modifications that can enhance genetic variation, restore endangered populations, and strengthen resilience against climate change and invasive species. This chapter reviews methodological frameworks, including guide design, delivery systems, regeneration protocols, and verification of off-target effects, as well as ecological and ethical considerations. Case studies are highlighted in both wild and cultivated species: American chestnut, ash, elm, Pacific yew, hemlock, and whitebark pine, alongside crops of global significance such as coffee, cacao, wild apple, and banana. Applications include resistance to blight, borers, Dutch elm disease, woolly adelgid, rust, rots, and endogenous viral elements. Finally, the chapter outlines future directions emphasizing biosafety, ecological validation, and equitable governance, underscoring the potential of CRISPR-based strategies to integrate conservation science with sustainable agricultural practices.},
}
@article {pmid41987615,
year = {2026},
author = {Eliwa, AI and Eldahshan, MM},
title = {CRISPR-Cas at a crossroads: from microbial immunity to precision biotechnology.},
journal = {Journal of immunoassay &amp; immunochemistry},
volume = {},
number = {},
pages = {1-24},
doi = {10.1080/15321819.2026.2658465},
pmid = {41987615},
issn = {1532-4230},
abstract = {Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) form RNA-guided adaptive immune systems in bacteria and archaea that mediate sequence-specific defense against invading genetic elements. Beyond their ecological role in restricting bacteriophage infection and horizontal gene transfer (HGT), CRISPR-Cas systems have been repurposed as programmable nucleases, enabling rapid, scalable, and precise genome engineering. Over the past decade, CRISPR platforms, most prominently Cas9, have transformed functional genomics, accelerated target discovery and drug development, and progressed from experimental tools to clinically evaluated gene and cell therapies. In parallel, growing attention has focused on both native and engineered roles of CRISPR-Cas in shaping HGT, plasmid ecology, and antimicrobial resistance (AMR), as AMR continues to expand globally. In this Review, we integrate advances spanning eukaryotic genome editing and prokaryotic antimicrobial applications. We summarize CRISPR-Cas classification and molecular mechanisms, highlighting spacer acquisition, guide RNA biogenesis, target recognition, and nucleic acid cleavage. We then examine how cellular DNA repair pathways influence editing outcomes and discuss strategies to enhance precision. We further review delivery strategies, such as conjugative plasmids, bacteriophages and phagemids, extracellular vesicles, and nanoparticles, together with evolutionary countermeasures encoded by mobile genetic elements, including anti-CRISPR proteins. Finally, we outline current limitations.},
}
@article {pmid41988391,
year = {2026},
author = {Tang, W and Yang, N and Shi, M},
title = {Applications and Challenges of CRISPR-Cas Technology for the Detection of Antimicrobial Resistance Genes.},
journal = {Infection and drug resistance},
volume = {19},
number = {},
pages = {578705},
pmid = {41988391},
issn = {1178-6973},
abstract = {The global rise of antimicrobial resistance (AMR) demands rapid and precise diagnostic tools capable of providing actionable results at the point of care. Traditional methods, including bacterial culture and PCR, face critical limitations: culture-based approaches require days to weeks for results, potentially delaying life-saving treatment decisions, while PCR-based methods, though faster, require expensive instrumentation, trained personnel, and are typically confined to centralized laboratory settings. CRISPR-Cas systems offer a promising approach for detecting bacterial drug resistance genes with high specificity, speed, and sensitivity. By utilizing Cas enzymes (eg, Cas12 and Cas13) and guide RNAs, CRISPR enables targeted recognition of resistance markers, typically requiring upstream nucleic acid amplification (eg, recombinase polymerase amplification or loop-mediated isothermal amplification) to achieve clinically relevant sensitivity. Platforms such as SHERLOCK and DETECTR facilitate rapid identification of markers associated with pathogens like MRSA and CRE directly from clinical samples, supporting point-of-care use. Integration with microfluidics and AI further expands its potential for personalized therapy and AMR surveillance. Despite ethical and regulatory challenges, CRISPR-Cas technology holds significant promise for advancing antimicrobial stewardship and public health responses to AMR.},
}
@article {pmid41989832,
year = {2026},
author = {Ceasar, SA and Pandey, H and Misra, V and Sharma, A and Kumar, R and Mall, AK},
title = {Biotechnological Improvement of Fiber Crops: Role of In Vitro Culture, Genetic Transformation, and Genome Editing.},
journal = {Journal of agricultural and food chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.jafc.5c14248},
pmid = {41989832},
issn = {1520-5118},
abstract = {Fiber crops face major challenges from climate instability, pests, and suboptimal fiber or oil quality. These challenges can be addressed using plant tissue culture and molecular breeding tools, including genetic transformation and CRISPR/Cas-mediated genome editing. Advances in in vitro regeneration have enabled efficient plant recovery in crops such as cotton, jute, mesta, flax, sunn hemp, and industrial hemp. Techniques including anther culture have facilitated the development of doubled haploid lines with improved fiber quality and uniformity. Genetic transformation and emerging genome-editing applications provide new opportunities for targeted trait improvement in fiber crops. This review covers in detail the application of in vitro regeneration, genetic transformation, and genome editing studies in fiber crops. This review also includes several insights for improving fiber crops by applying these tools. This review will be a rich resource for the details of in vitro regeneration, genetic transformation, and gene editing studies in fiber crops.},
}
@article {pmid41990079,
year = {2026},
author = {Schenstnyi, K and Zhang, Z and Liu, B and Nakamura, M and Schepler-Luu, V and Loo, EPI and Yang, B and Frommer, WB},
title = {Methylviologen resistance in loss-of-function mutants of the polyamine transporter gene OsLAT5.},
journal = {PloS one},
volume = {21},
number = {4},
pages = {e0346828},
pmid = {41990079},
issn = {1932-6203},
mesh = {*Oryza/genetics/drug effects ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Loss of Function Mutation ; *Membrane Transport Proteins/genetics ; Gene Editing ; Plants, Genetically Modified ; *Herbicide Resistance/genetics ; Herbicides/pharmacology ; },
abstract = {TALENs and CRISPR/Cas have become routine tools for genome editing. During stable plant transformation, genes coding for editing enzymes, e.g., Cas9, guide RNAs (gRNA), and selectable or screenable markers are integrated into the nuclear genome. Identification of successful transformants relies on selectable or screenable markers, typically genes providing resistance to herbicides or antibiotics. Selectable markers use a substantial portion of the T-DNA, hence reducing transfer efficiency by limiting the effective number of TALENs or guide/pegRNAs that can be used. Marker genes are frequently subject to gene silencing. Here, we generated loss-of-function mutations in PUT/LAT-type polyamine transporter family genes to confer resistance to methylviologen (MV) in rice. As proof of concept, CRISPR/Cas9 constructs with gRNAs were generated to target three close homologs, namely OsLAT1, OsLAT5, and OsLAT7. Loss of OsLAT5 (also known as OsPUT3 or OsPAR1) function was sufficient to confer resistance to MV in rice seeds, seedlings and calli. Loss-of-function alleles generated by editing of LAT5 can serve as a selectable marker at the seed germination stage. We discuss the potential utility of rice lat5 loss of function variants as selectable markers for genome editing.},
}
@article {pmid41990179,
year = {2026},
author = {Hartweger, H and Ruprecht, C and Yao, KH and Laffont, P and Lima Dos Reis, G and Zhou, P and Hägglöf, T and Binet, L and Loewe, M and Hong, JP and Xiao, T and Sefik, E and Hernandez, B and Gazumyan, A and Jankovic, M and Seaman, MS and Costa, G and Nelson, SA and Clark, J and Kanatani, S and Wilson, PC and Krammer, F and Levashina, EA and Julien, JP and Wardemann, H and Sinnis, P and Stamatatos, L and Flavell, RA and Nussenzweig, MC},
title = {B lymphocyte protein factories produced by hematopoietic stem cell gene editing.},
journal = {Science (New York, N.Y.)},
volume = {392},
number = {6795},
pages = {eadz8994},
doi = {10.1126/science.adz8994},
pmid = {41990179},
issn = {1095-9203},
mesh = {Animals ; Mice ; *B-Lymphocytes/immunology ; Broadly Neutralizing Antibodies/immunology ; CRISPR-Cas Systems ; *Gene Editing/methods ; Hematopoietic Stem Cell Transplantation ; *Hematopoietic Stem Cells/immunology ; HIV Antibodies/blood/immunology/biosynthesis ; HIV-1/immunology ; Malaria/prevention &amp; control/immunology/therapy ; Mice, Inbred C57BL ; Orthomyxoviridae Infections/prevention &amp; control/immunology ; Plasma Cells/immunology ; Precursor Cells, B-Lymphoid/immunology ; Antibodies, Protozoan/biosynthesis/blood/immunology ; },
abstract = {Long-term in vivo production of therapeutic proteins and development of vaccines that elicit protective levels of broadly neutralizing antibodies (bNAbs) against major pathogens face challenges. In this study, we report on an alternative gene editing approach using small numbers of hematopoietic stem and progenitor cells (HSPCs) to direct long-term, high-level expression of antibodies or cargo proteins. In mice, edited B lymphocytes derived from transplanted HSPCs were activated by cognate antigen, underwent clonal expansion, and developed into specific antibody-synthesizing or cargo protein-synthesizing plasma cells. These cells produced long-lasting, therapeutic levels of serum antibody against HIV-1, malaria, or an anti-influenza virus bNAb that mediated universal protection from heterologous lethal challenge. Our data provide a paradigm for cell therapy approaches to prevent or treat disease using self-amplifying B cell protein factories.},
}
@article {pmid41991098,
year = {2026},
author = {Lu, P and Guo, J and Jia, R},
title = {Alternative Splicing of Immune Checkpoints: Classifications, Mechanisms, and Therapeutic Implications for Overcoming Immune Checkpoint Blockade Resistance.},
journal = {Critical reviews in oncology/hematology},
volume = {},
number = {},
pages = {105339},
doi = {10.1016/j.critrevonc.2026.105339},
pmid = {41991098},
issn = {1879-0461},
abstract = {Immune checkpoint blockade (ICB) therapy has revolutionized oncology, yet its clinical efficacy remains limited due to primary and acquired resistance. Alternative splicing (AS), a fundamental post-transcriptional regulatory mechanism in eukaryotic gene expression, has been shown to profoundly remodel immune checkpoint molecules, driving immune evasion and ICB resistance. In this review, we systematically categorize immune checkpoint splicing based on splicing events, evolutionary conservation, altered domains, and functional impacts. We propose a new feature of immune checkpoint splicing, transmembrane exon splicing strategy. Mechanistically, we explain how dysregulated spliceosomes, cis-acting elements, and trans-acting factors within the tumor microenvironment orchestrate these splicing events, impacting ligand/receptor interactions and downstream immune signaling. Therapeutically, soluble isoforms serve as diagnostic/prognostic biomarkers. Engineered oncolytic viruses expressing soluble decoys offer novel combination strategies. Emerging therapeutic approaches, including antisense oligonucleotides, splicing modulators, RNA interference, and CRISPR/Cas systems, show promise for directly targeting aberrant splicing to overcome ICB resistance.},
}
@article {pmid41991300,
year = {2026},
author = {Ji, T and Wang, T and Yu, K and Gao, YZ and Gao, XZ},
title = {[Research progress in the application of RPA-CRISPR/Cas13a technology in the detection of pathogenic microorganisms].},
journal = {Zhonghua yu fang yi xue za zhi [Chinese journal of preventive medicine]},
volume = {60},
number = {4},
pages = {622-630},
doi = {10.3760/cma.j.cn112150-20250430-00378},
pmid = {41991300},
issn = {0253-9624},
support = {M2024086//2024 Research Project of Jiangsu Provincial Health Commission/ ; },
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Bacteria/isolation &amp; purification/genetics ; },
abstract = {Recombinase polymerase amplification (RPA) is an emerging method for nucleic acid amplification. It can be performed under low-temperature conditions, making it suitable for point-of-care testing. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 13 (CRISPR/Cas13a), a type Ⅵ CRISPR-Cas system, employs a single-subunit RNA-guided Cas endonuclease to target exogenous RNA and exhibits distinct trans-cleavage activity. The integration of CRISPR/Cas13a with RPA technology significantly enhances detection sensitivity and accuracy, demonstrating considerable potential for molecular diagnostics. The RPA-CRISPR/Cas13a platform has achieved notable success in areas such as food safety, environmental monitoring, and clinical diagnosis, and has become an important tool in modern biomedical research. This article provides an in-depth analysis of the fundamental principles of RPA-CRISPR/Cas13a technology and comprehensively reviews its applications in pathogenic microorganism detection, aiming to offer new insights for accurate pathogen diagnosis.},
}
@article {pmid41991526,
year = {2026},
author = {Omura, SN and Nakagawa, R and Kajimoto, S and Okazaki, S and Ishiguro, S and Mori, H and Onishi, K and Kashiwakura, Y and Hiramoto, T and Horinaka, K and Tanaka, M and Hirano, H and Jividen, K and Yamashita, K and Tsai, SQ and Yachie, N and Ohmori, T and Nishimasu, H and Nureki, O},
title = {Engineering a compact high-fidelity Staphylococcus aureus Cas9 variant with broader targeting range and mechanistic insights into its activation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {},
pmid = {41991526},
issn = {2041-1723},
support = {JP25ama121012//Japan Agency for Medical Research and Development (AMED)/ ; },
mesh = {*Staphylococcus aureus/genetics/enzymology ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; HEK293 Cells ; Cryoelectron Microscopy ; *Bacterial Proteins/genetics/metabolism ; Protein Engineering/methods ; },
abstract = {Staphylococcus aureus Cas9 (SaCas9) is smaller than the widely used Streptococcus pyogenes Cas9 (SpCas9) and has been harnessed for gene therapy using an adeno-associated virus vector. However, SaCas9 requires a longer NNGRRT (where N is any nucleotide and R is A or G) protospacer adjacent motif (PAM) for target DNA recognition, thereby restricting the targeting range. Although PAM-relaxed Cas9 variants have been developed, expanded targeting is often accompanied by compromised target specificity. Here, we report the rational engineering of eSaCas9-NNG, a SaCas9 variant that recognizes relaxed NNG PAMs while maintaining high target fidelity, thereby overcoming a fundamental trade-off in Cas9-based genome editing. eSaCas9-NNG efficiently induces indels and base conversions at endogenous sites bearing NNG PAMs in human cells and mice, with editing efficiencies comparable to those of other PAM-relaxed nucleases, including SpRY, SpG, and iGeoCas9, but with reduced off-target activity. We further determine the cryo-electron microscopy structures of eSaCas9-NNG in five distinct functional states, revealing the structural basis for its relaxed PAM recognition, improved target specificity, and nuclease activation. Overall, our findings demonstrate that eSaCas9-NNG could be used as a versatile genome editing tool for in vivo gene therapy, and improve our mechanistic understanding of the diverse CRISPR-Cas9 nucleases.},
}
@article {pmid41992430,
year = {2026},
author = {Pinos, D and García-Marín, E and Ramírez-Serrano, B and Benavent-Albarracín, L and Gamir, J and Crava, CM},
title = {Maintenance of Gut Microbial Balance via the Kynurenine Pathway Improves Larval Performance and Resistance to Bacillus thuringiensis in Spodoptera exigua.},
journal = {MicrobiologyOpen},
volume = {15},
number = {2},
pages = {e70289},
pmid = {41992430},
issn = {2045-8827},
support = {PID2020-118787RA-I00//Agencia Estatal de Investigaci&#xf3;n/ ; MRR/RYC2021-033098-I//Agencia Estatal de Investigaci&#xf3;n/ ; PID2024-162058OB-C32//Agencia Estatal de Investigaci&#xf3;n/ ; RGY0052/2022//Human Frontier Science Program/ ; },
mesh = {Animals ; *Gastrointestinal Microbiome ; Larva/microbiology/growth &amp; development ; *Spodoptera/microbiology/growth &amp; development ; *Kynurenine/metabolism ; *Bacillus thuringiensis/physiology ; Kynurenine 3-Monooxygenase/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {The gut microbiota is a key determinant of insect physiology, influencing nutrition, immunity, and interactions with plants and pathogens. In Lepidoptera, larval gut communities are dynamic, but a core microbiota, often dominated by Enterococcus species, persists across instars. In Spodoptera littoralis, the enzyme kynurenine 3-monooxygenase (KMO) regulates gut bacterial composition via 8-hydroxyquinoline-2-carboxylic acid (8-HQA), a secreted iron-chelating compound. To investigate whether this mechanism is conserved in Noctuidae, we generated Spodoptera exigua kmo[-/-] mutants using CRISPR/Cas9 and analyzed bacterial communities in foregut, midgut, hindgut, and oral secretions by 16S metabarcoding, using RNA-derived cDNA for gut samples and DNA for oral secretions due to lower microbial biomass. The kmo deletion abolished 8-HQA production, reduced bacterial diversity, and collapsed compartment-specific bacterial communities in the gut, while also being associated with Enterococcus dominance in oral secretions. Fitness assays revealed that kmo[-/-] larvae exhibited reduced weight gain on artificial diet, and higher mortality and delayed growth when fed on pepper leaves. Moreover, kmo[-/-] larvae were threefold more susceptible to Bacillus thuringiensis, consistent with an interaction between host physiological state, gut microbial homeostasis, and pathogen susceptibility. Dietary supplementation with 8-HQA partially mitigated, but did not fully rescue, growth deficits. Our results demonstrate that the kynurenine pathway and 8-HQA production are crucial for maintaining gut microbial homeostasis, particularly within Enterococcus, thereby supporting larval development, dietary adaptation, and pathogen resilience. These findings reveal a conserved mechanism in noctuid moths linking host metabolism, microbiota regulation, and ecological performance, emphasizing the interplay between host genetics, microbiota composition, and environmental stressors.},
}
@article {pmid41992887,
year = {2026},
author = {Ouyang, PD and Tang, JL and Wu, CC and Li, XY and Ke, SS and Zhang, JJ},
title = {The mechanism of synpo in intracerebral hemorrhage using a zebrafish model.},
journal = {Yi chuan = Hereditas},
volume = {48},
number = {4},
pages = {407-420},
doi = {10.16288/j.yczz.25-281},
pmid = {41992887},
issn = {0253-9772},
mesh = {Animals ; *Zebrafish/genetics ; *Cerebral Hemorrhage/genetics/metabolism ; Disease Models, Animal ; *Microfilament Proteins/genetics/metabolism ; Genome-Wide Association Study ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Genetic Predisposition to Disease ; },
abstract = {Intracerebral Hemorrhage (ICH) is a stroke subtype with high mortality, and its core pathological mechanism involves the disruption of cerebrovascular homeostasis. Genetic factors play a crucial role in ICH pathogenesis, underscoring the importance of identifying core regulatory factors and delineating the associated pathological network. Here, through genome-wide association study (GWAS), we identified synaptopodin (SYNPO) as a genetic susceptibility gene for ICH. SYNPO is an evolutionarily conserved actin-binding protein previously shown to be highly expressed in cerebrovascular endothelial cells, where it regulates the actin cytoskeleton to maintain endothelial junction stability. However, its functional role in ICH remains unclear. To investigate this, we conducted a synpo mutant zebrafish line using CRISPR/Cas9. Following epinephrine challenge, synpo mutant larvae displayed significantly elevated cerebrovascular leakage compared with wild-type controls, and adult mutants showed a markedly higher incidence of ICH. Transcriptomic profiling revealed significant downregulation of the key adhesion gene cdh2 in mutant brains. Subsequent rescue experiments confirmed that cdh2 mRNA supplementation effectively ameliorated the cerebrovascular leakage. In summary, our study unveils a pathway in which synpo maintains cerebrovascular homeostasis by positively regulating cdh2, demonstrating that the synpo-cdh2 axis serves as a key regulatory pathway in ICH. These findings provide insights into the genetic mechanisms underlying ICH and highlight potential therapeutic targets.},
}
@article {pmid41992908,
year = {2026},
author = {Cleanclay, WD and Adedoyin, ED and Zakari, S and Ogunlana, OO and Iweala, EEJ and Chinedu, SN},
title = {Advancing Malaria Vector Control: Insights Into Mosquito Immunity and Genetic Strategies.},
journal = {TheScientificWorldJournal},
volume = {2026},
number = {1},
pages = {e7634044},
pmid = {41992908},
issn = {1537-744X},
mesh = {Animals ; *Malaria/prevention &amp; control/transmission/immunology/parasitology ; *Mosquito Vectors/immunology/genetics/parasitology ; *Mosquito Control/methods ; *Anopheles/immunology/genetics/parasitology ; CRISPR-Cas Systems ; Humans ; RNA Interference ; Wolbachia/genetics ; Plasmodium ; },
abstract = {Malaria remains a major global health challenge, particularly in sub-Saharan Africa where Anopheles mosquitoes transmit the Plasmodium parasites. Resistance to insecticides remains an obstacle in spite of the efforts to control malaria vector. The interaction between Plasmodium parasites and mosquito vectors, with a focus on the immunity of mosquitoes and approaches to combat malaria, is examined in this review. This review explores the potential of genetic approaches including CRISPR-Cas9, Wolbachia, RNA interference (RNAi), and symbiont-based strategies for the control of malaria vector. The innate immune system of Anopheles mosquitoes that identify, recognize, and limit Plasmodium infection through pathogen recognition receptors, signaling pathways, and effector mechanisms like antimicrobial peptides and melanization is well developed. However, Plasmodium has developed several evasion mechanisms to establish infection. This led to various genetic modification techniques being designed to reduce vector population and transmission. Gene drive such as CRISPR-Cas9 can introduce genetic alterations to interfere with the transmission of malaria; Wolbachia interferes with vector competence, RNAi-mediated gene to target relevant genes involved in reproduction and survival. Self-limiting strategies such as RIDL and pgSIT genetically modified insect releasement to the environment. mosGILT is an emerging immune regulator which has shown relevance in blocking transmission. This review explores the potential of these genetic approaches in malaria vector control efforts, highlighting their advantages and imitations. Further research should explore mosquito immune genes and pathways in developing innovative and acceptable genetic vector control approaches.},
}
@article {pmid41993032,
year = {2026},
author = {Zhao, J and Sui, Z and Zhou, Y and Peng, Y and Xu, J},
title = {Rigidity-Responsive Fluorescence Polarization Detection of Aflatoxin B1 via Programmable RCA-Coupled CRISPR/Cas12a and a Conformation-Restricted Depolarization Reporter.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.6c00481},
pmid = {41993032},
issn = {1520-6882},
abstract = {Aflatoxin B1 (AFB1) is one of the most toxic and carcinogenic mycotoxins, and its trace-level determination in complex food matrices remains a major analytical challenge. Conventional chromatographic methods, while highly accurate, rely on expensive instrumentation and labor-intensive sample pretreatment, whereas most CRISPR/Cas-based biosensors depend on fluorescence intensity turn-on readouts that are vulnerable to matrix autofluorescence, photobleaching, and signal instability, limiting their reliability in real samples. Herein, we propose a rigidity-responsive fluorescence polarization (FP) biosensing strategy that integrates aptamer-based molecular recognition, rolling circle amplification (RCA), and CRISPR/Cas12a trans-cleavage for robust and matrix-tolerant AFB1 detection. In this system, target binding induces the release of a complementary DNA strand from an immobilized aptamer duplex, initiating padlock probe circularization and RCA to generate abundant Cas12a-activating amplicons. A rationally engineered conformation-restricted depolarization reporter (CRD-Reporter), in which the fluorophore is confined within a rigid duplex framework, provides an intrinsically high FP signal. Upon Cas12a activation, collateral cleavage disrupts the rigid architecture, releasing freely rotating fragments and producing a pronounced FP decrease. Unlike intensity-based CRISPR assays, the FP readout effectively suppresses background interference and signal fluctuations. The proposed assay exhibits a wide linear range from 0.003 to 300 ng/mL with a low detection limit of 0.00113 ng/mL, high specificity, and excellent accuracy in grains, peanuts, and tea samples. This work establishes a robust FP-based CRISPR sensing paradigm for reliable mycotoxin monitoring in complex food systems.},
}
@article {pmid41993526,
year = {2026},
author = {Knight, AL and Belato, HB and Dresser, CS and Pindi, C and Mercado, BJ and Lasekan, P and Luo, J and Arantes, PR and Jogl, G and Palermo, G and Lisi, GP},
title = {Orthosteric and allosteric effects of anti-CRISPR II-C1 inhibition on Geo Cas9 from integrated structural biophysics.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.64898/2026.04.08.717222},
pmid = {41993526},
issn = {2692-8205},
abstract = {Anti-CRISPRs (Acrs) are small protein inhibitors of CRISPR-Cas effectors that originate from the translated genetic material of bacteriophage. Harnessing the natural ability of Acrs to bind and disrupt CRISPR-Cas editing can provide enhanced spatiotemporal control of gene editing. Recent studies have revealed diverse structures and functions of Acrs, however, atomistic studies of the specific molecular mechanisms behind Acr inhibition are lacking. Here, we reveal how structure, function, and dynamics govern AcrIIC1 inhibition of Cas9 from G. stearothermophilus (Geo Cas9) via its HNH nuclease domain. An X-ray crystal structure of the Geo HNH-AcrIIC1complex reveals a conserved binding interface at the catalytic site and disruption of crucial electrostatic contacts known to modulate the thermostability of Geo Cas9. AcrIIC1 binding also rewires the intrinsic dynamics of the Geo HNH domain, stimulates millisecond motions that are absent from the unliganded nuclease, and attenuates the guide RNA affinity of Geo Cas9. Subsequent AcrIIC1 mutations in residues at its crystallographic binding interface uncouple Acr binding from inhibition, providing new insight into mechanism by which AcrIIC1 acts on Geo Cas9.},
}
@article {pmid41995110,
year = {2026},
author = {Dong, J and Liu, J and Li, Y and Sohail, H and Ji, Z and Chen, X and Xu, J},
title = {CsPNRC1 regulated the resistance of Fusarium wilt by interacting with Csfla15 to influence the cell wall structure of cucumber (Cucumis sativus L.).},
journal = {The Plant journal : for cell and molecular biology},
volume = {126},
number = {1},
pages = {e70871},
doi = {10.1111/tpj.70871},
pmid = {41995110},
issn = {1365-313X},
support = {32372690//National Natural Science Foundation of China/ ; JBGS [2021]018//"JBGS" Project of Seed Industry Revitalization in Jiangsu Province/ ; },
mesh = {*Cucumis sativus/microbiology/genetics/metabolism ; *Fusarium/physiology ; *Plant Proteins/metabolism/genetics ; *Cell Wall/metabolism ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/immunology/genetics ; Gene Expression Regulation, Plant ; *Mucoproteins/metabolism/genetics ; },
abstract = {Cucumber (Cucumis sativus L.) Fusarium wilt (FW), caused by Fusarium oxysporum f. sp. cucumerinum (Foc), is a devastating disease that significantly impacts cucumber production worldwide. The lack of natural resistant cultivars to FW has hindered the development of resistant cucumber varieties through conventional breeding methods. Interestingly, the disruption of susceptibility (S) genes has emerged as an effective alternative approach for enhancing crop resistance. Here, we identified a proline-rich nuclear receptor coactivator CsPNRC1 through transcriptomic analysis, and function as a susceptibility gene of cucumber FW using virus-induced gene silencing (VIGS), CRISPR/Cas- and overexpression-based methods. Seedlings overexpressing CsPNRC1 exhibited browning at the shoot bases 7 days after inoculation with Foc pathogens, while knockout seedlings demonstrated significantly greater resistance compared with wild-type (WT) plants. We identified CsPNRC1 interacting with a fasciclin-like arabinogalactan protein Csfla15 through yeast two-hybrid (Y2H) assays, bimolecular fluorescence complementation (BiFC), Co-immunoprecipitation (Co-IP), and luciferase complementation imaging (LCI) assay, and found that Csfla15 was regulated by CsPNRC1. Also, CsPNRC1 could influence the content of cell wall components pectin and cellulose in cucumber roots. Meanwhile, these findings provide valuable insights into the role of CsPNRC1 in cucumber's susceptibility to FW and highlight its potential use for resistance breeding.},
}
@article {pmid41995867,
year = {2026},
author = {Ventre, E and Forrow, A and Gadhiwala, N and Chakraborty, P and Angel, O and Schiebinger, G},
title = {Trajectory inference for a branching SDE model of cell differentiation via lineage tracing.},
journal = {Journal of mathematical biology},
volume = {92},
number = {5},
pages = {},
pmid = {41995867},
issn = {1432-1416},
mesh = {*Cell Differentiation/genetics ; *Cell Lineage/genetics ; *Models, Biological ; Cell Proliferation/genetics ; Computer Simulation ; Mathematical Concepts ; Humans ; Animals ; Computational Biology ; CRISPR-Cas Systems ; },
abstract = {A core challenge for modern biology is how to infer the trajectories of individual cells from population-level time courses of high-dimensional gene expression data. Birth and death of cells present a particular difficulty: existing trajectory inference methods cannot distinguish variability in net proliferation from cell differentiation dynamics, and hence require accurate prior knowledge of the proliferation rate. Building on Global Waddington-OT (gWOT), which performs trajectory inference with rigorous theoretical guarantees when birth and death can be neglected, we show how to use lineage trees available with recently developed CRISPR-based measurement technologies to disentangle proliferation and differentiation. In particular, when there is neither death nor subsampling of cells, we show that we extend gWOT to the case with proliferation with similar theoretical guarantees and computational cost, without requiring any prior information. In the case of death and/or subsampling, our method introduces a bias, that we describe explicitly and argue to be inherent to these lineage tracing data. We demonstrate in both cases the ability of this method to reliably reconstruct the landscape of a branching SDE from time-courses of simulated datasets with lineage tracing, outperforming even a benchmark using the experimentally unavailable true branching rates.},
}
@article {pmid41996895,
year = {2026},
author = {Pindi, C and Palermo, G},
title = {Deep learning and cryogenic electron microscopy modeling for gene editing dynamics.},
journal = {Current opinion in structural biology},
volume = {98},
number = {},
pages = {103270},
doi = {10.1016/j.sbi.2026.103270},
pmid = {41996895},
issn = {1879-033X},
abstract = {Advances in cryogenic electron microscopy (cryo-EM) data modeling and deep learning are reshaping our ability to interrogate and engineer genome-editing systems. Their synergistic integration enables high-resolution structural interpretation, quantitative mapping of conformational landscapes, and rational design across diverse CRISPR-Cas architectures. By coupling molecular dynamics with cryo-EM refinement, we uncover functionally relevant dynamic ensembles, while quantum mechanical methods resolve ambiguous features in low-resolution density maps. Emerging deep-learning frameworks including graph neural networks, extract interpretable communication pathways from large-scale simulations and provide methods that are broadly transferable across biomolecular systems. These advances propel the field beyond static structural snapshots toward a dynamic, predictive, and data-driven approach for understanding and designing genome-editing systems.},
}
@article {pmid41997664,
year = {2026},
author = {Karan, R and Prasannakumar, MK and Kour Khera, H and Harish, J and Patil, SS and Devanna, P and Manjunatha, C and Mishra, RK},
title = {Unified Primer Enabled Detection (UPED) system for Magnaporthe oryzae infecting rice: A comparative study from conventional PCR to CRISPR Cas12a based detection systems.},
journal = {Analytica chimica acta},
volume = {1404},
number = {},
pages = {345418},
doi = {10.1016/j.aca.2026.345418},
pmid = {41997664},
issn = {1873-4324},
mesh = {*Oryza/microbiology ; *CRISPR-Cas Systems ; *Polymerase Chain Reaction/methods ; Nucleic Acid Amplification Techniques ; *DNA Primers/genetics ; *Plant Diseases/microbiology ; Molecular Diagnostic Techniques/methods ; *Magnaporthe/genetics/isolation &amp; purification ; *Ascomycota/genetics/isolation &amp; purification ; Real-Time Polymerase Chain Reaction ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Rice blast, caused by Magnaporthe oryzae, is one of the most devastating fungal pathogens of rice. Early, rapid and accurate detection is critical for effective disease management and the prevention of outbreaks. Conventional and isothermal molecular methods vary widely in sensitivity and field applicability, making it difficult to understand the reliable detection. This study aimed to perform the first unified, side-by-side comparison of six molecular detection techniques using a common primer set to identify the most sensitive, specific and field deployable approach for M. oryzae detection.<br><br>RESULTS: In this study, we have evaluated six diagnostic methods such as PCR, qPCR, LAMP, RPA, and CRISPR-Cas12a integrated with LAMP or RPA targeting the multi copy Pot2 transposon in M. oryzae. Sensitivity assays using 10-fold genomic DNA dilutions (10[9] to 10[-1] copies/reaction) revealed that LAMP-CRISPR and RPA-CRISPR were most sensitive, detecting down to 1 copy/reaction. LAMP and qPCR detected down to 10[2] and 10[3] copies/reaction, while RPA and PCR were limited to 10[5] and 10[6] copies/reaction, respectively. All assays showed high specificity with no cross-reactivity to 10 non-target rice fungal pathogens. Field validation on 17 symptomatic samples confirmed that CRISPR-based methods outperformed traditional techniques, detecting positives missed by other platforms. This is the first systematic comparison applying the same primer set across multiple diagnostic methods for M. oryzae.<br><br>SIGNIFICANCE: Our findings demonstrate that CRISPR-Cas12a-based platforms combined with isothermal amplification offers high sensitivity and specificity. CRISPR based detection allows strong field applicability for M. oryzae detection. The use of a unified primer set allowed for a direct performance comparison. These results highlight the potential of CRISPR-based diagnostics to enhance early pathogen detection, enabling rapid interventions and improved management of rice blast disease.},
}
@article {pmid41997865,
year = {2026},
author = {George, G and Harding, HP and Kay, R and Ron, D and Ordoñez, A},
title = {Metabolite import by SLC33A1 is required for ATF6 activation during endoplasmic reticulum stress.},
journal = {Life science alliance},
volume = {9},
number = {6},
pages = {},
pmid = {41997865},
issn = {2575-1077},
mesh = {Animals ; *Activating Transcription Factor 6/metabolism/genetics ; *Endoplasmic Reticulum Stress/physiology ; CHO Cells ; Cricetulus ; Unfolded Protein Response/physiology ; Endoplasmic Reticulum/metabolism ; Golgi Apparatus/metabolism ; Humans ; CRISPR-Cas Systems ; Signal Transduction ; Acetylation ; Cricetinae ; *Membrane Transport Proteins/metabolism ; },
abstract = {The transcription factor ATF6α has a central role in adapting mammalian cells to ER stress via the unfolded protein response (UPR), prompting efforts to identify ATF6α modulators. Here, an unbiased genome-wide CRISPR-Cas9 screen performed in Chinese Hamster Ovary cells revealed that proteolytic processing of the ATF6α precursor to its active form was impaired in cells lacking the ER-resident solute carrier SLC33A1, a transporter previously implicated in acetyl-CoA import, sialylation, and Nε-lysine protein acetylation. Cells lacking SLC33A1 constitutively trafficked the ATF6α to the Golgi but exhibited impaired Golgi processing and activating proteolysis. IRE1α signalling was derepressed by SLC33A1 deficiency consistent with selective loss of ATF6α-mediated negative feedback in the UPR. Slc33a1-deleted cells accumulated unmodified sialylated N-glycans, precursors to acetylated glycans, likely reflecting impaired glycan processing. Deletion of ER-localised acetyltransferases NAT8 and NAT8B, which catalyse protein Nε-lysine acetylation in the secretory pathway, did not replicate the ATF6α processing defects observed in Slc33a1-deficient cells. Together, our findings highlight a role of SLC33A1-mediated metabolite transport in the post-ER ATF6α maturation, linking small-molecule metabolism to branch-specific signalling in the UPR.},
}
@article {pmid41999294,
year = {2026},
author = {Liu, S and Wang, L and Zhang, H and Jiang, F and Tang, F and Han, R and Guo, W and Gu, S and Chen, G and Zhang, D and Zhan, X},
title = {A novel rapid detection approach based on CRISPR-Cas13a for Dermatophagoides pteronyssinus and Dermatophagoides farinae (Acariformes: Pyroglyphidae).},
journal = {Journal of insect science (Online)},
volume = {26},
number = {2},
pages = {},
doi = {10.1093/jisesa/ieag029},
pmid = {41999294},
issn = {1536-2442},
support = {H202112//Commissioned Research Project and the Anhui Provincial Graduate Innovation and Entrepreneurship Practice Project/ ; 2022cxcysj180//Commissioned Research Project and the Anhui Provincial Graduate Innovation and Entrepreneurship Practice Project/ ; },
mesh = {Animals ; *Dermatophagoides pteronyssinus/genetics ; *CRISPR-Cas Systems ; *Dermatophagoides farinae/genetics ; },
abstract = {Dermatophagoides pteronyssinus (Trouessart) and Dermatophagoides farinae (Hughes) (Acariformes: Pyroglyphidae) are the prevalent kinds of house dust mites (HDMs). HDM is a common indoor pest, which mainly breeds in indoor dust and is an important allergen source causing a variety of allergic diseases. Effective detection of these HDMs is crucial in preventing the allergic diseases they cause. The objective of this study was to develop an innovative method for the rapid visualization of HDMs (D. pteronyssinus and D. farinae) using recombinase polymerase amplification (RPA) and lateral flow dipstick (LFD) in combination with CRISPR-Cas13a (RPA-Cas13a-LFD). To achieve heightened sensitivity in the detection of HDMs, Cas13a was incorporated into the RPA process and coupled with T7 transcripts. Based on this approach, a total of 2.23-102 copies/μl of HDM were detected within 35 min (detection limit of 2.23 copies/μl for D. farinae and 39.7 copies/μl for D. pteronyssinus). No cross-reactivity occurred with Aleuroglyphus ovatus (Troupeau) (Acariformes: Acaridae), Tyrophagus putrescentiae (Schrank) (Acariformes: Acaridae), Blomia tropicalis (van Bronswijk, de Cock &amp; Oshima) (Acariformes: Acaridae), Suidasia nesbitti (Hughes) (Acariformes: Acaridae), and Carpoglyphus lactis(Linnaeus) (Acariformes: Acaridae). The RPA-Cas13a-LFD methodology demonstrated high specificity and sensitivity in detecting HDM. Given its advantages, such as ease of operation, rapid detection, and time efficiency, it is well-suited for rapid field-based detection of HDMs, providing a new technical tool for detecting D. farinae and D. pteronyssinus.},
}
@article {pmid42000334,
year = {2026},
author = {Sarsaiya, S and Jain, A and Chen, J and Gong, Q},
title = {Unlocking non-model organisms with CRISPR-Cas: A roadmap for sustainable biotechnology.},
journal = {Biotechnology advances},
volume = {90},
number = {},
pages = {108890},
doi = {10.1016/j.biotechadv.2026.108890},
pmid = {42000334},
issn = {1873-1899},
abstract = {The reliance on model organisms in biotechnology has advanced our understanding of fundamental biology but has failed to capture the complexity of real-world ecosystems, limiting applications in agriculture, biomanufacturing, and environmental remediation. This review critically evaluates the challenges and opportunities of applying CRISPR-Cas genome editing to non-model organisms, structured around a framework that systematically addresses host-specific barriers, enabling technical solutions, and real-world applications. Key obstacles are first delineated, such as restrictive genetic tools, inefficient DNA repair pathways (including NHEJ-dominance, HDR-deficiency, and polyploidy), and delivery limitations. Subsequently, innovative solutions are explored, including the engineering of Cas variants with expanded PAM flexibility and reduced toxicity, the development of host-adapted delivery systems such as phage-based vectors and conjugative plasmids, and the integration of synthetic biology tools and machine learning for optimization. Alternative, DSB-free modalities-comprising base editing, prime editing, CRISPR-associated transposases (CAST), and recombinase-assisted engineering-are further expanded upon, offering enhanced precision and expanded capabilities for complex genetic modifications. Major findings indicate that these approaches can unlock the potential of non-conventional hosts to address global challenges, such as low-energy biomanufacturing, environmental bioremediation, and carbon capture. It is concluded that bridging the gap between foundational CRISPR research and its real-world applications is imperative. Future efforts should focus on democratizing tools via open-source platforms, advancing delivery systems, establishing ethical governance-with detailed considerations for environmental release, horizontal gene transfer, regional regulatory frameworks, and biosafety in extremophile engineering-and fostering sustainable innovation through synthetic biology integration to fully realize the transformative potential of genome editing in organisms beyond model organisms.},
}
@article {pmid42002643,
year = {2026},
author = {Marnet, K and Subramanian, H and Wiesler, M and Borst, A and Liedtke, D and Pattappa, G and Docheva, D and Nikolaev, VO and Stellzig-Eisenhauer, A and Eigenthaler, M and Herrmann, M},
title = {Live-cell imaging reveals decreased cAMP in a PFE-associated c.1050-3C&gt;G PTH1R cell model.},
journal = {Journal of molecular medicine (Berlin, Germany)},
volume = {104},
number = {1},
pages = {},
pmid = {42002643},
issn = {1432-1440},
support = {453600987//Deutsche Forschungsgemeinschaft/ ; D-361//Interdisziplin&#xe4;res Zentrum f&#xfc;r Klinische Forschung, Universit&#xe4;tsklinikum W&#xfc;rzburg/ ; CA22170//European Cooperation in Science and Technology/ ; },
mesh = {*Receptor, Parathyroid Hormone, Type 1/genetics/metabolism ; Humans ; *Cyclic AMP/metabolism ; Mutation ; Signal Transduction ; Fluorescence Resonance Energy Transfer ; Cyclic AMP-Dependent Protein Kinases/metabolism ; Cell Line ; Periodontal Ligament/metabolism ; CRISPR-Cas Systems ; Phosphorylation ; },
abstract = {Primary failure of eruption (PFE) is a rare autosomal disorder provoked by heterozygous mutations in the parathyroid hormone receptor 1 (PTH1R) gene. PTH1R is a G protein-coupled receptor (GPCR) which regulates intracellular signaling molecules like cAMP. By using CRISPR/Cas9, the pathogenic PTH1R variant, c.1050-3C>G, was introduced into the periodontal ligament (PDL-hTERT) cell line to investigate molecular mechanisms in a PFE in vitro model. The PDL-hTERT immortal cell line, derived from human primary PDL cells, is a well-established model for dental diseases and expresses PTH1R. We performed different functional assays to compare the behavior of the PDL-hTERT WT versus PTH1R-mutated cells. cAMP synthesis and PKA activation were compared between different cell lines by live-cell imaging using Förster Resonance Energy Transfer (FRET)-based biosensors. Phosphorylation of VASP was measured to validate and compare the PKA activation between the cell lines. In summary, our experiments show that the mutated cell line has no major phenotypic changes, but the PTH1R downstream signaling cascade is impaired. KEY MESSAGES: A rare autosomal disorder linked to mutations in the PTH1R gene, which encodes a G protein-coupled receptor regulating intracellular signaling, including cAMP production. The pathogenic PTH1R mutation (c.1050-3C>G) was introduced into a periodontal ligament (PDL hTERT) cell line to model PFE and study molecular mechanisms in vitro. Functional assay revealed that while the mutated cell line displayed no major phenotypic changes, the PTH1R downstream signaling cascade, including cAMP synthesis and PKA activation, was disrupted. Techniques like FRET-based biosensors and VASP phosphorylation assays highlighted specific impairments in signaling pathways in cells with the PTH1R mutations.},
}
@article {pmid42003551,
year = {2026},
author = {Otten, C and Kutnjak, M and Supina-Pavic, C and Pranjic, M and Anticevic, I and Medved, V and Popovic, M},
title = {ACRC/GCNA is an essential protease that repairs DNA-protein crosslinks during vertebrate development.},
journal = {Nucleic acids research},
volume = {54},
number = {7},
pages = {},
doi = {10.1093/nar/gkag324},
pmid = {42003551},
issn = {1362-4962},
support = {UIP-2017-05-5258//Croatian Science Foundation Installation Grant/ ; IPS-2020-01-4225//Slovenian-Croatian Bilateral Research Project grant/ ; KK.01.1.1.01.0003//European Structural and Investment Funds STIM - REI project/ ; HRZZ-IP-2024-05-9425//Croatian Science Foundation/ ; //NextGenerationEU/ ; },
abstract = {DNA-protein crosslinks (DPCs) are toxic DNA lesions that block all DNA transactions including replication and transcription, and the consequences of impaired DNA-protein crosslink repair (DPCR) are severe. At the cellular level, impaired DPCR leads to the formation of double strand breaks, genomic instability, and cell death, while at the organismal level, it is associated with cancer, aging, and neurodegeneration. Despite its importance, the mechanisms of DPCR at the organismal level are largely unknown. Proteases play a central role in DPCR, as they remove proteinaceous part of the DPCs, while the peptide remnant crosslinked to DNA is subsequently removed by other repair factors. We characterized the role of putative protease ACRC/GCNA (ACidic Repeat Containing/Germ Cell Nuclear Antigen) in DPCR at the organismal level. For this purpose, we have created new animal models with CRISPR/Cas system: two zebrafish lines with inactive Acrc. We were able to overcome the early embryonic lethality caused by Acrc inactivation by injecting Acrc-WT messenger RNA and have created a viable animal model to study the role of Acrc in adult tissues. We identified histone H3, topoisomerases 1 and 2, Dnmt1, Parp1, Polr3a, and Mcm2 as putative DPC substrates of Acrc. We have shown that Acrc is essential for vertebrate development, and that the mechanism behind it is DPC removal.},
}
@article {pmid41064856,
year = {2025},
author = {Jiang, Z and Su, L and Chen, C and He, R and Jiang, L and Shu, Y and Dai, D and Wu, M and Guo, A and Liu, J and Liu, S and Liu, Z},
title = {Atf3 Deficiency Promotes Mesodermal Commitment and Enhances Endothelial Differentiation in Embryonic Stem Cells.},
journal = {Arteriosclerosis, thrombosis, and vascular biology},
volume = {45},
number = {12},
pages = {2226-2242},
pmid = {41064856},
issn = {1524-4636},
mesh = {*Activating Transcription Factor 3/genetics/deficiency/metabolism ; Animals ; *Cell Differentiation ; Mice, Knockout ; Mice ; *Endothelial Cells/metabolism ; *Mouse Embryonic Stem Cells/metabolism ; *Mesoderm/metabolism/cytology ; Cells, Cultured ; CRISPR-Cas Systems ; Cell Lineage ; Signal Transduction ; },
abstract = {BACKGROUND: Ischemic diseases have become a major threat to global health, with endothelial cell (EC) damage closely associated with their pathogenesis and progression. Cell therapies targeting endothelial repair have thus become a treatment approach of great interest, yet the procurement of clinically approved ECs for these applications has not been fully established. Modulating the expression of Atf3 (activating transcription factor 3) represents a potential strategy for deriving ECs from stem cells; however, its precise function in the development and differentiation of ECs from stem cells remains elusive. In the present study, we sought to elucidate the potential role of Atf3 in the differentiation of embryonic stem cells into ECs.<br><br>METHODS: CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) system was used to knockout Atf3 (Atf3KO [Atf3 knockout]) in mouse embryonic stem cells. EC differentiation was initially induced using the hanging drop method to promote embryoid bodies formation, followed by embryoid bodies attachment onto culture slides. The expression changes of EC markers during differentiation were assessed by RNA sequencing, Western blotting, immunofluorescence staining, flow cytometry, and reverse transcription quantitative polymerase chain reaction. Functional comparisons of differentiated ECs were performed by assessing LDL (low-density lipoprotein) uptake and NO production. Potential molecular mechanisms were further explored via bioinformatic analysis of RNA sequencing data.<br><br>RESULTS: Atf3KO led to a significant upregulation in the expression levels of progenitor and mesoderm cell markers on days 3 and 6 of differentiation. By day 9, the expression of mature EC markers also exhibited a notable increase. Moreover, Atf3KO enhanced the functional properties of differentiated Atf3KO ECs. In addition, our findings revealed that the activation of the Rap1 (Ras-related protein 1) signaling pathway, triggered by Atf3KO, contributed to ECs development and maturation.<br><br>CONCLUSIONS: Atf3KO directs embryonic stem cells toward the mesodermal lineage and activates the Rap1 signaling pathway, thereby promoting ECs development. These findings highlight a key role of Atf3 in regulating early stage of vascular endothelial development.},
}
@article {pmid41064997,
year = {2025},
author = {Chen, X and Ye, Q and Liang, Q and Li, J and Huang, Y and Xia, Q and Xiao, J and Liao, C and Lau, CH and Zhu, H},
title = {CRISPR-based platforms for detecting tumor-associated genetic materials in clinical samples.},
journal = {Bioanalysis},
volume = {17},
number = {19},
pages = {1227-1241},
pmid = {41064997},
issn = {1757-6199},
mesh = {Humans ; MicroRNAs/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Biomarkers, Tumor/genetics ; Mutation ; },
abstract = {Tumor-associated genetic markers are useful for early cancer screening, diagnosis, and treatment monitoring. However, traditional detection methods are complex in operation procedures, time-consuming, and the equipment costs are expensive. CRISPR/Cas systems are becoming emerging detection tools for tumor detection due to their programmability, rapid reaction, high targeting specificity, and the ability to amplify the signals. CRISPR/Cas has made breakthroughs in the detection of tumor-associated genetic materials including gene mutations, DNA methylation, miRNA, lncRNA, and circRNA detection. Herein, we critically discuss these advancements and describe the key concepts of each CRISPR/Cas system for detecting tumor-associated genetic materials. The significance of these tumor-associated genetic materials in cancer diagnosis and prognosis is highlighted.},
}
@article {pmid41065057,
year = {2025},
author = {Anjali, and Punetha, M and Kumar, A and Tripathi, MK and Kishor Kumar, DG and Khanna, S and Nanda, R and Yadav, P and Sharma, S and Maurya, VP and Singh, G and Chouhan, VS},
title = {Application of CRISPR/Cas9 for GDF9 Gene Editing in Caprine Granulosa Cells: Effects on Receptor Signalling and FGF2 Response.},
journal = {Reproduction in domestic animals = Zuchthygiene},
volume = {60},
number = {10},
pages = {e70128},
doi = {10.1111/rda.70128},
pmid = {41065057},
issn = {1439-0531},
support = {NASF/GTR-8004/2019-20//ICAR - National Agricultural Science Fund/ ; },
mesh = {Animals ; Female ; *Granulosa Cells/metabolism ; *Growth Differentiation Factor 9/genetics/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/veterinary ; *Goats/genetics ; *Fibroblast Growth Factor 2/pharmacology/metabolism ; Signal Transduction ; Receptors, FSH/metabolism/genetics ; },
abstract = {Fecundity-related genes, such as GDF9, play a critical role in regulating ovulation, fertilisation and early embryonic development. This study aimed to elucidate the functional role of GDF9 in caprine granulosa cells by employing CRISPR/Cas9-mediated gene editing. The CRISPR/Cas9 system, incorporating single guide RNA (sgRNA) and Cas9 endonuclease, was used to specifically disrupt the GDF9 gene. Successful GDF9 knockout was confirmed via the T7 Endonuclease I (T7E1) cleavage assay. Subsequent analyses assessed the impact of GDF9 disruption on the expression of GDF9 and its associated receptors-BMPR-1A, BMPR-1B and BMPR-II. Additionally, the study examined the modulatory effects of fibroblast growth factor 2 (FGF2) on receptor expression. FGF2 treatment led to increased mRNA expression of BMPR-1A, BMPR-1B and BMPR-II in wild-type granulosa cells. Furthermore, follicle-stimulating hormone receptor (FSHR) levels were significantly upregulated, whereas luteinising hormone receptor (LHR) expression decreased following FGF2 stimulation in wild-type cells. In contrast, GDF9-knockout cells showed elevated expression of both FSHR and LHR. The study also investigated the impact of GDF9 deletion on the expression of key steroidogenic genes, particularly StAR. The combined presence of GDF9 and FGF2 synergistically enhanced StAR expression. Cellular responses to FGF2 included a downregulation of CASPASE 3, indicating reduced apoptosis and an upregulation of PCNA, suggesting increased cell proliferation. In conclusion, this study provides novel insights into the regulatory role of GDF9 in ovarian granulosa cell function and highlights the utility of CRISPR/Cas9 technology for functional genomics in caprine species. The findings have significant implications for enhancing reproductive performance through targeted gene modulation.},
}
@article {pmid41066575,
year = {2025},
author = {Groessl, S and Kalis, R and Snaebjornsson, MT and Wambach, L and Haider, J and Andersch, F and Schulze, A and Palm, W and Zuber, J},
title = {Acidosis orchestrates adaptations of energy metabolism in tumors.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6769},
pages = {eadp7603},
doi = {10.1126/science.adp7603},
pmid = {41066575},
issn = {1095-9203},
mesh = {*Energy Metabolism/genetics ; *Acidosis/metabolism/genetics ; Humans ; *Pancreatic Neoplasms/metabolism/genetics ; Mitochondria/metabolism ; Cell Line, Tumor ; *Adaptation, Physiological/genetics ; Animals ; Mice ; Stress, Physiological ; CRISPR-Cas Systems ; MAP Kinase Signaling System ; Mitochondrial Dynamics ; },
abstract = {Malignant tumors are characterized by diverse metabolic stresses, including nutrient shortages, hypoxia, and buildup of metabolic by-products. To understand how cancer cells adapt to such challenges, we conducted sequential CRISPR screens to identify genes that affect cellular fitness under specific metabolic stress conditions in cell culture and to then probe their relevance in pancreatic tumors. Comparative analyses of hundreds of fitness genes revealed that cancer metabolism in vivo was shaped by bioenergetic adaptations to tumor acidosis. Mechanistically, acidosis suppressed cytoplasmic activity of extracellular signal-regulated kinase (ERK), thereby preventing oncogene-induced mitochondrial fragmentation and promoting fused mitochondria. The resulting boost in mitochondrial respiration supported cancer cell adaptations to various metabolic stresses. Thus, acidosis is an environmental factor that alters energy metabolism to promote stress resilience in cancer.},
}
@article {pmid41067151,
year = {2025},
author = {Li, C and Meng, C and Wang, X and Yuan, C and Chen, Q and Yang, Y and Zhang, Z and Duan, Y and Dong, Q and Sun, X},
title = {CRISPR/Cas9-targeted mutagenesis of the PpTCP4 gene increased tiller number in Poa pratensis.},
journal = {Plant physiology and biochemistry : PPB},
volume = {229},
number = {Pt C},
pages = {110588},
doi = {10.1016/j.plaphy.2025.110588},
pmid = {41067151},
issn = {1873-2690},
mesh = {*CRISPR-Cas Systems/genetics ; *Poa/genetics/growth &amp; development ; *Mutagenesis ; Gene Editing ; Plants, Genetically Modified/genetics ; *Plant Proteins/genetics/metabolism ; *Genes, Plant ; },
abstract = {Poa pratensis, commonly known as Kentucky bluegrass, is a widely used cool-season grass species for turf in lawns and recreational areas worldwide. However, the lack of genome editing platforms has impeded research into enhancing forage crops suitable for land unsuitable for other crops. In this study, we successfully established an Agrobacterium-mediated genetic transformation system for P. pratensis. Furthermore, we successfully produced PpTCP4-edited plants using the CRISPR/Cas9 system. This research represents a significant advancement in the genetic transformation and gene editing of P. pratensis, providing a foundational technology for future investigations into gene function and breeding in P. pratensis.},
}
@article {pmid41067593,
year = {2026},
author = {Wijerathna, HMSM and Jung, S and Lee, J},
title = {Enhancing Edwardsiella piscicida resistance through CRISPR/Cas9-mediated deletion of toll-like receptor 5a (tlr5a) in zebrafish.},
journal = {Fish &amp; shellfish immunology},
volume = {168},
number = {},
pages = {110915},
doi = {10.1016/j.fsi.2025.110915},
pmid = {41067593},
issn = {1095-9947},
mesh = {Animals ; *Toll-Like Receptor 5/genetics/immunology ; Edwardsiella/physiology ; *Fish Diseases/immunology/microbiology/genetics ; *Zebrafish/immunology/genetics ; *Enterobacteriaceae Infections/immunology/veterinary/genetics ; CRISPR-Cas Systems ; *Immunity, Innate/genetics ; *Disease Resistance/genetics ; *Zebrafish Proteins/genetics/immunology ; },
abstract = {Toll-like receptor 5 (TLR5), a pattern recognition receptor that detects bacterial flagellin, plays a critical role in innate immune responses. Teleost fish possess two paralogs, tlr5a and tlr5b, of which the functional role of tlr5a during flagellated bacterial infections remains unclear. In this study, we investigated the involvement of tlr5a in the immune response against Edwardsiella piscicida infection in zebrafish (Danio rerio). To this end, we generated tlr5a-deficient (tlr5a[-/-]) zebrafish using CRISPR/Cas9 technology. Survival analysis following E. piscicida challenge revealed significantly higher survival rates and reduced bacterial loads in tlr5a[-/-] larvae and adults compared to wild-type (WT) controls. tlr5a[-/-] fish exhibited significantly lower expression of pro-inflammatory cytokines (tnfa, il6, and il-1β), chemokine (il8), and pathway genes (nfκb, myd88, and mapk14a) at multiple time points post-infection (0, 6, 12, 24, 48, and 72 hpi). DCFH-DA staining and Sudan Black/Neutral Red staining revealed elevated reactive oxygen species (ROS) levels and immune cell recruitment, respectively, indicating reduced ROS production and diminished neutrophil and macrophage infiltration in tlr5a[-/-] larvae. Antioxidant gene expression analysis revealed reduced levels of cat and nrf2 in tlr5a[-/-] larvae compared to WT. The results of this study indicate that tlr5a knockout attenuates excessive inflammation and improves resistance against E. piscicida infection, likely by reducing bacterial adhesion and suppressing NF-κB-mediated pro-inflammatory pathways. This study highlights tlr5a as a potential immunomodulatory target to enhance disease resistance in teleost aquaculture.},
}
@article {pmid41068553,
year = {2025},
author = {Wang, YM and Xu, T and Duan, JQ and Zhao, L and You, D and Lai, SY and Qing, Y and Ge, LP and Liu, ZH and Sun, J and Zeng, X and Xu, ZW and Zhu, L},
title = {An Integrated One-Tube RPA-CRISPR/Cas13d Assay Coupled with Lateral Flow for Rapid PRRSV-1 Detection.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {42},
pages = {27080-27088},
doi = {10.1021/acs.jafc.5c07919},
pmid = {41068553},
issn = {1520-5118},
mesh = {*CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; Animals ; *Porcine respiratory and reproductive syndrome virus/genetics/isolation &amp; purification ; Swine ; *Porcine Reproductive and Respiratory Syndrome/virology/diagnosis ; RNA, Viral/genetics ; },
abstract = {Rapid and scalable diagnostic technologies are essential for controlling infectious diseases. We present STEP (Single-Tube Extraction-free Platform for CRISPR/Cas13d detection), a streamlined, equipment-minimal CRISPR-based platform enabling rapid, sensitive, and accurate viral RNA detection. STEP integrates isothermal RPA amplification with CRISPR-based nucleic acid cleavage, providing multimodal readouts including lateral flow strips, in-tube fluorescence, and fluorescence quantification. Lab-free extraction reagents and lyophilized formulations enhance user friendliness of STEP and stability for point-of-care testing (POCT), reduce cost, and eliminate cold-chain requirements. Optimization of baseline time and the ability to operate at both ambient and body temperatures minimize temporal and equipment constraints. Clinical evaluation showed 100% sensitivity and specificity versus RT-qPCR, delivering a sample-to-answer workflow within 35 min. STEP provides a robust platform for decentralized infectious disease diagnostics and rapid public health response, combining speed, user friendliness, and minimal instrumentation requirements.},
}
@article {pmid41068986,
year = {2025},
author = {Wu, J and Jang, H and Kwak, H and Son, M and Jiang, W and Hwang, HY and Jo, DH and Kim, D and Kim, HH and Kim, JH},
title = {Engineered virus-like particle-assembled Vegfa-targeting Cas9 ribonucleoprotein treatment alleviates neovascularization in wet age-related macular degeneration.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {346},
pmid = {41068986},
issn = {1474-760X},
support = {2022M3A9F3017506//National Research Foundation of Korea/ ; 2022M3A9E4017127//National Research Foundation of Korea/ ; 202200004004//Kun-hee Lee Child Cancer &amp; Rare Disease Project/ ; 18-2023-0010//Seoul National University Hospital/ ; GTL24021-000//National Research Council of Science and Technology/ ; },
mesh = {CRISPR-Associated Protein 9/genetics ; *Vascular Endothelial Growth Factor A/genetics ; NIH 3T3 Cells ; Animals ; Mice ; *Gene Editing ; Gene Therapy Agents ; Disease Models, Animal ; *Choroidal Neovascularization/therapy ; Ribonucleoproteins/genetics ; Mice, Inbred C57BL ; Male ; *CRISPR-Cas Systems ; *Wet Macular Degeneration/therapy ; },
abstract = {BACKGROUND: Age-related macular degeneration, particularly the wet form, is a leading cause of vision loss, characterized by vascular endothelial growth factor A (VEGFA) overproduction. Engineered virus-like particles (eVLPs) combine the efficiency of viral systems with the transient nature of non-viral platforms to offer a potential solution for delivering VEGFA-targeting genome editing enzymes in a safe and efficient manner. Here, we investigate the therapeutic efficacy of eVLPs for transient delivery of Vegfa-targeting Cas9 ribonucleoprotein in a laser-induced choroidal neovascularization mouse model of wet age-related macular degeneration.<br><br>RESULTS: We find that Cas9-eVLPs enables efficient intracellular delivery in vitro, achieving up to 99% insertion and deletion frequency at Vegfa target locus and significant VEGFA protein downregulation in NIH/3T3 cells. A single subretinal injection of Cas9-eVLPs into the mouse retinal pigment epithelium effectively disrupts Vegfa expression, achieving an average indel efficiency of 16.7%. Compared to control groups, the laser-induced choroidal neovascularization mouse model exhibits significantly reduced choroidal neovascularization formation following Cas9-eVLPs intervention, and decreased VEGFA protein levels are detected in the retinal pigment epithelium. Furthermore, the retinal anatomical and functional toxicity are not affected after treatment.<br><br>CONCLUSIONS: eVLPs exhibit the potential as a safe and efficient delivery platform for Cas9 ribonucleoproteins, achieving precise Vegfa downregulation and significant reduction in choroidal neovascularization in a mouse model of wet age-related macular degeneration. With transient delivery of gene editing enzymes, high editing efficiency, and minimal risk of genomic integration, eVLPs present a promising alternative to conventional delivery systems for advancing genome editing therapies in retinal diseases.},
}
@article {pmid41069170,
year = {2026},
author = {Zhu, Z and Xue, J and Cao, J and Zhang, Z and Gu, T and Sun, Y and Wang, H},
title = {One-pot assay for rapid detection of heterozygous herbicide resistance in Digitaria ciliaris var. chrysoblephara by combining CRISPR/Cas and LAMP.},
journal = {Pest management science},
volume = {82},
number = {2},
pages = {1331-1342},
doi = {10.1002/ps.70279},
pmid = {41069170},
issn = {1526-4998},
support = {CX(22)5003//Jiangsu Agricultural Science and Technology Innovation Fund/ ; 2023YFD1401100//National Key R&amp;D Program of China/ ; },
mesh = {*Herbicide Resistance/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Polymorphism, Single Nucleotide ; *Herbicides/pharmacology ; Acetyl-CoA Carboxylase/genetics ; Heterozygote ; *Genetic Techniques ; *Plant Weeds/genetics/drug effects ; Mutation ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Resistance to the acetyl-CoA carboxylase (ACCase) inhibitor herbicide cyhalofop-butyl in Digitaria ciliaris var. chrysoblephara is mainly caused by a mutation at the W2027C or W2027S site; however, the existing methods for this mutation site have insufficient detection performance and are difficult to achieve integrated detection in the field.<br><br>RESULTS: In this work, we have developed and optimized a One-Pot single-nucleotide polymorphism (SNP) detection for herbicide resistance based on CRISPR/Cas recognition coupled with the loop-mediated isothermal amplification (LAMP), named OpCas-LAMP. By designing specific CRISPR/Cas guide RNAs and LAMP primers, the OpCas-LAMP can accurately identify with 1% heterozygous mutants of the W2027S or W2027C mutations ACCase gene in D.ciliaris var. chrysoblephara. The optimized reaction system exhibits optimal amplification efficiency at 65&#xb0;C, effectively distinguishing within 60 min (30-min LAMP detection after 30 min CRISPR/Cas pre-cleavage) between homozygous mutant (HM), heterozygous mutant (HT) and wild-type (WT).<br><br>CONCLUSION: This method enables real-time one-pot field detection by integrated with miniaturized detection devices, significantly enhancing its practicality and potential for widespread application. This work provides a novel technical approach for detecting herbicide resistance for global weed resistance monitoring and management. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid41069986,
year = {2025},
author = {Pandey, V and Sharma, S and Pokharel, YR},
title = {Exploring CRISPR-Cas: The transformative impact of gene editing in molecular biology.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102717},
pmid = {41069986},
issn = {2162-2531},
abstract = {This review traces the evolution of clustered regularly interspaced short palindromic repeats (CRISPR) technology from a prokaryotic immune mechanism to a versatile tool for precise genome engineering. We compare CRISPR with traditional gene-editing methods like RNA interference (RNAi), zinc finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), emphasizing its advantages in target specificity, multiplexing, and ease of design. We examine various Cas enzyme classes, engineered variants, and their applications in dissecting genetic alterations at the cellular level. The review further explores CRISPR's expanding role in developing disease models using tissues, organoids, and animal systems, enhancing our understanding of disease mechanisms. Finally, we discuss CRISPR's emerging applications in diagnostics and its transformative impact on immunotherapy and cell-based cancer treatments.},
}
@article {pmid41070887,
year = {2025},
author = {Zhang, Y and Wu, Y and Guo, A and Liu, Y and Sun, Q and Zou, X and Sun, Z},
title = {Fluorescent biosensors for the detection of foodborne pathogenic bacteria in food: a comprehensive review.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {41},
pages = {8298-8316},
doi = {10.1039/d5ay01025j},
pmid = {41070887},
issn = {1759-9679},
mesh = {*Biosensing Techniques/methods ; *Food Microbiology/methods ; *Bacteria/isolation &amp; purification ; *Food Contamination/analysis ; Foodborne Diseases/microbiology ; Humans ; Food Safety ; CRISPR-Cas Systems ; },
abstract = {Foodborne pathogenic bacterial contamination poses a major challenge to global food safety and public health, making the development of rapid, sensitive, and specific detection technologies critically important. Conventional methods are limited by their long turnaround time, complex operations, and reliance on large-scale instruments, making them unsuitable for on-site rapid detection. Fluorescent biosensors, which combine highly specific biological recognition elements with highly sensitive fluorescent signal output, demonstrate significant advantages in detecting foodborne pathogens. This review systematically summarizes recent advances in fluorescent biosensors for the detection of common foodborne pathogenic bacteria, with a focus on the application of signal amplification strategies such as functional nanomaterials, amplification techniques, CRISPR/Cas systems, and Argonaute proteins. Furthermore, it analyzes performance metrics including multiplex pathogen detection, real-time quantification, anti-interference capability, and on-site applicability. Finally, future development trends and challenges are discussed, aiming to provide insights for the innovation of food safety monitoring technologies.},
}
@article {pmid41071356,
year = {2025},
author = {Asaoka, Y and Tarumoto, S and Hirose, Y and Iwamoto, A and Tokunaga, M and Matsuura, T and Takemoto, Y and Yamashita, H and Furutani-Seiki, M and Sugahara, K},
title = {LOXHD1b knockout alters swimming behavior in zebrafish.},
journal = {Cell and tissue research},
volume = {402},
number = {3},
pages = {243-254},
pmid = {41071356},
issn = {1432-0878},
support = {23H04710//Japanese Ministry of Education, Culture, Sports, Science, and Technology/ ; 20K09733//Japanese Ministry of Education, Culture, Sports, Science, and Technology/ ; },
mesh = {Animals ; *Zebrafish/physiology/genetics ; *Swimming/physiology ; *Zebrafish Proteins/metabolism/genetics ; *Gene Knockout Techniques ; Lateral Line System/metabolism ; Hair Cells, Auditory/metabolism ; CRISPR-Cas Systems/genetics ; Humans ; Larva ; },
abstract = {Since human inner ear hair cells do not regenerate, the current treatments of hereditary deafness depend on hearing aids or cochlear implant. However, uncovering the functions of genes responsible for hereditary hearing loss is not only useful for their diagnosis but also for developing therapies. The pathogenetic mechanism of human non-syndromic deafness DFNB77 without morphological defects in the inner year caused by LOXHD1 mutations is not fully understood. We introduced zebrafish because the lateral line hair cells are structurally and physiologically similar to the human inner ear hair cells and mutations involved in non-symptomatic hearing loss can be assessed by their swimming behavior. The knock-out (KO) of LOXHD1b gene which is expressed in the lateral line hair cells was generated using the CRISPR-Cas9 system in zebrafish, and its morphological and functional changes were evaluated. As with human patients the LOXHD1b KO zebrafish larvae did not exhibit detectable morphological defects, but showed prolonged water flow sensing time. These results suggest that LOXHD1b plays pivotal roles for the hair cell neural activity and its KO zebrafish mutant serves as a useful model for revealing the molecular mechanisms linking LOXHD with hair cell function and for a drug screening to rescue the swimming phenotype.},
}
@article {pmid41071600,
year = {2025},
author = {Zhang, W and Feng, ZY and Feng, Z and Lian, R and Liu, Z and Zhang, J},
title = {A CRISPR-Customizable Copper-Coordinated DNA Nanoplatform Potentiates Cuproptosis Through Circadian and Metabolic Pathway Manipulation.},
journal = {ACS nano},
volume = {19},
number = {41},
pages = {36701-36717},
doi = {10.1021/acsnano.5c12641},
pmid = {41071600},
issn = {1936-086X},
mesh = {*Cuproptosis ; *Circadian Rhythm/genetics ; *Copper/administration &amp; dosage ; DNA/chemistry ; *Metabolic Networks and Pathways/genetics ; Nanospheres/administration &amp; dosage/chemistry ; *CRISPR-Cas Systems ; *Neoplasms/genetics/therapy ; *ARNTL Transcription Factors/genetics ; Ribonucleoproteins/genetics ; Gene Silencing ; *Chronotherapy/methods ; Humans ; MCF-7 Cells ; Animals ; Mice ; HEK293 Cells ; NIH 3T3 Cells ; },
abstract = {Despite the promise of cuproptosis in antitumor therapy, developing strategies to enhance its therapeutic efficacy within the tumor microenvironment remains a challenge. Inspired by the chronotherapy that manipulate circadian rhythms to enhance drug effectiveness, herein we report for a CRISPR-customized copper-DNA nanoplatform (Cu-RNP) that synergistically induces multimodal cell death, including potentiated cuproptosis, by manipulating circadian and metabolic pathways. Cu-RNP integrates coordination-driven self-assembly of Cu[2+]-DNA nanospheres with Cas13d/crRNA ribonucleoproteins targeting BMAL1. Upon cellular internalization, the acidic and reducing endo/lysosomal environment triggers Cu-RNP disassembly, releasing RNP to silence BMAL1 and disrupt circadian oscillations, leading to WEE1 downregulation and p21 upregulation, thereby inducing apoptosis. Simultaneously, liberated Cu[2+] generates cytotoxic hydroxyl radicals for chemodynamic therapy (CDT) and concurrently depletes GSH, promoting mitochondrial copper overload for cuproptosis. Importantly, we demonstrate that silencing BMAL1 disrupts circadian rhythms, inhibits glycolysis, enhances mitochondrial respiration, and redirects metabolic flux to the TCA cycle, thereby amplifying the cell's vulnerability to copper-induced cuproptosis. In vitro and in vivo results demonstrate that Cu-RNP sensitizes cancer cells to cuproptosis and elicit strong antitumor response through the synergistic combination of cuproptosis, CDT, apoptosis, and circadian-metabolic modulation. This study demonstrates a mechanistic link between BMAL1-regulated circadian rhythms and cuproptosis sensitivity, suggesting a potential treatment strategy for multimodal, cuproptosis-potentiating cancer therapies.},
}
@article {pmid41071884,
year = {2025},
author = {Guo, Y and Xu, M and Xue, H and Ding, X and Wong, AM and Lin, N and Pu, D and Wong, AM and Wang, X and Zhao, H and Wong, N},
title = {Genome-wide CRISPR screen identifies splicing factor SF3B4 in driving hepatocellular carcinoma.},
journal = {Science advances},
volume = {11},
number = {41},
pages = {eadw7181},
pmid = {41071884},
issn = {2375-2548},
mesh = {*Carcinoma, Hepatocellular/genetics/pathology/metabolism/drug therapy ; Humans ; *Liver Neoplasms/genetics/pathology/metabolism/drug therapy ; Animals ; Mice ; *RNA Splicing Factors/genetics/metabolism ; Ferroptosis/genetics ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems ; Cell Line, Tumor ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Phenylurea Compounds/pharmacology ; Drug Resistance, Neoplasm/genetics ; T-Box Domain Proteins/genetics/metabolism ; Quinolines ; },
abstract = {Although genome sequencings have recognized many cancer-associated genes in hepatocellular carcinoma (HCC), distinguishing their functional effect remains challenging. Leveraging on a genome-wide CRISPR knockout (KO) screening, we uncovered spliceosome factors as major survival essential genes in HCC and up-regulations of ferroptosis suppressors [particularly glutamate-cysteine ligase catalytic subunit (GCLC)] in lenvatinib resistance. Our KO screen in patient-derived HCC organoid showed splicing factor 3b subunit 4 (SF3B4) to be top-ranked, conferring prosurvival signal in HCC organoid and driving tumorigenic potentials in both hepatic progenitor organoids and hydrodynamic tail vein injection HCC murine model. The combined RNA immunoprecipitation sequencing, long-read isoform sequencing, and transcriptome revealed characteristic splicing landscape regulated by SF3B4 and identified T-box transcription factor 3 (TBX3) variant TBX3+2a as a potent downstream effector. Our findings highlighted vital roles of SF3B4 in HCC cell survival and tumor progression, and the phenomenon of ferroptosis resistance in patients unresponsive to first-line agent lenvatinib.},
}
@article {pmid41072226,
year = {2026},
author = {Li, L and Li, M and Wang, S and Dong, Y},
title = {Development of a CRISPR/Cas12a-assisted fluorescent aptasensor for simultaneous detection of zearalenone and ochratoxin A.},
journal = {Talanta},
volume = {298},
number = {Pt B},
pages = {128937},
doi = {10.1016/j.talanta.2025.128937},
pmid = {41072226},
issn = {1873-3573},
mesh = {*Ochratoxins/analysis ; *Zearalenone/analysis ; *Aptamers, Nucleotide/chemistry/genetics/metabolism ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; Food Contamination/analysis ; Spectrometry, Fluorescence ; Fluorescence ; *Fluorescent Dyes/chemistry ; },
abstract = {Mycotoxins, such as zearalenone (ZEN) and ochratoxin A (OTA), represent significant hazards to both human and animal health, necessitating strict monitoring and regulation of mycotoxin levels in food, feed, and environment. In this study, a simple and efficient CRISPR/Cas12a-assisted fluorescent aptasensor is presented for the simultaneous detection of ZEN and OTA. Utilizing a Bifunctional aptamer (B-APT), this biosensor achieves dual recognition of the both targets, subsequently converting their concentrations into observable fluorescent signals through the Cas12a/crRNA cis-cleavage activity. Rational modifications of the complementary strands specific to the two targets enable distinct emission wavelengths under the same excitation light, facilitating simultaneous and independent quantitative determination of ZEN and OTA. Under optimized conditions, the CRISPR/Cas12a-aptasensor demonstrates robust detection capabilities for individual ZEN and OTA targets, as well as their mixture, yielding consistent standard curves. This methodology exhibits reliable detection of ZEN and OTA concentrations spanning from 0.25 nM to 256 nM and 1 nM to 1024 nM, with respective limit of detection (LOD) values of 190 pM and 931 pM. Furthermore, this method showcases exceptional selectivity and considerable recovery rates (89.17 %-109.88 % for ZEN and 101.19 %-106.51 % for OTA) in corn oil samples, underscoring its efficacy as an advanced tool for ZEN and OTA detection and offering valuable insights into the simultaneous detection of diverse targets.},
}
@article {pmid41072237,
year = {2026},
author = {Xin, M and Liu, J and Zhou, H and Bu, S and Hao, Z and Sun, H and Lu, J and Feng, X and Jiang, X and Wang, Q and Wan, J},
title = {An H1N1 virus biosensor based on enzyme activity-gated PER-CRISPR/Cas12a cascade signal amplification.},
journal = {Enzyme and microbial technology},
volume = {192},
number = {},
pages = {110759},
doi = {10.1016/j.enzmictec.2025.110759},
pmid = {41072237},
issn = {1879-0909},
mesh = {*Influenza A Virus, H1N1 Subtype/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Influenza, Human/diagnosis/virology ; SARS-CoV-2/isolation &amp; purification/genetics ; RNA, Viral/analysis/genetics ; Nucleic Acid Amplification Techniques/methods ; DNA, Viral/analysis/genetics ; DNA, Single-Stranded ; Bacterial Proteins ; },
abstract = {The rapid and accurate detection of the H1N1 influenza virus is a key link in epidemic prevention and control. This study innovatively constructed a cascade signal amplification biosensor based on DNA polymerase activity regulation, aiming to achieve ultra-sensitive and highly specific detection of viral nucleic acids. This biosensor has the following significant advantages: (i) Molecular lock-key regulation mechanism: A functional DNA inhibitor is designed to form a complex with Taq DNA polymerase, and the target H1N1 RNA is specifically recognized to release enzyme activity inhibition, converting the target presence information into a PER reaction initiation signal. (ii) Cascade signal amplification system: The single-stranded DNA generated by PER activates Cas12a trans-cleavage activity, achieving a three-level signal amplification of enzyme activity activation → nucleic acid synthesis → CRISPR cleavage. The biosensor exhibits a linear detection range between 1 pM and 1 μM, with a detection limit of 25 fM. Moreover, the platform showed high versatility and could be readily adapted for the detection of other pathogens such as SARS-CoV-2 by simply modifying the nucleic acid sequences of the inhibitor and activator. This study not only provides a new tool for the screening of H1N1 influenza virus, but also offers a novel strategy for the development of next-generation molecular detection technologies suitable for point-of-care diagnostics, indicating considerable application potential.},
}
@article {pmid41072406,
year = {2025},
author = {Henriques, WS and Bowman, J and Hall, LN and Gauvin, CC and Wei, H and Kuang, H and Zimanyi, CM and Eng, ET and Santiago-Frangos, A and Wiedenheft, B},
title = {Structures reveal how the Cas1-2/3 integrase captures, delivers, and integrates foreign DNA into CRISPR loci.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {12},
pages = {2058-2070.e6},
pmid = {41072406},
issn = {1878-4186},
support = {R00 GM147842/GM/NIGMS NIH HHS/United States ; R24 GM154192/GM/NIGMS NIH HHS/United States ; R35 GM134867/GM/NIGMS NIH HHS/United States ; P30 GM140963/GM/NIGMS NIH HHS/United States ; U24 GM129539/GM/NIGMS NIH HHS/United States ; K99 GM147842/GM/NIGMS NIH HHS/United States ; },
mesh = {*Pseudomonas aeruginosa/enzymology/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Integrases/chemistry/metabolism/genetics ; CRISPR-Cas Systems ; Models, Molecular ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacterial Proteins/chemistry/metabolism/genetics ; Protein Binding ; DNA/metabolism/chemistry ; },
abstract = {Cas1 and Cas2 are the hallmark proteins of prokaryotic adaptive immunity. However, these two proteins are often fused to other proteins and the functional association of these fusions often remain poorly understood. Here we purify and determine structures of Cas1 and the Cas2/3 fusion proteins from Pseudomonas aeruginosa at distinct stages of CRISPR adaptation. Collectively, these structures reveal a prominent, positively charged channel on one face of the integration complex that captures short fragments of foreign DNA. Foreign DNA binding triggers conformational changes in Cas2/3 that expose new DNA binding surfaces necessary for homing the DNA-bound integrase to specific CRISPR loci. The length of the foreign DNA substrate determines if Cas1-2/3 docks completely onto the CRISPR repeat to successfully catalyze two sequential transesterification reactions required for integration. Together, these structures clarify how the Cas1-2/3 proteins orchestrate foreign DNA capture, site-specific delivery, and integration of new DNA into the bacterial genome.},
}
@article {pmid41072420,
year = {2025},
author = {Zhao, G and Liu, Y and Zhang, G and Wang, J and Zou, Z and Wang, Y and Xu, S and Han, D and Xu, Z and Chen, Z and Yang, M and Zeng, Z and Wu, Y and Zhou, X and Huang, J},
title = {Engineered hypercompact Fanzor-ωRNA system with enhanced genome editing activity.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4138-4151.e4},
doi = {10.1016/j.molcel.2025.09.031},
pmid = {41072420},
issn = {1097-4164},
mesh = {Humans ; *Gene Editing/methods ; HEK293 Cells ; Animals ; Dependovirus/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *Genome, Human ; CRISPR-Cas Systems ; Mice ; },
abstract = {As programmable RNA-guided DNA nucleases found in eukaryotes, Fanzors have shown promise in genome editing with their compact size. A wide variety of RuvC-containing Fanzor nucleases have been identified in various eukaryotes and their viruses. However, low editing efficiency limits the application of Fanzor in mammalian genome editing. In this study, we introduce SpuFz1 V4, an engineered RNA-guided DNA endonuclease with vigorous editing activity in the human genome. Furthermore, we demonstrate the substantial potential of the Fanzor system as a base editor. SpuFz1 V4 is currently the most active RNA-guided DNA nuclease of eukaryotic origin, belonging to the Fanzor1 family. Due to its compact size, SpuFz1 V4 can be efficiently delivered via a single adeno-associated virus (AAV) into the retina, achieving robust in vivo genome editing, which has the potential to be applied in both basic research and disease treatment applications.},
}
@article {pmid41072860,
year = {2025},
author = {Patel, RR and Arun, PP and Singh, SK and Singh, M},
title = {Overcoming antimicrobial resistance: Phage therapy as a promising solution to combat ESKAPE pathogens.},
journal = {International journal of antimicrobial agents},
volume = {66},
number = {6},
pages = {107640},
doi = {10.1016/j.ijantimicag.2025.107640},
pmid = {41072860},
issn = {1872-7913},
mesh = {*Phage Therapy/methods ; Humans ; *Drug Resistance, Multiple, Bacterial ; *Bacteriophages/physiology ; Anti-Bacterial Agents/pharmacology/therapeutic use ; Biofilms/drug effects ; *Bacterial Infections/therapy/microbiology ; *Cross Infection/therapy/microbiology ; *Bacteria/drug effects/virology ; },
abstract = {OBJECTIVE: The global escalation of antimicrobial resistance (AMR) has intensified the search for alternative therapies, with bacteriophage (phage) therapy re-emerging as a promising solution. This review aims to critically evaluate the therapeutic potential of phage therapy against multidrug-resistant (MDR) ESKAPE pathogens, which are major contributors to hospital-acquired infections. The review discusses the distinct antibacterial strategies of phage namely, targeted lysis, enzymatic biofilm disruption, and synergy with antibiotics.<br><br>METHODS: A comprehensive literature review was conducted focusing on the mechanisms of phage antibacterial activity and the molecular regulation of lytic and lysogenic life cycles, highlighting the therapeutic importance of the lytic-lysogenic switch. It also explores the integration of nanotechnology-based delivery systems that enhances therapeutic efficacy. Recent advances in phage engineering, including CRISPR-Cas technologies and synthetic biology approaches, were also analyzed to understand their contribution to optimizing phage efficacy.<br><br>RESULTS: Phage therapy demonstrates multiple antibacterial mechanisms, including targeted bacterial lysis, enzymatic biofilm disruption, and synergistic interactions with antibiotics. Advanced delivery systems such as liposomes, hydrogels, nanofibers, and nanoemulsions enhance phage stability and retention by overcoming physiological barriers like gastric acidity, enzymatic degradation, and immune clearance. Clinical evidence from compassionate use cases and early-phase trials highlights the safety and therapeutic promise of phage therapy.<br><br>CONCLUSIONS: Phage therapy represents a versatile and sustainable approach to combat multidrug-resistant infections. While regulatory, resistance, and scalability challenges remain, continued integration of microbiology, nanotechnology, and clinical research may enable its transition from experimental to mainstream therapeutic application in the post-antibiotic era.},
}
@article {pmid41072992,
year = {2025},
author = {Shi, S and Qin, F and Wu, J and Yang, J and Zhang, X and Wang, S and Wen, W and Wu, Z},
title = {Ultrasensitive single-particle collision electrochemical platform employing CRISPR/Cas12a for ctDNA biosensing.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344590},
doi = {10.1016/j.aca.2025.344590},
pmid = {41072992},
issn = {1873-4324},
mesh = {CRISPR-Cas Systems ; Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Limit of Detection ; *Circulating Tumor DNA/analysis/blood/chemistry/genetics ; Silver/chemistry ; Metal Nanoparticles/chemistry ; Humans ; },
abstract = {Circulating tumor DNA (ctDNA) is a characteristic tumor biomarker used for cancer diagnosis, treatment, and prognosis. However, the low concentration of ctDNA in peripheral blood and the interference of complex matrices with signals make the detection of ctDNA extremely challenging. Single-particle collision electrochemistry (SPCE) has been widely used in bioanalysis due to its advantages such as high throughput, simple operation, high sensitivity, and low detection limit. In this work, a novel SPCE biosensor for the rapid detection of ctDNA was developed by combining CRISPR/Cas12a with excellent cleavage activity and magnetic beads (MBs) with good separation and enrichment capabilities. The trans-cleavage ability of CRISPR/Cas12a can only be triggered in the presence of target ctDNA to effectively cleave the ssDNA2 on the surface of Ag NPs-ssDNA2 within 1 h, thereby activating the collision activity of silver nanoparticles (Ag NPs). ctDNA was quantified by the collision frequency of Ag NPs. The detection limit of the developed SPCE biosensor for ctDNA was as low as 4.2 fM, and the linear range was 10 fM-1 nM. In addition, MBs allow the biosensor to detect ctDNA in complex samples by directly sampling from complex matrices, with excellent sensitivity and specificity, demonstrating the great potential of the developed SPCE biosensor in the detection of patient samples.},
}
@article {pmid41073014,
year = {2025},
author = {Cao, R and Wang, S and Guo, Q and Xie, H and Wang, Y and Li, T and Chang, F and Shi, H and Ding, S and Min, X and Duan, X},
title = {DNAzyme-driven SDA reaction regulates CRISPR/Cas12a for highly sensitive and selective analysis of underexpressed miRNA.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344620},
doi = {10.1016/j.aca.2025.344620},
pmid = {41073014},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/metabolism ; *DNA, Catalytic/metabolism/chemistry ; *CRISPR-Cas Systems ; Humans ; *Biosensing Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Male ; Prostatic Neoplasms/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Underexpressed microRNA (miRNA) exerts a pivotal influence across a spectrum of physiological and pathological processes, with their role being particularly pronounced in the incipient stages of tumorigenesis. However, there are several challenges to analyzing these underexpressed miRNAs for their lower abundance and relative decreases in some cancers. Here, we developed a novel fluorescence biosensor based on the DNAzyme-driven strand displacement amplification (SDA) regulating CRISPR/Cas12a for the sensitive and selective detection of underexpressed miRNA, using prostate cancer-associated miR-222 as a proof-of-concept. This study innovatively expanded the application of DNAzyme substrates, designed as templates to trigger SDA and CRISPR/Cas12a reaction, which could effectively generate a positive signal output for detecting underexpressed miRNA. In the absence of miR-222, DNAzyme formation was blocked, allowing the complete substrate to activate SDA, which generated ssDNA that triggered CRISPR/Cas12a trans-cleavage activity to produce a strong fluorescent signal. In contrast, intact DNAzymes (in the presence of miR-222) cleaved the substrates into short DNA fragments, preventing SDA and CRISPR/Cas12a activation, thereby maintaining the sensor in a low fluorescent state. The biosensor demonstrated a linear detection range spanning from 0.1 pmol/L to 1 nmol/L, accompanied by a detection limit of 33.5 fmol/L. Moreover, it exhibited excellent specificity and anti-interference capacity, enabling the successful detection of miR-222 in blood samples. This "DNAzyme-SDA-CRISPR" fluorescence strategy offers a effective, programmability and scalable solution for detecting underexpressed miRNAs in early cancer screening, which is expected to become a powerful tool in early tumor diagnostics and precision therapy.},
}
@article {pmid41073016,
year = {2025},
author = {Nie, Y and Wang, W and Wang, N and Yuan, M and Huang, L and Sun, Y and Li, K and Liu, Z and Mu, Y},
title = {PCR-CRISPR/Cas12a-based fluorescence and lateral flow dipstick platforms for efficient screening of CD71 biallelic mutants.},
journal = {Analytica chimica acta},
volume = {1376},
number = {},
pages = {344622},
doi = {10.1016/j.aca.2025.344622},
pmid = {41073016},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Receptors, Transferrin/genetics ; *Polymerase Chain Reaction/methods ; *Mutation ; *Antigens, CD/genetics ; *Alleles ; Fluorescence ; *CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas9 technology plays a pivotal role in gene editing and has been extensively utilized in gene function studies, disease modeling, and gene therapy. However, efficient and accurate detection of CRISPR/Cas9-induced mutants remains a challenge due to the complexity, time-consuming nature, and high cost of existing detection methods. Meanwhile, CRISPR/Cas12a systems have been widely applied in molecular diagnostics due to the non-specific trans-cleavage activity of Cas12a, yet their application in detecting CRISPR/Cas9-induced mutations remains limited. In this study, we developed a PCR-CRISPR/Cas12a-based method to enable the rapid and accurate screening of CD71 biallelic mutants. The detection system was mainly composed of CRISPR RNA specific to the CD71 gene-editing site, Cas12a protein, target DNA, and ssDNA probes for fluorescence or lateral flow dipstick assays. The system demonstrated high specificity in distinguishing CD71 biallelic mutants, with validation through TA cloning confirming its accuracy. Additionally, the method exhibited high sensitivity, establishing it as an efficient tool for biallelic mutated cell clone screening. These findings underscore the potential of PCR-CRISPR/Cas12a as a rapid, sensitive, and cost-effective approach for the precise identification of biallelic mutants, contributing to advancements in gene-editing research and molecular diagnostics.},
}
@article {pmid41073413,
year = {2025},
author = {Zhang, H and Li, M and Wang, G and Zhu, K and Guo, G and Fu, H and Hu, C and Chu, Z and Hu, J and Wu, Q and Chen, Y and Qiu, D and Xie, J and Li, D and Li, B and Li, W and Dong, L and Hou, Y and Cui, X and Huang, B and Liu, Y and Li, Y and Li, H and Yuan, C and Dong, L and Liu, Z and Lu, P},
title = {Paired NLRs originated from Triticum dicoccoides coordinately confer resistance to powdery mildew in wheat.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9040},
pmid = {41073413},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Ascomycota/physiology/pathogenicity ; *Plant Proteins/genetics/metabolism ; *NLR Proteins/genetics/metabolism ; Haplotypes ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Wheat has evolved diverse resistance genes against powdery mildew, typically controlled by single-gene-encoded proteins. Here, we report the map-based cloning of PmWR183, a resistance locus encoding two adjacent NLR proteins (PmWR183-NLR1 and PmWR183-NLR2) from wild emmer wheat. Stable transformation and CRISPR/Cas9 knockout experiments demonstrate that the two NLRs function cooperatively: neither gene alone confers resistance, but their co-expression restores immunity, while disruption of either gene abolishes resistance. PmWR183 mediates a developmental stage-dependent response, with susceptibility at the seedling stage and strong resistance at the adult stage. Protein interaction assays reveal constitutive association of PmWR183-NLR1 and PmWR183-NLR2, supporting their cooperative role. Geographical and haplotype analyses show the locus originates from wild emmer and is rare in cultivated wheat, exhibiting at least nine haplotypes. Together, our findings uncover a rare NLR gene pair conferring effective resistance to powdery mildew, providing valuable resources for wheat breeding.},
}
@article {pmid41073543,
year = {2025},
author = {Shi, Z and Cheng, TL},
title = {UGI relocation inside Cas9 reduces Cas9 dependent off target effects in cytosine base editors.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {35518},
pmid = {41073543},
issn = {2045-2322},
support = {20ZR1403100//Shanghai Natural Science Foundation/ ; },
mesh = {*Gene Editing/methods ; *Cytosine/metabolism ; *CRISPR-Associated Protein 9/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Uracil-DNA Glycosidase/antagonists &amp; inhibitors/metabolism ; Humans ; HEK293 Cells ; },
abstract = {Cytosine base editors (CBEs) achieve precise C-to-T conversions by addition of uracil DNA glycosylase inhibitor (UGI) with Cas9 nickase (nCas9) and cytidine deaminase, and the conventional fusion at the nCas9 carboxyl terminus effectively inhibits uracil excision repair to enhance editing efficiency. However, despite potent on-target activity, classical CBEs exhibit significant Cas9-dependent DNA off-target effects that necessitate optimization for future applications. Here we present a strategic UGI relocation through internal fusion within the nCas9 architecture. This spatial reorganization maintains comparable on-target editing efficiency while substantially reducing Cas9-dependent DNA off-target activity. Our findings establish an alternative engineering paradigm to develop high-fidelity CBEs, offering an improved platform for widespread genome editing applications.},
}
@article {pmid41073589,
year = {2025},
author = {Das, A and Debnath, S and Pramanik, S and Monshi, FI and Rahimi, M},
title = {Bio-digital feedback loop systems: a synergistic integration of predictive genomics, genome editing, and AI-driven phenomic synthesis for next-generation edible and medicinal mushroom breeding.},
journal = {Antonie van Leeuwenhoek},
volume = {118},
number = {11},
pages = {168},
pmid = {41073589},
issn = {1572-9699},
mesh = {*Gene Editing/methods ; *Genomics/methods ; *Agaricales/genetics ; *Artificial Intelligence ; CRISPR-Cas Systems ; *Phenomics/methods ; *Breeding/methods ; },
abstract = {Edible mushrooms face persistent challenges in yield optimization, bioactive compound production, and climate resilience that conventional breeding methods struggle to address. Traditional approaches such as cross-breeding, protoplast fusion, and mutagenesis are limited by genetic noise, laborious screening, and unstable trait inheritance. This review proposes a transformative paradigm built upon converging advances in molecular biology and data science: the bio-digital feedback loop (BDFL) framework, integrating multi-omics, CRISPR-engineered chassis strains, and predictive phenomics for precision mushroom breeding. Our framework employs multi-omics to decipher gene networks governing critical traits, such as substrate degradation enzymes, developmental synchrony regulators, and secondary metabolite pathways. CRISPR-Cas9 and synthetic biology tools then deploy these insights to verify and design modular gene circuits in pre-engineered "plug-and-play" chassis strains, enabling conflict-free stacking of desirable traits. Artificial intelligence serves as the linchpin, not only automating high-throughput phenotyping through advanced imaging but also accelerating the entire breeding cycle by predicting trait heritability from omics data and optimizing the design of CRISPR guide RNAs and genetic constructs for efficient editing. The BDFL we describe iteratively refines strains by feeding phenomics data back into AI algorithms, enabling rapid trait optimization cycles. This transcends the trial-and-error limitations of classical methods, accelerating development of climate-smart mushrooms for circular bioeconomies including strains engineered to thrive on agricultural waste, overproduce immunomodulatory compounds, or resist emerging pathogens. The integration of predictive genomics, AI-driven phenomics, and CRISPR-edited chassis strains heralds a new era of precision mycology, where mushrooms are computationally designed as sustainable solutions for global food security, pharmaceutical innovation, and ecological resilience, ultimately transforming fungi into programmable biological factories tailored to address pressing agricultural and ecological challenges.},
}
@article {pmid41074026,
year = {2025},
author = {Frey, T and Kandolf-Zumpf, C and Kaempf, A and Schaffer, K and Hollenstein, M and Lampl, A and Kovarik, JJ and Strobl, J and Stary, G and Eckl-Dorna, J and Schmidt, R and Schmetterer, KG},
title = {T cell receptor associated transmembrane adaptor 1 (TRAT1) modulates human Th17 and Treg responses via PI3-kinase and STAT dependent mechanisms.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {431},
pmid = {41074026},
issn = {1478-811X},
support = {P34728-B//Austrian Science Funds/ ; P34728-B//Austrian Science Funds/ ; P34728-B//Austrian Science Funds/ ; },
mesh = {Humans ; *T-Lymphocytes, Regulatory/metabolism/immunology/cytology ; *Th17 Cells/metabolism/immunology/cytology ; Signal Transduction ; *Phosphatidylinositol 3-Kinases/metabolism ; *STAT Transcription Factors/metabolism ; *Adaptor Proteins, Signal Transducing/metabolism ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Adaptor proteins associated with the T cell receptor (TCR) play critical roles in regulating immune responses by Translating receptor engagement into intracellular signals. T cell Receptor Associated Transmembrane Adaptor 1 (TRAT1) has been implicated in modulating TCR complex stability, but its functional role in human effector and regulatory CD4[+] T cell subsets remains poorly understood. This study aimed to elucidate the role of TRAT1 in regulating T cell activation and differentiation, particularly in helper T cells function and regulatory T cells.<br><br>METHODS: Primary human CD4&#x207a; T cells, including thymus-derived and induced regulatory T cells (Treg), were genetically modified by CRISPR/Cas9-mediated gene deletion or retro-/lentiviral overexpression of TRAT1. Functional assays, flow cytometry, cytokine quantification, and RNA sequencing were performed to evaluate modulation of T cell functions. Mechanistic studies included pathway inhibition using small molecules and phospho-protein analysis. The influence of TRAT1 on Treg function was further assessed in a CAR Treg context in an immune organoid model of allo-rejection.<br><br>RESULTS: Thymus-derived, TGFb-induced and FOXP3-transgenic Treg displayed reduced expression of TRAT1 compared to effector T cells, which showed pronounced up-regulation of TRAT1 following activation. In effector T cells, deletion of TRAT1 led to increased signaling through the phosphoinositide 3-kinase pathway resulting in enhanced proliferation and increased expression of activation markers. However, this was accompanied by reduced production of interleukin-17, which was linked to elevated activity of STAT6 as shown by inhibition experiments using small molecule inhibitors. Overexpression and CRISPR/Cas9-mediated knockout of TRAT1 in Treg enhanced suppression of CD4&#x207a; target cells via up-regulation of LAP/GARP but reduced suppression of CD8&#x207a; target cells, an effect confirmed in HLA-A2-specific CAR Treg in a human organoid model of allo-rejection.<br><br>CONCLUSIONS: TRAT1 acts as a dual regulator of human CD4&#x207a; T cell function, limiting effector activation through modulation of intracellular signaling and supporting regulatory T cell-mediated suppression. These findings reveal a novel mechanism of immune regulation with potential implications for the development of cell-based immunotherapies.},
}
@article {pmid41074741,
year = {2025},
author = {Karmakar, A and Hota, A and Tanga, S and Kumar, V and Das, P and S, AE and Thapa, M and Maji, B},
title = {Engineered Thermostable Chemically Responsive GlowCas9 System for Real-Time Therapeutic Monitoring Applications.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {64},
number = {49},
pages = {e202511707},
doi = {10.1002/anie.202511707},
pmid = {41074741},
issn = {1521-3773},
mesh = {*Gene Editing ; *CRISPR-Cas Systems ; Humans ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Animals ; Temperature ; *Protein Engineering ; Genetic Therapy ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has revolutionized gene therapy applications due to its ease of design and efficiency, albeit accompanied by off-target effects. Spatiotemporally regulated Cas9 offers safer gene editing methods due to its lower off-target mediated genotoxicities; however, probing the Cas9 in real time for assessing the delivery efficiency, gene editing timeframe, and spatial target information remains elusive. Here, we report an engineered Cas9 system, GlowCas9, that can be probed chemically in real-time across all assay systems, including cell lysate, native gel, ex vivo, and in vivo. Importantly, we rationally engineered the Cas9 system to attain remarkable thermostability with enhanced gene editing activities. Besides its sensitive reporter and enhanced gene editing activity, the GlowCas9 system exhibits precision HDR-based gene knock-in ability in cells and significantly outperformed the WTCas9. Overall, we report a new thermostable engineered SpCas9 system for real-time theratracking applications compatible with diverse assay formats, including live ex vivo and in vivo. This GlowCas9 system will add a new dimension of non-invasive real-time tracking in gene therapy development, both ex vivo and in vivo.},
}
@article {pmid41074985,
year = {2025},
author = {Gupta, I and Sharma, JG and Kaul, T},
title = {Nanoparticle-driven CRISPR-Cas9 genome editing: a new frontier in crop improvement.},
journal = {Molecular biology reports},
volume = {52},
number = {1},
pages = {1015},
pmid = {41074985},
issn = {1573-4978},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Crops, Agricultural/genetics ; *Nanoparticles/chemistry ; Genome, Plant/genetics ; Plants, Genetically Modified/genetics ; Nanotechnology/methods ; Genetic Engineering/methods ; Agriculture/methods ; },
abstract = {The agricultural sector has experienced unpredictable and extreme climatic aberrations, which have severely hampered food production. However, applying advanced nanotechnological approaches in agriculture will be crucial for ensuring more secure and sustainable food production. The revolutionizing phyto-nanotechnology enables the precise delivery of biomolecules i.e., nucleotides and proteins, and the modulated release of agrochemicals, including fungicides and pesticides. In addition, CRISPR-Cas-based genetic engineering holds great promise for food security, agriculture, and environmental sustainability. However, its application in plants faces challenges, including cargo delivery, germline transformation, species independence, HDR efficiency, and overall editing effectiveness. Nanomaterials offer innovative and effective solutions to overcome these challenges by enhancing genome-editing tools precision, efficiency, and delivery mechanisms. This review examines the key limitations of CRISPR-mediated plant genome editing and how nanoparticle technologies can overcome them. We highlight essential nanotech innovations that enhance genome modification, paving the way for a faster, more versatile genomic toolbox in plant biotechnology.},
}
@article {pmid41075360,
year = {2026},
author = {Rahimi, M and Schreiber, M and Habermann, H and Haake, L and Miskel, D and Tesfaye, D and Held-Hoelker, E and Brenig, B and Benedetti, C and Hoelker, M},
title = {Efficient genome editing of bovine in vitro derived zygotes via Cas9 RNP-electroporation using extended-stored bovine ovaries.},
journal = {Theriogenology},
volume = {250},
number = {},
pages = {117703},
doi = {10.1016/j.theriogenology.2025.117703},
pmid = {41075360},
issn = {1879-3231},
mesh = {Animals ; Cattle/embryology ; Female ; *Gene Editing/veterinary/methods ; *Electroporation/veterinary/methods ; *Zygote/physiology ; *Ovary/physiology ; *CRISPR-Cas Systems ; Fertilization in Vitro/veterinary ; CRISPR-Associated Protein 9 ; Ribonucleoproteins/genetics ; },
abstract = {Bovine embryos serve as a relevant model for human preimplantation development and are increasingly used in genome editing research. Although CRISPR-Cas9 electroporation in zygotes offers a promising approach, challenges with respect to editing efficiency and mosaicism remain a challenge. However, traditional in vitro fertilization (IVF) workflows enforce highly inconvenient electroporation times of zygotes usually around midnight. This study therefore aimed to investigate the feasibility of using bovine zygotes derived from extended-stored slaughterhouse derived ovaries (Phosphate buffered saline, 14-18 °C, 20-22 h) with particular attention to the strength of the voltage field during electroporation and the Cas9-RNP concentration with respect to embryonic development and genome editing efficiency. Oocytes obtained under these conditions demonstrated in vitro developmental competence similar to those derived from fresh ovaries, with comparable cleavage and blastocyst formation rates. Electroporation conducted 10 h post-insemination at voltages of 15V, 20V, and 25V revealed that 25V yielded the highest blastocyst editing rate (40.7 %) while maintaining acceptable developmental rates. Increasing voltage to 30V and 35V further enhanced blastocyst editing rate (66.7 % and 67.9 %, respectively), with a greater proportion of blastocyst showing a full edit. While higher voltages reduced developmental rates, the optimal voltage for electroporation was found to be 30V for bovine zygotes derived from extended stored ovaries. Using that setting, contrasting Cas9-RNP concentrations (6 μM vs. 3 μM) were tested. A concentration of 6 μM resulted in higher editing rates compared to 3 μM (81.5 % vs. 60.0 %), although the distribution of monoallelic, biallelic, and mosaic modifications did not differ significantly. Taken together, this study confirmed for the first time that zygotes from extend-stored bovine ovaries are suitable for genome editing via CRISPR Cas9-RNP electroporation. Mosaicism, however, remained prevalent, underscoring the need for strategies to improve the precision and consistency of embryo editing.},
}
@article {pmid41075867,
year = {2025},
author = {Zhang, D and Liu, H and Zhong, Y},
title = {Monoclonal antibodies production in microbial systems: Current status, challenges and perspectives.},
journal = {New biotechnology},
volume = {90},
number = {},
pages = {163-173},
doi = {10.1016/j.nbt.2025.10.005},
pmid = {41075867},
issn = {1876-4347},
mesh = {*Antibodies, Monoclonal/biosynthesis/genetics ; *Metabolic Engineering ; Humans ; CRISPR-Cas Systems ; Animals ; Gene Editing ; Synthetic Biology ; },
abstract = {Monoclonal antibodies (mAbs) serve as indispensable tools in diagnostics, clinical therapeutics, and biomedical research. However, their large-scale production faces significant challenges due to the high costs and lengthy timelines associated with conventional mammalian cell-based expression systems. Microbial expression platforms have emerged as a transformative alternative, offering cost-effectiveness, rapid cultivation cycles, and superior genetic tractability for industrial-scale monoclonal antibodies production. Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9-mediated gene editing enable precise metabolic engineering of host strains to enhance protein folding, secretion efficiency, and translational accuracy. Synthetic biology approaches facilities the reconstruction of mammalian glycosylation pathways in microbial systems, yielding monoclonal antibodies with near-native structural integrity. Furthermore, AI (artificial intelligence)-driven optimization of expression vectors, promoter systems, and culture conditions, combined with high-throughput screening of engineered strains, significantly accelerates the identification of high-yield production clones. This review comprehensively examines current progress in microbial expression systems, strain engineering strategies, and fermentation optimization for enhanced monoclonal antibodies production, while critically discussing existing limitations and potential solutions to advance the field.},
}
@article {pmid41075940,
year = {2026},
author = {Zhang, J and Dai, P and Weng, Z and Xu, R and Li, Y and Liu, X and Lei, J},
title = {Efficient CRISPR/Cas-based gene editing in cotton induced by cotton leaf crumple virus.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {33-43},
doi = {10.1016/j.jbiotec.2025.10.001},
pmid = {41075940},
issn = {1873-4863},
mesh = {*Gossypium/genetics/virology ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Luteoviridae/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Plant viral vectors can replicate autonomously and spread within host cells, making them an ideal tool for the delivery of CRISPR/Cas gene-editing elements. Here, we constructed a cotton CRISPR/Cas system mediated by cotton leaf crumple virus (CLCrV) as a delivery vector. We first inoculated Pro35s::Cas9 and ProUbi::Cas9 cotton with sgRNAs designed to knock out GhAGL16, GhPDS, and GhCLA1 target genes via the CLCrV vector and then compared the effects of these two transformation receptors on the editing efficiency of the same target genes. We next explored the feasibility of simultaneous multi-target editing in cotton via pooled virus inoculation. Finally, we used a cotton line overexpressing nCas9-TadA7.10 as the transformation receptor to explore the feasibility of CLCrV-mediated adenine base editing and verify the specificity of gene editing in this system. Mutation detection and deep sequencing revealed that the Pro35s::Cas9 and ProUbi::Cas9 cotton lines did not differ significantly in editing efficiency, and both could be used as successful receptors for the CLCrV-mediated Cas9 system. Pooled inoculation with CLCrV-sgRNAs enabled the simultaneous editing of multiple target genes in Pro35s::Cas9 and ProUbi::Cas9 cotton, although this approach had somewhat lower editing efficiency than inoculation with single sgRNAs. The CLCrV-mediated adenine base-editing system enabled A-to-G conversion at target sites in cotton GhPEBP and showed high gene-editing specificity. In summary, this study establishes an efficient CLCrV-mediated CRISPR system in cotton, providing a powerful technical tool for editing of multiple target genes and base editing.},
}
@article {pmid41076478,
year = {2025},
author = {Wu, M and Wang, F and Wang, Y and Wu, YX and Tian, BY and Ou, XY and Xu, QC and Wu, XY and Han, C and Liu, WL and Xing, S},
title = {Mismatch-introduced crRNA guided PCR-CRISPR/Cas12a platform improves EGFR point mutation detection in single tumor cell.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {727},
pmid = {41076478},
issn = {1436-5073},
support = {2021B1515230006//the Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; ErbB Receptors/genetics ; *Point Mutation ; *CRISPR-Cas Systems ; *Polymerase Chain Reaction/methods ; *Single-Cell Analysis/methods ; Cell Line, Tumor ; Neoplastic Cells, Circulating ; DNA Mutational Analysis/methods ; Base Pair Mismatch ; },
abstract = {Dynamic monitoring of epidermal growth factor receptor (EGFR) mutations is essential for the early identification of resistance and treatment adaptation. Single-cell heterogeneity analysis is crucial for precision cancer medicine, yet sensitive and specific detection methods for individual tumor cells remain challenging. Here, we develop a PCR-CRISPR/Cas12a platform enhanced by the incorporation of mismatched base in crRNA at specific site for single-cell point mutation detection. This platform demonstrated high specificity and sensitivity, detecting point mutation at a frequency of 0.1% and in as low as 1.02 ng of genomic DNA, which represents an improvement over the amplification-refractory mutation system PCR (ARMS-PCR). Notably, the accuracy of the platform is highly consistent with next-generation sequencing (NGS), as evidenced by Kappa test values surpassing 0.9. By utilizing a conical-pore membrane with optimized porosity for single circulating tumor cell (CTC) enrichment, our platform enables point mutations detection in individual tumor cells, offering potential enhancements in precision and reliability for EGFR mutation analysis. This novel methodology holds potential for more accurate and personalized cancer treatment strategies.},
}
@article {pmid41077083,
year = {2025},
author = {Luo, J and Lu, Y and Zhang, Z and Zhang, Q and Shang, Y and Yuan, Y and Duan, X and Zhang, L and Tang, Y and Hu, H and Tong, X and Dai, F},
title = {A conserved Piezo-mRPL2/Jun axis controls reproduction via mitochondrial bioenergetics.},
journal = {Insect biochemistry and molecular biology},
volume = {185},
number = {},
pages = {104417},
doi = {10.1016/j.ibmb.2025.104417},
pmid = {41077083},
issn = {1879-0240},
mesh = {*Mitochondria/physiology ; *Mechanotransduction, Cellular/physiology ; Bombyx ; CRISPR-Cas Systems ; Oxidative Stress ; Phosphorylation ; Transgenes ; Male ; Animals ; Ion Channels ; *Energy Metabolism ; Mice ; *Ribosomal Proteins/metabolism ; *Mitochondrial Proteins/metabolism ; Insect Proteins/metabolism ; Fertility ; },
abstract = {Piezo channels are essential mechanosensors, but their roles in reproduction remain poorly understood. Here, we identify a critical function for BmPiezo in silkworm fertility and embryonic diapause, mediated through a conserved downstream regulatory axis. CRISPR/Cas9-mediated knockout of BmPiezo resulted in male sterility and loss of embryonic diapause, accompanied by dysregulation of ribosome-associated genes and oxidative phosphorylation, and reduced ATP production. We identified mitochondrial ribosomal protein L2 (BmmRPL2) and transcription factor BmJun as key downstream effectors, required for spermatogenesis and diapause, respectively. Homozygous disruption of either gene caused embryonic lethality. Cross-species expression analysis revealed strong expression of Piezo2 and mRPL2 in early human spermatids. Remarkably, transgenic expression of murine Piezo2 in BmPiezo-/-silkworms reactivated the Piezo-mRPL2/Jun axis, restored ATP levels, and rescued reproductive defects. These findings define a conserved pathway that connects mechanical sensing to mitochondrial bioenergetics in reproductive regulation, advancing our understanding of fertility mechanisms across species.},
}
@article {pmid41078118,
year = {2026},
author = {Wang, Y and Yang, J and Hou, H and Song, L and Cheng, X and Liu, YX},
title = {Advancing Plant Microbiome Research Through Host DNA Depletion Techniques.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1189-1203},
pmid = {41078118},
issn = {1467-7652},
support = {32470055//National Natural Science Foundation of China/ ; U23A20148//National Natural Science Foundation of China/ ; CAAS-BRC-CB-2025-01//Basic Research Center for Crop Biosafety Sciences/ ; CAAS-ZDRW202308//Agricultural Science and Technology Innovation Program/ ; },
mesh = {*Microbiota/genetics ; *Plants/microbiology/genetics ; *Metagenomics/methods ; *DNA, Plant/genetics/isolation &amp; purification ; CRISPR-Cas Systems ; },
abstract = {Plants provide ecological habitats for diverse microorganisms, making accurate metagenomic sequencing essential for understanding the complex interactions that support plant growth, development and disease resistance. However, host DNA contamination poses a major challenge in plant microbiome studies, obscuring microbial genetic signatures and complicating the accurate analysis of microbial genomes. This review provides a comprehensive overview of current host DNA depletion strategies, including physical separation (e.g., filtration, gradient centrifugation), selective lysis and enzymatic treatments targeting plant cell walls. Advanced techniques such as targeted sequence capture with magnetic beads, methylation-based enrichment and nanopore selective sequencing offer additional options for host DNA removal. Despite these advances, current methods still face challenges in efficiency, specificity and applicability, emphasising the need for tailored strategies and the exploration of novel approaches for microbial enrichment. Innovations like CRISPR-Cas9 and chromatin immunoprecipitation-based host DNA depletion methods are proposed to provide novel directions for addressing current limitations. The development and refinement of host depletion techniques tailored to plant systems are crucial for enabling high-resolution, cost-effective metagenomic studies. These efforts promise to deepen our understanding of microbial diversity and functionality, ultimately accelerating microbiome-based innovations in crop improvement, sustainable agriculture and ecosystem resilience.},
}
@article {pmid41080519,
year = {2025},
author = {Chaturvedi, A and Ranjan, R},
title = {Strategies for plant-virus disease management from gene editing to nanotechnology.},
journal = {Physiology and molecular biology of plants : an international journal of functional plant biology},
volume = {31},
number = {8},
pages = {1293-1308},
pmid = {41080519},
issn = {0971-5894},
abstract = {Plant viruses are a global agricultural threat and can result in large financial losses. The globalization of agriculture and its international trading are the major causes of viruses and their vectors expanding to new environmental niches. Conventional methods are not effective in managing virus infection. To mitigate the virus spread, one of the cutting-edge biotechnological approaches, CRISPR/Cas is a robust tool. CRISPR/Cas is a powerful genome editing technology, and provides a highly specific viral genome targeting. Additionally, nanotechnology is a cutting-edge method for mitigating plant viruses. Nanoparticles in biosensors aid in the early identification of plant viruses, hence preventing the spread of disease in the future. Moreover, nanoparticles can also be used as a flexible delivery system. Nanoparticle-mediated delivery of dsRNA ensures minimal off-target while maintaining biosafety. This review explores the genome editing approach and nanotechnological strategies for ensuring sustainable agriculture practices for virus disease management, focusing on biosafety, efficacy, and practical applicability. It also aims to provide a clear insight into the limitations and strengths of each approach.},
}
@article {pmid41081480,
year = {2025},
author = {Ma, F and Zheng, Q and Sun, Y and Zhang, N and Xu, W},
title = {Toward Tissue-Free Plant Engineering: Emerging Platforms for Sustainable Horticultural Transformation.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {42},
pages = {26457-26471},
doi = {10.1021/acs.jafc.5c06803},
pmid = {41081480},
issn = {1520-5118},
mesh = {*Plants, Genetically Modified/genetics/growth &amp; development/metabolism ; *Crops, Agricultural/genetics/growth &amp; development ; Transformation, Genetic ; *Genetic Engineering/methods ; Agrobacterium/genetics ; },
abstract = {Genetic transformation in horticultural crops is being reshaped by the emergence of nontissue culture technologies that bypass entrenched barriers of genotype dependence, regeneration inefficiency, and sterile culture requirements. This review surveys recent in planta methods, including regenerative activity-dependent in Planta injection delivery (RAPID), cut-dip-budding (CDB), virus-based delivery, nanoparticle-mediated transformation, and Agrobacterium rhizogenes-induced regeneration, and evaluates their operational versatility across species. We further examine their integration with developmental regulators (BABY BOOM [BBM], WUSCHEL [WUS]), visual markers (RUBY), and CRISPR/Cas systems to enhance transformation efficiency and precision. Case studies across fruit, vegetable, and ornamental crops illustrate broad applicability and growing technical maturity. Despite these advances, unresolved challenges in biosafety, reproducibility, and regulatory alignment remain. We advocate a new transformation paradigm that is rapid, genotype-independent, and environmentally compatible, enabling scalable and more accessible broadly applicable crop improvement in horticultural biotechnology.},
}
@article {pmid41081521,
year = {2025},
author = {Wu, H and Lin, X and Tian, C and Xiong, D},
title = {Establishment of an RAA-CRISPR/Cas12a assay based on CpSge1 for rapid detection of Cryphonectria parasitica.},
journal = {Microbiology spectrum},
volume = {13},
number = {11},
pages = {e0107925},
pmid = {41081521},
issn = {2165-0497},
support = {2023YFD1401301//National Key Research and Development Program/ ; },
mesh = {*Ascomycota/genetics/isolation &amp; purification ; *Plant Diseases/microbiology ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Fagaceae/microbiology ; Fungal Proteins/genetics ; Recombinases/metabolism/genetics ; China ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Chestnut blight disease caused by Cryphonectria parasitica is a serious branch disease that occurs worldwide, especially in Europe and North America. In recent years, chestnut blight disease has also been severe and even posed a great threat to the healthy development of chestnut orchards in some areas of China. Accurate and rapid detection of C. parasitica during the initial stages of the disease is helpful to take corresponding prevention and control measures in advance. In this study, we selected the CpSge1 (Gti1/Pac2 transcription factor family) of C. parasitica as the detection target and established a rapid and visual detection system of C. parasitica that combined the recombinase-aided amplification (RAA) and CRISPR/Cas12a, called CpSge1-RAA-CRISPR/Cas12a. The system allows for the specific detection of C. parasitica in approximately 60 mins, with visualization of results. The detection sensitivity of this system was found to be 1 pg/µL. We combined the RAA-CRISPR/Cas12a with a lateral flow dipstick, which also showed specific, high sensitivity, and fast characters. In conclusion, the RAA-CRISPR/Cas12a assay has great potential to be a method for early diagnosis and on-site detection of C. parasitica, especially for areas where specialized equipment is lacking.IMPORTANCEA rapid, highly sensitive, and visualized detection system of Cryphonectria parasitica was established by using the RAA-CRISPR/Cas12a method based on the C-terminal variable regions of a fungal-specific transcription factor CpSge1. The detection system was performed at a constant temperature condition of 37°C, which provides important support for the diagnosis of chestnut blight diseases in the field.},
}
@article {pmid41081710,
year = {2025},
author = {Serreze, DV and Tousey-Pfarrer, M and Racine, JJ},
title = {Humanized Mouse Models for Type 1 Diabetes.},
journal = {Current protocols},
volume = {5},
number = {10},
pages = {e70224},
pmid = {41081710},
issn = {2691-1299},
support = {R01 DK095735/DK/NIDDK NIH HHS/United States ; R01 DK136472/DK/NIDDK NIH HHS/United States ; },
mesh = {*Diabetes Mellitus, Type 1/immunology/genetics ; Animals ; *Disease Models, Animal ; Mice, Inbred NOD ; Humans ; Mice ; CRISPR-Cas Systems ; },
abstract = {T cell-mediated autoimmune type 1 diabetes (T1D) is under complex polygenic control in both humans and the NOD mouse model. However, in both species, particular major histocompatibility complex (MHC; designated HLA in humans) haplotypes provide the primary T1D risk factor. Both MHC/HLA class I and II variants interactively contribute to T1D by respectively driving autoreactive CD8 and CD4 T cell responses that cooperatively destroy insulin-producing pancreatic β cells. While NOD mice have provided important insights to the pathogenic basis of T1D, the model has so far provided only a limited means to identify possible clinically translatable disease intervention approaches. This highlights a need to humanize NOD mice in ways that their pathogenic basis of T1D development becomes more similar to that characterizing the disease course in patients. In this review, we discuss the use of CRISPR/Cas9-generated murine-MHC-deficient NOD mice as a platform for introduction of patient-relevant HLA and T cell receptor molecules. These mice provide ever-improving models for development of clinically applicable interventions for T1D and other autoimmune diseases. © 2025 The Author(s) Current Protocols published by Wiley Periodicals LLC.},
}
@article {pmid41081763,
year = {2025},
author = {Wang, X and Feng, S and Chen, H and Zhou, B and Fan, T and Qin, Y and Zhao, L and Jiang, Y and Chen, Y},
title = {Development of an Aptamer/CRISPR-Cas12a-Based Dual-Modal Biosensor for Fusobacterium nucleatum Detection in Non-Invasive Colorectal Cancer Screening.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23360-23369},
doi = {10.1021/acs.analchem.5c04132},
pmid = {41081763},
issn = {1520-6882},
mesh = {*Fusobacterium nucleatum/isolation &amp; purification ; *Colorectal Neoplasms/diagnosis/microbiology ; *Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Aptamers, Nucleotide/chemistry ; *Early Detection of Cancer/methods ; Feces/microbiology ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Colorectal cancer (CRC) is the third most common cancer and leading cause of cancer-related deaths worldwide. However, current CRC screening methods are complex, invasive, and tend to exhibit low sensitivity. Recent evidence has highlighted gut microbiota dysbiosis, especially elevated Fusobacterium nucleatum levels, as a promising biomarker for CRC. In this study, a sensitive and specific detection platform was developed for F. nucleatum by combining a highly specific aptamer with rolling circle amplification (RCA) and the CRISPR/Cas12a technology. The aptamer enables specific target recognition, while RCA amplifies the target signal, and the Cas12a-mediated cleavage of a fluorescence-quenching substrate generates a quantifiable fluorescence or grayscale signal. Using a microplate reader, this assay achieved a limit of detection (LOD) of 3.68 CFU/mL; furthermore, by incorporating smartphone-assisted ImageJ grayscale analysis, it elevated the LOD to 4.30 CFU/mL, thereby enabling a dual-mode output along with on-site applicability. Additionally, the strong correlation between the two signals allowed for mutual validation. Upon application to clinical fecal samples, the developed method sensitively distinguished CRC patients from healthy controls, and its results correlated with the quantitative polymerase chain reaction results. This triple-synergistic platform, integrating aptamer specificity, RCA amplification, and CRISPR/Cas12a sensitivity, enables the noninvasive, ultrasensitive detection of F. nucleatum, supporting early CRC screening, prognosis monitoring, and microbiome-targeted therapy. Moreover, this approach overcomes the challenges of detecting low-abundance bacteria in early stage CRC and advances the precision of microbiome-based diagnostics for CRC.},
}
@article {pmid41082037,
year = {2025},
author = {Kim, HJ and Kwon, MY and Song, S and Cheon, SW and Kim, HJ},
title = {Engineered Lactiplantibacillus plantarum and Levilactobacillus brevis utilizing ribonucleoprotein-mediated editing for inactivation of hemolysin gene.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {373},
pmid = {41082037},
issn = {1573-0972},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/metabolism ; *Hemolysin Proteins/genetics ; Probiotics ; Bacterial Proteins/genetics ; Plasmids/genetics ; *Lactiplantibacillus plantarum/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Lactobacillus/genetics ; },
abstract = {Lactiplantibacillus plantarum and Levilactobacillus brevis are widely used probiotics with significant potential as chassis organisms for probiotic engineering. However, their bioengineering remains underdeveloped compared to that of other probiotic bacteria due to the limited availability of genetic tools. Although CRISPR-Cas systems have shown promise for genome editing in Lactobacillus species, strain- or site-specific targeting challenges must be overcome to enhance their broader applicability. This study aimed to develop a novel editing system with reduced dependency on plasmids and antibiotics in L. plantarum WCFS1, L. plantarum SPC 72 - 1 and L. brevis SPC-SNU 70 - 2 using a Cas9-gRNA ribonucleoprotein (RNP) complex. Although the hlyIII gene has been annotated as a hemolysin-related gene in several Lactobacillus genomes, no functional hemolytic activity has been definitively demonstrated to date. In this study, hlyIII was selected as a target to evaluate genome editing efficiency and to assess its potential relevance to strain safety. To construct ΔhlyIII strains, the RNP complex targeting hlyIII was separately transformed with recombinase RecE/T and double-stranded donor DNA. As a result, ΔhlyIII mutants were obtained under optimized electroporation conditions. Sequencing analysis revealed a 50 bp deletion and the introduction of a stop codon in hlyIII across all mutant strains. The hemolytic activity test showed a reduction in free hemoglobin levels in the ΔhlyIII strains compared to the wild type: 27.0%, 74.3%, and 5.0% in L. plantarum WCFS1, L. plantarum SPC 72 - 1, and L. brevis SPC-SNU 70 - 2, respectively. These results suggest strain-dependent differences in hemolytic activity and indicate that inactivation of hlyIII may contribute to reduced hemolysis, although further validation is needed to clarify its functional role. In conclusion, the hlyIII gene was successfully edited in L. plantarum and L. brevis using Cas9-gRNA ribonucleoprotein-mediated editing, demonstrating the feasibility of this genome editing platform for application in probiotic strains.},
}
@article {pmid41082119,
year = {2026},
author = {Chear, S and Chiam, A and Talbot, J and Thorne, BN and Wilkinson, EJ and Hewitt, AW and Cook, AL},
title = {Generation of Donor-Specific iPSC for Modelling Lysosomal Storage Disorders.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2976},
number = {},
pages = {151-173},
pmid = {41082119},
issn = {1940-6029},
mesh = {Humans ; *Lysosomal Storage Diseases/pathology/genetics/metabolism ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Fibroblasts/metabolism/cytology ; *Cell Culture Techniques/methods ; CRISPR-Cas Systems ; Cells, Cultured ; Tissue Donors ; },
abstract = {iPSC technology has enabled the generation of human cell-based models of lysosomal storage disorders and has provided disease-relevant systems to undertake drug discovery or pre-clinical testing of gene- or cell-based therapies. Here, we provide a protocol to generate iPSCs derived from people with lysosomal storage disorders and illustrate expected results using a CLN2 disease donor-specific skin fibroblast culture. Protocol steps include lipofection of episomal plasmids, picking of putative iPSC colonies following live cell TRA-1-60 immunofluorescence, and quality control steps such as immunofluorescence for expression of undifferentiated cell markers, germ layer differentiation, and confirmation of pathological variant genotype. The iPSC generated by this protocol can be differentiated to several cell lineages and can be used with CRISPR/Cas technology to generate isogenic disease models.},
}
@article {pmid41082121,
year = {2026},
author = {Kim, WD and Huber, RJ},
title = {Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2976},
number = {},
pages = {189-207},
pmid = {41082121},
issn = {1940-6029},
mesh = {*Dictyostelium/genetics/metabolism/enzymology ; *Lysosomes/enzymology/metabolism ; Protein Transport ; *Lysosomal Storage Diseases/genetics/metabolism/enzymology ; Gene Editing/methods ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Protozoan Proteins/metabolism/genetics ; },
abstract = {Non-mammalian models are powerful systems for enhancing our understanding of lysosomal function and lysosomal storage diseases. The social amoeba Dictyostelium discoideum is an excellent model organism for studying lysosomal function, as its genome encodes many proteins associated with lysosomal disease. Methods for gene knockout are straightforward in D. discoideum and include restriction enzyme-mediated integration (REMI) mutagenesis, homologous recombination via the Cre-loxP system, and CRISPR/Cas9-mediated gene editing, which collectively allow researchers to study protein function (e.g., lysosomal enzymes) in a genetically tractable biomedical model system. Additionally, activity assays for conserved lysosomal enzymes are well-established in D. discoideum. In this chapter, we outline methods for studying the intracellular localization and secretion of conserved lysosomal proteins in D. discoideum.},
}
@article {pmid41082405,
year = {2025},
author = {Yang, L and Fang, Y and Lian, Y and Kong, Z and Miao, J and Chen, Y and Li, W and Chen, F and Zhang, B and Chen, Y and Bian, Y},
title = {Aloe-Emodin Targeting FOXC2 Disrupts NETs Formation and EMT-Driven Postoperative Peritoneal Adhesion Through TGF-β1-Smad2/3 Pathway.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {48},
pages = {e11013},
pmid = {41082405},
issn = {2198-3844},
support = {81704084//National Natural Science Foundation of China/ ; 0121/2022/A3//Science and Technology Development Fund, Macau SAR/ ; 0127/2023/RIA2//Science and Technology Development Fund, Macau SAR/ ; SJCX24_1123//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; SJCX25_0992//Postgraduate Research &amp; Practice Innovation Program of Jiangsu Province/ ; //333 High-Level Talents Cultivation Project of Jiangsu Province/ ; },
mesh = {*Forkhead Transcription Factors/metabolism/genetics ; *Epithelial-Mesenchymal Transition/drug effects ; *Transforming Growth Factor beta1/metabolism ; Animals ; Humans ; Tissue Adhesions/metabolism ; Smad2 Protein/metabolism ; Signal Transduction/drug effects ; Smad3 Protein/metabolism ; *Extracellular Traps/metabolism/drug effects ; Mice ; Peritoneum/metabolism/pathology ; Rats ; Male ; *Postoperative Complications/metabolism ; },
abstract = {Postoperative peritoneal adhesion (PPA) develops through TGF-β1-driven fibrotic remodeling, characterized by neutrophil extracellular trap (NETs)-induced aberrant epithelial-to-mesenchymal transition (EMT) deposition. Although aloe-emodin (AE) exhibits anti-fibrosis potential, its molecular mechanisms remain elusive. Forkhead box protein C2 (FOXC2) is a critical regulator of fibrotic tissue formation, yet its role in PPA is unknown. Here, it is demonstrated that FOXC2 expression is elevated in human ileostomy tissue, PPA rodent model, and TGF-β1-exposed peritoneal mesothelial cells (PMCs), where it orchestrates NETs formation and extracellular matrix (ECM) remodeling. Mechanically, CRISPR/Cas-based knockdown and overexpression of FOXC2 alter EMT changes in PMCs, which is achieved via TGF-β1-Smad2/3 signaling. FOXC2 functions as a dual mediator and amplifier through the TGF-β1-Smad2/3 pathway feedback loop to drive EMT alterations. Its overexpression further induces neutrophil recruitment and NETs formation, exacerbating EMT in PMCs. Notably, AE ameliorates FOXC2-driven peritoneal fibrosis by impeding NETs formation and EMT changes through the TGF-β1-Smad2/3 pathway. Moreover, AE binds directly to FOXC2, and the Ser125 residue is critical for the binding of FOXC2 to AE. These findings identify FOXC2 as a pivotal effector in fibrotic responses during PPA formation and reveal that AE targeting the Ser125 residue of FOXC2 may be a promising therapeutic approach to attenuate PPA.},
}
@article {pmid41083470,
year = {2025},
author = {McCallion, O and Du, W and Glaser, V and Milward, K and Short, S and Bilici, M and Cross, A and Stark, H and Franke, C and Kath, J and Valkov, M and Yang, M and Amini, L and Künkele, A and Polansky, JK and Schmueck-Henneresse, M and Volk, HD and Reinke, P and Wagner, DL and Hester, J and Issa, F},
title = {HLA matching or CRISPR editing of HLA class I/II enables engraftment and effective function of allogeneic human regulatory T cell therapy in a humanized mouse transplantation model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9090},
pmid = {41083470},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; 211122/Z/18//Wellcome Trust (Wellcome)/ ; 825392//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; FS/ 12/72/29754//British Heart Foundation (BHF)/ ; },
mesh = {Animals ; Humans ; *T-Lymphocytes, Regulatory/immunology/transplantation ; Mice ; Skin Transplantation ; Gene Editing/methods ; Graft Survival/immunology ; CRISPR-Cas Systems ; Transplantation, Homologous ; *Histocompatibility Antigens Class I/genetics/immunology ; Adoptive Transfer ; Forkhead Transcription Factors/metabolism/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Regulatory T cells (Tregs) hold promise for treating autoimmune disease and transplant rejection, yet generation of autologous products for adoptive transfer can suffer donor variability and slow turnaround, limiting their use in urgent indications. We therefore examine whether allogeneic, pre-manufactured ('off-the-shelf') Tregs could overcome these barriers. In a human skin-xenograft model, HLA-mismatched Tregs are swiftly eliminated by recipient CD8[+] T cells and fail to protect grafts. Stringent matching of HLA class I and II restores efficacy but is clinically impractical. Using non-viral CRISPR editing we disrupt B2M and CIITA while inserting an HLA-E-B2M fusion, generating hypo-immunogenic Tregs that evade both T and NK cell attack. Engineered cells retain FOXP3 stability and potent in vitro suppression, and after a single low-dose infusion, prolong human skin graft survival in a humanized mouse model comparably to autologous Tregs. Histology and spatial transcriptomics reveal minimal cytotoxic infiltration and enrichment of immunoregulatory and tissue-repair programmes. Multiplex HLA engineering thus enables ready-to-use allogeneic Tregs that withstand host immune attack for adoptive transfer.},
}
@article {pmid41083939,
year = {2025},
author = {Menelih, A and Girma, A and Aemiro, A},
title = {Advancing nutritional quality in oilseed crops through genome editing: a comprehensive review.},
journal = {GM crops &amp; food},
volume = {16},
number = {1},
pages = {709-732},
pmid = {41083939},
issn = {2164-5701},
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics/metabolism ; *Nutritive Value ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; *Plant Oils/metabolism ; Seeds/genetics ; Fatty Acid Desaturases/genetics/metabolism ; Genome, Plant ; },
abstract = {Genome editing has emerged as a powerful approach to enhancing the nutritional quality of oilseed crops. Clustered regularly interspaced short palindromic repeats case9 (CRISPR/Cas9) is the predominant editing tool, while transcription activator-like effector nucleases (TALENs) and base editors are used less commonly. Key fatty acid desaturase genes such as FAD2 and FAD3 are prime targets because of their critical functions in fatty acid desaturation. This review summarizes recent progress in editing genes associated with oil composition and related traits across oilseed species. Visual data representations including, Sankey diagrams, heat maps, and crop-trait matrices illustrate shared editing priorities and emerging trait targets across crops. Despite its promise, genome editing still faces challenges in transformation efficiency, field-level validation, and regulatory acceptance. This review underscores the increasing impact of target gene editing on nutritional trait improvement and its potential to accelerate the development of healthier, more sustainable oilseed varieties.},
}
@article {pmid41084330,
year = {2026},
author = {Huppertz, F and Caturelli, MS and Lehmann, LS and Kurth, F and Maier, AG and Matuschewski, K},
title = {Plasmodium falciparum gametogenesis essential protein 1 (GEP1) is a transmission-blocking target.},
journal = {FEBS letters},
volume = {600},
number = {2},
pages = {239-250},
pmid = {41084330},
issn = {1873-3468},
support = {IRTG2290//Deutsche Forschungsgemeinschaft/ ; },
mesh = {*Plasmodium falciparum/genetics/drug effects/metabolism/growth &amp; development ; Animals ; *Protozoan Proteins/genetics/metabolism ; *Gametogenesis/genetics/drug effects ; Mice ; Humans ; *Malaria, Falciparum/transmission/parasitology ; Female ; Male ; Xanthurenates/pharmacology ; Anopheles/parasitology ; CRISPR-Cas Systems ; Polymorphism, Single Nucleotide ; },
abstract = {Transmission of Plasmodium parasites to Anopheles mosquitoes relies on rapid activation of mature gametocytes in the midgut, triggered by a temperature drop and xanthurenic acid. In Plasmodium yoelii, the gametogenesis essential protein 1 (GEP1) was linked to xanthurenic acid (XA)-dependent gamete activation. We characterized GEP1 in Plasmodium falciparum using CRISPR-Cas9 to create PfGEP1 loss-of-function lines. These lines failed to undergo male or female gametogenesis, even when stimulated by XA or a temperature drop. The defect persisted despite treatment with the phosphodiesterase inhibitor Zaprinast. Analysis of field samples revealed two GEP1 single-nucleotide polymorphisms (V241L and S263P) in 12% and 20% of 49 cases. Our findings confirm GEP1's essential role in gamete activation, highlight an XA-independent function, and support its potential as a transmission-blocking target. Impact statement For sustainable malaria control, transmission-blocking drug targets are urgently needed. Work in murine models showed that GEP1 is a candidate. We show complete block of life cycle progression of the human malarial parasite Plasmodium falciparum when GEP1 is deleted, warranting targeted drug development to achieve gamete-free mosquito blood meals.},
}
@article {pmid41084819,
year = {2025},
author = {Gao, Z and Yang, X and Ren, X and Ma, H and Wu, D and Du, Y and Fan, Q and Ma, Q and Wei, Q},
title = {Ultrasensitive Detection of m[6] A-Modified RNA Using CRISPR/Cas12a-Integrated Iontronic Biosensor with Hydrophobized Nanochannels: Toward Early Cancer Diagnosis by Machine Learning.},
journal = {Analytical chemistry},
volume = {97},
number = {42},
pages = {23609-23621},
doi = {10.1021/acs.analchem.5c05377},
pmid = {41084819},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; Humans ; *Machine Learning ; *Adenosine/analogs &amp; derivatives/analysis/chemistry ; *CRISPR-Cas Systems ; *Early Detection of Cancer/methods ; *RNA, Long Noncoding/genetics/analysis ; Hydrophobic and Hydrophilic Interactions ; *Neoplasms/diagnosis ; Limit of Detection ; Aluminum Oxide/chemistry ; },
abstract = {N[6] -methyladenosine (m[6] A), the most prevalent internal modification in eukaryotic RNAs, has emerged as a focal point of intensive research in recent years owing to its pivotal regulatory roles in carcinogenesis, progression, and metastasis. However, conventional methods for site-specific detection of m[6] A modifications are plagued by operational complexity, pose challenges for quantitative assessment of methylation levels, and exhibit elevated false-positive rates, severely limiting their utility in clinical and mechanistic studies. In this study, we engineered an ultrasensitive iontronic biosensor leveraging a hydrophobized anodic aluminum oxide (AAO) nanochannel platform, synergistically integrating the precise target recognition capability of the CRISPR/Cas12a system with the efficient signal amplification of the clamped hybridization chain reaction (CHCR). This integration enables ultrasensitive and specific detection of m[6] A-modified RNA with a low detection limit of 32 aM. Validation experiments targeting MALAT1 and HOTAIR lncRNAs demonstrated that the sensor achieves exceptional specificity in qualitative analysis of m[6] A modifications. Furthermore, combinatorial detection of these two lncRNAs enables robust discrimination between cancer patients and healthy individuals. Through in-depth mining of latent data patterns via machine learning, the random forest (RF) model yielded a cancer diagnostic accuracy of 96.7%. This study establishes a novel and potent paradigm for early cancer diagnosis, with far-reaching implications for epitranscriptomic research and clinical translation.},
}
@article {pmid41084820,
year = {2025},
author = {Ma, AX and Zhang, Q and Zhang, MY and Liu, SL and Wang, ZG and Pang, DW},
title = {One-step naked-eye fluorescence detection of viruses using quantum dot-magnetic beads coupled with CRISPR/Cas12a.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {89},
pages = {17432-17435},
doi = {10.1039/d5cc03645c},
pmid = {41084820},
issn = {1364-548X},
mesh = {*Quantum Dots/chemistry ; *CRISPR-Cas Systems ; Humans ; Fluorescence ; *CRISPR-Associated Proteins/metabolism/chemistry ; *Simplexvirus/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {This work constructs quantum dot-magnetic beads coupled with CRISPR/Cas12a for naked-eye detection of herpes simplex virus. The system integrates the high brightness of quantum dots and trans-cleavage activity of Cas12a, enabling rapid, simple, and accurate viral detection.},
}
@article {pmid41084992,
year = {2025},
author = {Han, J and Ganguly, R and Yi, JY and Yun, H and Jung, SY and Sung, C and Lee, CS},
title = {Osmotically Tunable Microdroplets Enable Amplification-Free CRISPR Detection of Gene Doping.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {48},
pages = {e15861},
pmid = {41084992},
issn = {2198-3844},
mesh = {Humans ; *Doping in Sports/methods ; *CRISPR-Cas Systems/genetics ; *Erythropoietin/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Gene doping is an increasing challenge in sports, demanding highly sensitive and specific detection tools beyond the limitations of the current amplification-dependent methods. Here, an innovative amplification-free clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) 12a assay integrated with osmotically tunable double emulsion (DE) droplets is reported for rapid and ultrasensitive gene doping detection. Target DNA and CRISPR/Cas12a complexes are encapsulated within DE droplets, where osmotic shrinkage rapidly concentrates the reaction components, thereby enhancing the fluorescent signal intensity without nucleic acid amplification. This platform enables the detection of the human erythropoietin (hEPO) gene at unprecedented attomolar levels within 30 min, achieving a 25-fold improvement in sensitivity compared with that of nonshrinkable formats. Notably, the assay demonstrated a robust and specific performance in complex serum samples with minimal matrix interference. This novel approach offers a rapid, reliable, and inherently contamination-free solution for gene doping surveillance with broad potential for versatile amplification-free nucleic acid diagnostics.},
}
@article {pmid41085033,
year = {2025},
author = {Levassor, L and Whitford, CM and Petersen, SD and Blin, K and Weber, T and Frandsen, RJN},
title = {StreptoCAD: An Open-Source Software Toolbox Automating Genome Engineering Workflows in Streptomycetes.},
journal = {ACS synthetic biology},
volume = {14},
number = {11},
pages = {4355-4372},
pmid = {41085033},
issn = {2161-5063},
mesh = {*Streptomyces/genetics ; *Software ; CRISPR-Cas Systems/genetics ; Genome, Bacterial/genetics ; Plasmids/genetics ; Gene Editing/methods ; *Genetic Engineering/methods ; Workflow ; },
abstract = {Streptomycetes hold immense potential for discovering novel bioactive molecules for applications in medicine or sustainable agriculture. However, high-throughput exploration is hampered by the current Streptomyces genetic engineering methods that involve the manual design of complex experimental molecular biological engineering strategies for each targeted gene. Here, we introduce StreptoCAD, an open-source software toolbox that automates and streamlines the design of genome engineering strategies in Streptomyces, supporting various CRISPR-based and gene overexpression methods. Once initiated, StreptoCAD designs all necessary DNA primers and CRISPR guide sequences, simulates plasmid assemblies (cloning) and the resulting modification of the genomic target(s), and further summarizes the information needed for laboratory implementation and documentation. StreptoCAD currently offers six design workflows, including the construction of overexpression libraries, base-editing, including multiplexed CRISPR-BEST plasmid generation, and genome engineering using CRISPR-Cas9, CRISPR-Cas3, and CRISPRi systems. In addition to automating the design process, StreptoCAD further secures compliance with the FAIR principles, ensuring reproducibility and ease of data management via standardized output files. To experimentally demonstrate the design process and output of StreptoCAD, we designed and constructed a series of gene overexpression strains, and performed CRISPRi knockdowns in Streptomyces Gö40/10, underscoring the tool's efficiency and user-friendliness.. This tool simplifies complex genetic engineering tasks and promotes collaboration through standardized workflows and design parameters. StreptoCAD is set to transform genome engineering in Streptomyces, making sophisticated genetic manipulations accessible for all and accelerating natural product discovery.},
}
@article {pmid41085147,
year = {2025},
author = {Liu, WJ and Wang, LY and Ma, F and Zhang, CY},
title = {MnO2 Nanosponge-Accelerated Cas12a Trans-Cleavage: Breaking the Kinetic Barrier for In Vivo RNA Imaging.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {12},
number = {48},
pages = {e11942},
pmid = {41085147},
issn = {2198-3844},
mesh = {*Manganese Compounds/chemistry ; Humans ; *Oxides/chemistry ; *CRISPR-Cas Systems/genetics ; Kinetics ; *RNA/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics ; Biosensing Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {CRISPR/Cas12a system has emerged as a promising tool for in vitro biosensing, but its in vivo applications are hindered by its inefficient intracellular delivery and suboptimal trans-cleavage kinetics. To address these challenges, a Cas12a@MnO2 nanosponge (hMNS) nanoprobe is constructed, in which hMNS as both a degradable carrier and an accelerator of CRISPR/Cas12a system for efficient imaging of RNA in living cells. The Cas12a@hMNS nanoprobe is obtained via a one-step co-assembly process. It not only facilitates synchronous cellular uptake and glutathione (GSH)-responsive release of CRISPR/Cas12a components, but also supplies adequate Mn[2+] cofactors to improve the trans-cleavage activity of Cas12a. This dual-function probe can break the kinetic barrier of conventional CRISPR/Cas12a systems due to its unique characteristics of effective cellular internalization, rapid intracellular release, and accelerated signal gain, enabling sensitive detection of mRNA down to 63.6 pM without pre-amplification. Moreover, the Cas12a@hMNS nanoprobe can profile endogenous mRNA at the single-cell level, discriminate breast cancer tissues from healthy counterparts, and real-time visualize mRNA dynamics in living cells with exceptional spatiotemporal precision. Importantly, the elongation-blocked (EB) activator-modulated CRISPR/Cas12a system can be extended to detect various intracellular biomarkers, holding promising applications in clinical diagnosis, treatment, and surveillance.},
}
@article {pmid41085803,
year = {2025},
author = {de Souza, HCA and Panzenhagen, P and Portes, AB and Dos Santos, AMP and Fidelis, J and Junior, CAC},
title = {CRISPR-Cas systems in combating antimicrobial resistance: which system to choose? A systematic review.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {381},
pmid = {41085803},
issn = {1573-0972},
support = {FinanceCode001//Coordena&#xe7;&#xe3;o de Aperfei&#xe7;oamento de Pessoal de N&#xed;vel Superior/ ; 313119/2020-1//Conselho Nacional de Desenvolvimento Cient&#xed;fico e Tecnol&#xf3;gico/ ; E26/202.227/2018//Funda&#xe7;&#xe3;o Carlos Chagas Filho de Amparo &#xe0; Pesquisa do Estado do Rio de Janeiro/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; *Bacteria/genetics/drug effects ; *Drug Resistance, Bacterial/genetics ; Humans ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Antimicrobial resistance (AMR) poses a growing threat to global public health, progressively compromising the efficacy of available antimicrobials. Technologies based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) have emerged as promising tools for controlling resistant pathogens, offering high specificity and versatility. However, a comprehensive and systematic synthesis of CRISPR strategies applied to AMR remains limited. From February 12, 2025, we conducted a systematic review of the PubMed, Embase, and Scopus databases, using the following search strategy: Population (resistant bacteria or plasmid-mediated resistance), Intervention (CRISPR, including variants such as CRISPR-Cas9, Cas3, Cas12, Cas13, and CRISPR interference [CRISPRi]), and Outcomes (bacterial resensitization or plasmid curing). The CRISPR-Cas9 system was the most frequently employed (75.7%), with conjugation identified as the primary delivery method. We identified the advantages and limitations of each system, highlighting CRISPRi and CRISPR-Cas13a as alternatives to overcome the constraints of direct genome editing. Delivery efficiency remains a central challenge, although nanocarrier- and bacteriophage-based methodologies show promising potential. We also propose a decision map that guides the selection of the most appropriate CRISPR-Cas system and delivery strategy, considering factors such as therapeutic objective, gene location, methodology efficiency, application environment, and clinical feasibility. This review provides an updated and structured synthesis of CRISPR strategies applied to AMR, emphasizing their potential translational and clinical applications. The findings can inform the development of CRISPR-based therapeutics, guide the design of preclinical studies, and support future strategies for combating multidrug-resistant infections in clinical settings.},
}
@article {pmid41086252,
year = {2025},
author = {Libri, AB and Wang, J and Marton, T and Yu, W and Dossin, F and Balmus, G and Reina-San-Martin, B and Frock, R and Lescale, C and Deriano, L},
title = {Senataxin promotes recombination fidelity during antigen receptor gene diversification.},
journal = {Science signaling},
volume = {18},
number = {908},
pages = {eadv8801},
doi = {10.1126/scisignal.adv8801},
pmid = {41086252},
issn = {1937-9145},
mesh = {Animals ; *V(D)J Recombination/genetics ; Mice ; DNA End-Joining Repair ; *RNA Helicases/genetics/metabolism ; Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; DNA Breaks, Double-Stranded ; Multifunctional Enzymes ; CRISPR-Cas Systems ; Mice, Knockout ; *DNA Helicases/genetics/metabolism ; Humans ; *Receptors, Antigen, T-Cell/genetics ; Precursor Cells, B-Lymphoid/metabolism/immunology ; },
abstract = {Antigen receptor diversity depends on the assembly of variable (V), diverse (D), and joining (J) exons in genes encoding immunoglobulins (Igs) and T cell receptors (TCRs). During V(D)J recombination, DNA double-strand breaks (DSBs) introduced by the RAG1/2 nuclease complex are repaired by the process of nonhomologous end-joining (NHEJ). We hypothesized that functional redundancies between NHEJ and the chromatin DSB response, which depends on the kinase ATM, potentially masked the activity of additional factors that regulate V(D)J recombination. We performed targeted CRISPR-Cas9 knockout screens for genes implicated in V(D)J recombination in pro-B cells that were either untreated or treated with an ATM inhibitor. We found that loss of the RNA/DNA helicase senataxin (SETX) impaired V(D)J recombination and led to the formation of aberrant hybrid joints between coding ends and signal ends, both in vitro and in mice. The loss of SETX in a background deficient in the NHEJ factor XLF or in which ATM was inhibited led to substantial impairment of V(D)J recombination and to the presence of unsealed coding ends. SETX limited aberrant activation-induced cytidine deaminase (AID)-induced DNA end-joining between Igh-containing alleles during the process of class-switch recombination. Together, our findings reveal a previously uncharacterized role for SETX in promoting recombination fidelity during antigen receptor gene diversification.},
}
@article {pmid41086891,
year = {2025},
author = {Sun, J and Huang, Y and Bai, J and Wang, J and Langford, PR and Zhang, Y and Li, G},
title = {A CRISPR/Cas12a-based DNAzyme chemiluminescence platform for rapid detection of all Streptococcus suis and individually S. suis serotypes 7 and 9.},
journal = {International journal of biological macromolecules},
volume = {330},
number = {Pt 4},
pages = {148280},
doi = {10.1016/j.ijbiomac.2025.148280},
pmid = {41086891},
issn = {1879-0003},
mesh = {*Streptococcus suis/genetics/isolation &amp; purification/classification ; *DNA, Catalytic/metabolism/genetics ; Serogroup ; *CRISPR-Cas Systems/genetics ; *Luminescent Measurements/methods ; Swine ; Animals ; Biosensing Techniques/methods ; Luminescence ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Streptococcus suis is a zoonotic pathogen that can infect pigs and humans with causing meningitis, sepsis, endocarditis, and arthritis. S. suis serotypes 7 and 9 cause substantial economic losses to the swine industry and pose a major threat to public health, thus, accurate and rapid detection is important for the prevention and control of epidemic disease. In this study, we developed a platform, combining recombinase polymerase amplification (RPA) with a CRISPR/Cas12a detection system to rapidly detect all S. suis serotypes and individually differentiates serotypes 7 and 9. This was achieved by targeting recN of S. suis and serotype-specific cpsH genes of serotypes 7 and 9, respectively. Both fluorescence and G4 DNAzyme chemiluminescence visualization biosensing methods had high specificity and sensitivity, no cross-reaction was found with common pig pathogens, closely related Streptococcus spp., or other S. suis serotypes. Compared to traditional identification techniques, these two methods are rapid and convenient. Notably, the G4 DNAzyme chemiluminescence method provides a clear, direct visual interpretation of results without the need for specialized equipment, which is particularly advantageous for point-of-care testing. Thus, this platform has the potential to significantly enhance diagnostic capabilities and ultimately benefit both animal and public health.},
}
@article {pmid41086995,
year = {2025},
author = {Jang, SH and Song, HG and Jung, J and Gee, HY},
title = {Recent preclinical and clinical advances in gene therapy for hereditary hearing loss.},
journal = {Molecules and cells},
volume = {48},
number = {12},
pages = {100285},
pmid = {41086995},
issn = {0219-1032},
mesh = {*Genetic Therapy/methods ; Humans ; *Hearing Loss/therapy/genetics ; Animals ; Gene Editing ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Dependovirus/genetics ; Gene Transfer Techniques ; Clinical Trials as Topic ; },
abstract = {Hereditary hearing loss is a genetically heterogeneous condition that affects millions of people worldwide and has limited curative treatment options. Recent advancements in gene therapy have opened promising avenues for correcting the underlying genetic defects in the inner ear. This review summarizes the key developments in vector platforms, delivery strategies, target genes, preclinical models, and clinical trials relevant to both gene supplementation and gene editing approaches, as well as future directions. Adeno-associated virus vectors have emerged as the leading platform for inner ear gene transfer, owing to their safety and efficacy. Clinical programs, such as those targeting OTOF variants, are currently underway and are supported by robust preclinical data. Additionally, genome editing technologies, including CRISPR/Cas9-mediated nonhomologous end joining, base editing, and prime editing, offer variant-specific therapeutic potential. Despite these advances, challenges remain in expanding the therapeutic window, ensuring long-term safety, and establishing ethical and regulatory frameworks for their use.},
}
@article {pmid41087678,
year = {2025},
author = {Kaplan, EG and Steger, RJ and Shah, ST and Drepanos, LM and Griffith, AL and Reint, G and Doench, JG},
title = {Activity-based selection for enhanced base editor mutational scanning.},
journal = {Nature genetics},
volume = {57},
number = {11},
pages = {2920-2929},
pmid = {41087678},
issn = {1546-1718},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Tumor Suppressor Protein p53/genetics ; },
abstract = {Base editing is a CRISPR-based technology that enables high-throughput, nucleotide-level functional interrogation of the genome that is essential for understanding the genetic basis of human disease and informing therapeutic development. Base editing screens have emerged as a powerful experimental approach, yet significant cell-to-cell variability in editing efficiency introduces noise that may obscure meaningful results. Here we develop a co-selection method that enriches for cells with high base editing activity, substantially increasing editing efficiency at a target locus. We evaluate this activity-based selection method against a traditional screening approach by tiling guide RNAs across TP53, demonstrating its enhanced capacity to pinpoint specific mutations and protein regions of functional importance. We anticipate that this modular selection method will enhance the resolution of base editing screens across many applications.},
}
@article {pmid41088816,
year = {2025},
author = {Mo, T and Ren, HY and Zhang, XX and Lu, YW and Teng, ZQ and Zhang, X and Dai, LP and Hou, L and Zhao, N and He, J and Qin, T},
title = {Phenotypic Function of Legionella pneumophila Type I-F CRISPR-Cas.},
journal = {Biomedical and environmental sciences : BES},
volume = {38},
number = {9},
pages = {1105-1119},
doi = {10.3967/bes2025.107},
pmid = {41088816},
issn = {2214-0190},
mesh = {*Legionella pneumophila/genetics/physiology/pathogenicity ; *CRISPR-Cas Systems ; Biofilms/growth &amp; development ; Phenotype ; Bacterial Proteins/genetics/metabolism ; Gene Deletion ; },
abstract = {OBJECTIVE: CRISPR-Cas protects bacteria from exogenous DNA invasion and is associated with bacterial biofilm formation and pathogenicity.<br><br>METHODS: We analyzed the type I-F CRISPR-Cas system of Legionella pneumophila WX48, including Cas1, Cas2-Cas3, Csy1, Csy2, Csy3, and Cas6f, along with downstream CRISPR arrays. We explored the effects of the CRISPR-Cas system on the in vitro growth, biofilm-forming ability, and pathogenicity of L. pneumophila through constructing gene deletion mutants.<br><br>RESULTS: The type I-F CRISPR-Cas system did not affect the in vitro growth of wild-type or mutant strains. The biofilm formation and intracellular proliferation of the mutant strains were weaker than those of the wild type owing to the regulation of type IV pili and Dot/Icm type IV secretion systems. In particular, Cas6f deletion strongly inhibited these processes.<br><br>CONCLUSION: The type I-F CRISPR-Cas system may reduce biofilm formation and intracellular proliferation in L. pneumophila.},
}
@article {pmid41090155,
year = {2025},
author = {Li, JM and Huang, J and Liao, Y and Hu, T and Wang, CL and Zhang, WZ and Huang, CW},
title = {Gene and RNA Editing: Revolutionary Approaches to Treating Diseases.},
journal = {MedComm},
volume = {6},
number = {10},
pages = {e70389},
pmid = {41090155},
issn = {2688-2663},
abstract = {Gene editing and RNA editing technologies are advancing modern medicine by enabling precise manipulation of genetic information at the DNA and RNA levels, respectively. The third-generation gene editing tools, particularly Clustered regularly interspaced shortpalindromic repeats (CRISPR)/CRISPR-associated (Cas) system, have transformed genetic disease treatment with high efficiency, precision, and cost effectiveness, while RNA editing, via adenosine deaminase acting on RNA (ADAR) enzymes and CRISPR-Cas13, offers reversible regulation to avoid genomic integration risks. Despite advancements, challenges persist in delivery efficiency, tissue specificity, and long-term safety, limiting their clinical translation. This review systematically discusses the molecular mechanisms and technological evolution of these tools, focusing on their promising applications in treating nervous system disorders (e.g., Alzheimer's, Parkinson's), immune diseases (e.g., severe combined immunodeficiency, lupus), and cancers. It compares their technical attributes, analyzes ethical and regulatory issues, and highlights synergies between the two technologies. By bridging basic research and clinical translation, this review provides critical insights for advancing precision medicine, reshaping disease diagnosis, prevention, and treatment paradigms.},
}
@article {pmid41090337,
year = {2025},
author = {Cherdantsev, AI and Kulagin, KA and Polyakova, AN and Karpov, VL and Sosnovtseva, AO and Karpov, DS},
title = {[DNA Double-Strand Break Repair System by a Mechanism of Non-Homologous End Joining Provides Resistance to DNA-Damaging and Oxidizing Stresses in the Yeast Debaryomyces hansenii].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {4},
pages = {616-628},
doi = {10.31857/S0026898425040083},
pmid = {41090337},
issn = {0026-8984},
mesh = {*DNA End-Joining Repair ; *DNA Breaks, Double-Stranded ; *Oxidative Stress/genetics ; CRISPR-Cas Systems ; *Debaryomyces/genetics/metabolism ; *Fungal Proteins/genetics/metabolism ; Osmotic Pressure ; },
abstract = {The unconventional halotolerant yeast Debaryomyces hansenii is of great importance in biotechnology and the food industry, and in basic research it serves as a model for studying the molecular mechanisms of resistance to increased salinity and osmotic stress. We have previously established an efficient method for editing the D. hansenii genome using the CRISPR/Cas9 system. In turn, this has stimulated further investigation of the structure and physiological role of DNA double-strand break repair pathways in D. hansenii. The aim of the present work was to evaluate the involvement of key components of the DNA double-stranded break repair system by the non-homologous end joining (NHEJ) mechanism in the resistance of D. hansenii to DNA-damaging compounds and compounds that induce oxidative, high salinity, and osmotic stress. Using the CRISPR/Cas9 system, mutant strains with knockout of the DEHA2F10208g (DhKU70), DEHA2B01584g (DhKU80) , and DEHA2G04224g (DhLIG4) genes encoding key components of NHEJ were obtained. It was found that mutant strains, unlike the wild-type strain, are sensitive to chemical compounds that damage DNA, as well as to compounds that cause oxidative stress. Osmotic and high salinity stresses and vanillin do not cause significant changes in the rate of colony formation of mutant strains. Unexpectedly, mutant strains exhibit increased resistance to caffeine compared to the wild-type strain. The data indicate that the NHEJ systems of D. hansenii play a significant role in the response to DNA-damaging and oxidative types of stress. The importance of the NHEJ system in the processes of maintaining yeast cell homeostasis should be taken into account when creating strains producing valuable substances.},
}
@article {pmid41090360,
year = {2025},
author = {Wei, W and Zhu, W and Silver, S and Armstrong, AM and Robbins, FS and Rameshbabu, AP and Walz, K and Quan, Y and Du, W and Kim, Y and Indzhykulian, AA and Shu, Y and Liu, XZ and Chen, ZY},
title = {Single-dose genome editing therapy rescues auditory and vestibular functions in adult mice with DFNA41 deafness.},
journal = {The Journal of clinical investigation},
volume = {135},
number = {20},
pages = {},
pmid = {41090360},
issn = {1558-8238},
support = {R01 DC017166/DC/NIDCD NIH HHS/United States ; R01 DC020190/DC/NIDCD NIH HHS/United States ; R01 DC021795/DC/NIDCD NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing ; Mice ; *Genetic Therapy ; Humans ; Dependovirus/genetics ; *Deafness/therapy/genetics ; Disease Models, Animal ; *Vestibule, Labyrinth/physiopathology ; Mutation, Missense ; CRISPR-Cas Systems ; },
abstract = {Genome editing has the potential to treat genetic hearing loss. However, current editing therapies for genetic hearing loss have shown efficacy only in hearing rescue. In this study, we evaluated a rescue strategy using adeno-associated virus (AAV) type 2-mediated delivery of Staphylococcus aureus Cas9-sgRNA in the mature inner ear of the P2rx2V61L/+ mouse model of autosomal dominant deafness-41 (DFNA41), a dominant, delayed-onset, and progressive hearing loss in humans. We demonstrate that local injection in adult mice results in efficient and specific editing that abolishes the mutation without notable off-target effects or AAV genome integration. Editing effectively restores long-term auditory and vestibular function. Editing further protects P2rx2V61L/+ mice from hypersensitivity to noise-induced hearing loss, a phenotype also observed in patients with DFNA41. Intervention in mice at a juvenile stage broadens the frequency range rescued, highlighting the importance of early intervention. An effective and specific gRNA for the human P2RX2 V60L mutation has been identified. Our study establishes the feasibility of editing to treat DFNA41 caused by P2RX2 V60L mutation in humans and opens an avenue for using editing to rescue hearing and vestibular function while mitigating noise-induced hearing loss.},
}
@article {pmid41090767,
year = {2025},
author = {Slattery, JR and Naung, NY and Kalinna, BH and Pal, M},
title = {CRISPR-Powered Liquid Biopsies in Cancer Diagnostics.},
journal = {Cells},
volume = {14},
number = {19},
pages = {},
pmid = {41090767},
issn = {2073-4409},
mesh = {Humans ; Liquid Biopsy/methods ; *Neoplasms/diagnosis/genetics ; Biomarkers, Tumor/genetics ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; },
abstract = {Liquid biopsies promise major advantages for cancer screening and diagnosis. By detecting biomarkers in peripheral blood samples, liquid biopsies reduce the need for invasive techniques and provide important genetic information integral to the emerging molecular classification of cancers. Unfortunately, the concentrations of most biomarkers, particularly circulating tumour nucleic acids, are vanishingly small-beyond the sensitivity and specificity of most assays. Clustered Regularly Interspaced Short Palindromic Repeats diagnostics (herein labelled 'CRISPR-Dx') use gene editing tools to detect, rather than modify, nucleic acids with extremely high specificity. These tools are commonly combined with isothermal nucleic acid amplification to also achieve sensitivities comparable to high-performance laboratory-based techniques, such as digital PCR. CRISPR assays, however, are inherently well suited to adaptation for point-of-care (POC) use, and unlike antigen-based POC assays, are significantly easier and faster to develop. In this review, we summarise current CRISPR-Dx platforms and their analytical potential for cancer biomarker discovery, with an emphasis on enhancing early diagnosis, disease monitoring, point-of-care testing, and supporting cancer therapy.},
}
@article {pmid41091245,
year = {2025},
author = {Yang, S and Jiao, X and Liu, J and Liu, Y and Wang, M and Li, S and Qiao, J},
title = {CRISPR-Cas opens a new era of antimicrobial therapy as a powerful gene editing tool.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {388},
pmid = {41091245},
issn = {1573-0972},
support = {X2024739//College Students' Innovation and Entrepreneurship Training Program of Shandong Second Medical University/ ; ZR2020MH305//Natural Science Foundation of Shandong Province, China/ ; },
}
@article {pmid41091748,
year = {2025},
author = {Yew, WN and Dean, CJ and Chan, DKH},
title = {STAG2 mutations in the normal colon induce upregulation of oncogenic pathways in neighbouring wildtype cells.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {pone.0332499.exml},
pmid = {41091748},
issn = {1932-6203},
mesh = {Humans ; Organoids/metabolism ; *Colon/metabolism/pathology/cytology ; *Mutation ; Up-Regulation ; Signal Transduction/genetics ; CRISPR-Cas Systems ; Coculture Techniques ; Gene Editing ; Proto-Oncogene Proteins p21(ras)/metabolism/genetics ; *Carcinogenesis/genetics ; *Colorectal Neoplasms/genetics/pathology ; Tumor Necrosis Factor-alpha/metabolism ; },
abstract = {While driver mutations in the normal colon have been described, characterizing the role and function of these driver mutations in relation to colorectal oncogenesis remains incomplete. Here, we investigated the role of STAG2 mutants in the normal colon using patient-derived wildtype organoids. Using CRISPR-Cas9 gene editing, we generated STAG2 mutants, and co-cultured these mutants with wildtype organoids, mimicking the presence of such STAG2 mutants in the normal colon. We sought to determine the transcriptional impact of co-culture using scRNAseq. Surprisingly, we uncovered a possible cell-cell interaction between STAG2 mutants and wildtype organoids, in which wildtype organoids in co-culture with STAG2 mutants upregulated known oncogenic pathways. This included the upregulation of TNFα-signaling, as well as KRAS-signaling in wildtype organoids. These results suggested that STAG2 mutant cells exert a pro-oncogenic effect in a cell interactive manner, instead of via a cell autonomous approach. In conclusion, our findings demonstrate a novel mechanism of colorectal oncogenesis which can support further investigation.},
}
@article {pmid41092254,
year = {2025},
author = {Yin, K and Tsai, CJ},
title = {Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70527},
pmid = {41092254},
issn = {1365-313X},
support = {//Georgia Research Alliance/ ; DE-SC0023166//U.S. Department of Energy/ ; DE-SC0023338//U.S. Department of Energy/ ; ERKP886//U.S. Department of Energy/ ; },
mesh = {*Gene Editing/methods ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Multifactorial Inheritance/genetics ; Plants, Genetically Modified/genetics ; *Genetic Engineering/methods ; },
abstract = {Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.},
}
@article {pmid41093192,
year = {2025},
author = {Chou, SJ and Wang, CH and Chang, YL and Fang, WC and Hwang, DK and Hsiao, YJ and Luo, YH and Lo, WL and Viet, NQ and Tang, KY and Lan, YT and Hsu, CC and Chen, SJ and Lin, TC and Yang, YP and Chiou, SH},
title = {Dual delivery of supramolecular nanoparticle-carried minicircle donor DNA with Cas9/gRNA improved HITI knock-in efficiency in X-linked juvenile retinoschisis.},
journal = {International journal of biological macromolecules},
volume = {332},
number = {Pt 2},
pages = {148300},
doi = {10.1016/j.ijbiomac.2025.148300},
pmid = {41093192},
issn = {1879-0003},
mesh = {Humans ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Retinoschisis/genetics/therapy ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *DNA, Circular/genetics ; Gene Editing/methods ; *Gene Knock-In Techniques/methods ; Gene Transfer Techniques ; CRISPR-Associated Protein 9/genetics ; Genetic Therapy/methods ; HEK293 Cells ; Eye Proteins/genetics ; Plasmids/genetics ; },
abstract = {X-linked retinoschisis (XLRS) is a hereditary mutation of the RS1 gene and is characterized by early-onset maculopathy with severe visual impairment. Current gene therapy utilizing CRISPR-associated protein 9 (Cas9) is ongoing; however, the optimization of nonviral/nanoparticle CRISPR/Cas9-based therapeutics for gene delivery into nondivided retinal neurons remains undetermined. Minicircles DNA, a circular DNA molecule lacking bacterial backbone sequences, has gained recognition for improving transfection efficiency and biosafety. Here, we developed a supramolecular nanoparticle (SMNP)-editing platform in which SMNPs carrying CRISPR/Cas9 integrated minicircle donor DNA (mc dDNA) to achieve highly efficient and precise gene knock-in. To increase the efficiency of RS1 gene knock-in, we replaced the conventional mc dDNA with a single flanking-Cas9/cut site. Furthermore, using homology-independent targeted integration (HITI) as an editing-nondivided cell strategy, SMNP-carried CRISPR/Cas9 could effectively facilitate the dual delivery of mc-RS1/GFP dDNA and Cas9/gRNA plasmids. Compared with the delivery of the Cas9/gRNA plasmid alone (~20 %), the delivery of mc-RS1/GFP dDNA via SMNPs had significantly higher transfection efficiency (90 %). Further flow cytometry analysis revealed that 5.99 % of the FACS-positive cells were detected in the mc-RS1/GFP dDNA group, markedly exceeding the 2.21 % with long-term expression in the conventional dDNA group. Moreover, when XLRS/iPSC-derived retinal neuron organoids were used as a patient-based disease model, compared with conventional plasmid-based delivery, robust RS1 expression with integration sustained transgene expression in XLRS/iPSC-derived retinal organoids. Collectively, these findings indicated that SMNP-mediated dual delivery of the Cas9/gRNA plasmid and mc-RS1/GFP dDNA substantially enhanced RS1-targeted integration with long-term transgene expression, providing safer and effective gene therapy for the treatment of XLRS.},
}
@article {pmid41093194,
year = {2025},
author = {Yang, G and Rao, Q and Dong, W and Yin, X and Shen, R and Wang, R and Deng, X and Peng, X and Tao, Y and Li, S and Wang, X and Tang, Y and Du, D},
title = {Rapid and sensitive one-pot CRISPR-Cas12a detection platform for common fungal species in ocular infections using multiplex crRNAs.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 1},
pages = {148279},
doi = {10.1016/j.ijbiomac.2025.148279},
pmid = {41093194},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Fungi/genetics/isolation &amp; purification ; *Eye Infections, Fungal/microbiology/diagnosis/genetics ; DNA, Fungal/genetics ; },
abstract = {Corneal fungal infection is a major cause of visual impairment and can cause blindness if not diagnosed promptly. Traditional diagnostic methods are often time-consuming and require specialized expertise, UNDERSCORING the urgent need for rapid, sensitive, and user-friendly diagnostic tools. In this study, we utilized multiplex crRNAs in the Cas12a system to develop a highly sensitive and specific platform for the molecular detection of common fungal species causing corneal infections in a single reaction. We enhanced detection sensitivity through recombinase-aided amplification (RAA) and designed a streamlined tube with insert one-pot protocol to minimize aerosol contamination risk. Remarkably, our platform achieved a limit of detection of just 4 copies of fungal genomic DNA per reaction in only 32 min. It allows for direct detection from simulated tear samples, providing a potential for convenient and non-invasive testing option. Furthermore, our platform effectively tests clinical samples such as aqueous humor (AH) and vitreous humor (VH), demonstrating superior sensitivity compared to traditional PCR-based methods. These results highlight the potential of our platform as an on-site diagnostic solution for both clinical and non-clinical settings, contributing to the preservation of visual function.},
}
@article {pmid41093250,
year = {2025},
author = {Buck-Wiese, M and Liechocki, S and Erfle, H and Starkuviene, V},
title = {Comparative analysis of antibody-mediated loss-of-function versus gene knock-out and knock-down.},
journal = {SLAS discovery : advancing life sciences R &amp; D},
volume = {37},
number = {},
pages = {100283},
doi = {10.1016/j.slasd.2025.100283},
pmid = {41093250},
issn = {2472-5560},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; *Gene Knockdown Techniques/methods ; *Antibodies ; Talin/genetics/metabolism ; RNA Interference ; RNA, Small Interfering/genetics ; Gene Expression Profiling ; Cell Adhesion/genetics ; },
abstract = {In this study we compare three methods for manipulating cell function: RNA interference (RNAi), CRISPR-Cas9 gene knock-out, and antibody-mediated loss-of-function. We have focused on analyzing changes in cell-matrix adhesion via targeting two key regulators, Talin1 (TLN1) and Kindlin-2 (KD2). Adhesion-relevant phenotypic assays revealed distinct temporal onset dynamics for each method. RNAi and CRISPR-Cas9 effectively reduced target mRNA and protein levels. In contrast, antibody transfection induced phenotypic changes without altering target expression, suggesting direct intracellular antibody-target interaction. Transcriptome analysis demonstrated that antibody transfection and CRISPR-Cas9 induced fewer deregulated mRNAs than RNAi. Furthermore, transfected antibodies and sgRNAs shared 30 % and 70 % of deregulated transcripts to their negative controls, respectively. Whereas only 10 % of overlap was recorded between targeting and control siRNAs. Our findings emphasize the importance of considering method-specific temporal dynamics of on-target phenotype appearance and off-target manifestation. Additionally, they highlight intracellular delivered antibodies as a valuable alternative for validating and complementing genetic approaches.},
}
@article {pmid41093508,
year = {2025},
author = {Wei, Z and Huang, D and Luo, H and Zhang, J and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {A multi-level signal conversion architecture for enzyme sensing: Integrating MXene nanoplatforms with CRISPR-driven electrochemiluminescence.},
journal = {Analytica chimica acta},
volume = {1377},
number = {},
pages = {344636},
doi = {10.1016/j.aca.2025.344636},
pmid = {41093508},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Luminescent Measurements/methods ; *CRISPR-Cas Systems ; Humans ; *Flap Endonucleases/analysis/metabolism ; Nanocomposites/chemistry ; Gold/chemistry ; Limit of Detection ; DNA/chemistry ; Nitrites ; Titanium ; Transition Elements ; },
abstract = {Precise and ultrasensitive detection of flap endonuclease 1 (FEN1), a key DNA repair enzyme implicated in cancer diagnostics, remains challenging due to its subtle structural cleavage activity. Herein, we present a cascade-amplified electrochemiluminescence (ECL) biosensor based on a Ti3C2 MXene-supported Ru (bpy)3[2+]/Au nanocomposite integrated with a CRISPR-Cas13a system and DNA walker circuitry. Upon specific recognition and cleavage of a 5'-flap substrate by FEN1, a nicked DNA product is circularized and transcribed via T7 RNA polymerase, yielding RNA activators that trigger Cas13a-mediated collateral cleavage. This event releases a blocked DNA walker, which reorganizes Fc-labeled DNA on the electrode surface and restores the ECL signal suppressed by resonance energy transfer. The system achieves a detection limit as low as 1.48 fU/mL and exhibits a dynamic range spanning five orders of magnitude. Compared to fluorescence-based CRISPR detection systems, the ECL-based platform offers low background, high signal-to-noise ratios, and operational simplicity using standard electrochemical instrumentation, supporting practical deployment in clinical diagnostics. Furthermore, the platform demonstrates high selectivity against other nucleases and proteins, along with excellent performance in spiked human serum samples. This work presents a robust and modular strategy for accurate enzyme activity profiling with promising applications in early-stage disease diagnostics.},
}
@article {pmid41093525,
year = {2025},
author = {Wei, Z and Luo, H and Huang, D and Wei, P and Zhang, J and Wei, J and Tang, Q and Huang, L and Zhang, K and Liao, X},
title = {Structure-specific electrochemiluminescent biosensor for FEN1 detection via dumbbell probe-mediated transcription and CRISPR/Cas13a-induced G-quadruplexes cleavage.},
journal = {Analytica chimica acta},
volume = {1377},
number = {},
pages = {344662},
doi = {10.1016/j.aca.2025.344662},
pmid = {41093525},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *G-Quadruplexes ; *Flap Endonucleases/analysis/blood/metabolism ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Luminescent Measurements/methods ; *DNA Probes/chemistry/genetics ; Limit of Detection ; Transcription, Genetic ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5'-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti3C2/AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10[-9] U μL[-1] and a wide dynamic range (1 × 10[-8] to 1 × 10[-5] U μL[-1]), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.},
}
@article {pmid41093834,
year = {2025},
author = {Shibue, K and Kahraman, S and Castillo-Quan, JI and De Jesus, DF and Hu, J and Morita, H and Blackwell, TK and Yi, P and Kulkarni, RN},
title = {Genome-wide CRISPR Screen Identifies Sec31A as a Key Regulator of Alpha Cell Survival.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9159},
pmid = {41093834},
issn = {2041-1723},
support = {DK067536//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 AG054215/AG/NIA NIH HHS/United States ; P30 DK036836/DK/NIDDK NIH HHS/United States ; R01AG054215//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; JP22K16404//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; K99 DK135927/DK/NIDDK NIH HHS/United States ; R00 DK135927/DK/NIDDK NIH HHS/United States ; R35GM122610//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; Humans ; Mice ; Caenorhabditis elegans/metabolism/genetics ; *Vesicular Transport Proteins/genetics/metabolism ; *Glucagon-Secreting Cells/metabolism/cytology ; Cell Survival/genetics ; Endoplasmic Reticulum Stress/genetics ; CRISPR-Cas Systems ; Receptor, Insulin/metabolism ; Endoplasmic Reticulum/metabolism ; Signal Transduction ; Insulin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Insulin-Secreting Cells/metabolism ; Glucagon/metabolism ; },
abstract = {Glucagon, secreted by pancreatic alpha cells, is essential for maintaining normal blood glucose levels. In type 1 and advanced type 2 diabetes, alpha cells often fail to respond to low glucose, yet the mechanisms underlying their stress resistance remain unclear. To investigate this, we performed a genome-wide CRISPR screen and identify Sec31A, a gene involved in transporting proteins from the endoplasmic reticulum (ER), as a key regulator of alpha cell survival under stress. We show that loss of Sec31A enhances survival in stressed mouse alpha cells and in C. elegans. In human islets, SEC31A expression increases in alpha cells under inflammatory stress, and this upregulation is reversed by reducing ER stress. Functional studies in lab-grown human islet clusters reveal distinct responses in alpha versus beta cells following Sec31A suppression. We also find that Sec31A interacts with the insulin receptor, suggesting a link between stress adaptation and insulin signaling in alpha cells.},
}
@article {pmid41093849,
year = {2025},
author = {Shang, M and Li, Y and Cao, Q and Ren, J and Zeng, Y and Wang, J and Gonzalez, RVL and Zhang, X},
title = {A motif preferred adenine base editor with minimal bystander and off-targets editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9153},
pmid = {41093849},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; Humans ; Mice ; *Adenine/metabolism/chemistry ; *Nucleotide Motifs/genetics ; HEK293 Cells ; CRISPR-Cas Systems ; },
abstract = {47% of hereditable diseases are caused by single C•G-to-T•A base conversions, which means efficient A-to-G base editing tools (ABEs) have great potential for the treatment of these diseases. However, the existing efficient ABEs, while catalyzing targeted A-to-G conversion, cause high A or C bystander editing and off-target events, which poses safety concerns for their clinical applications. To overcome this shortcoming, we have developed ABE8e-YA (ABE8e with TadA-8e A48E) for efficient and accurate editing of As in YA motifs with YAY > YAR (Y = T or C, R = A or G) hierarchy through structure-oriented rational design. Compared with ABE3.1, which is currently the only ABE version with a YAC motif preference, ABE8e-YA exhibits an average A-to-G editing efficiency improvement of an up to 3.1-fold increase in the indicated YA motif while maintaining reduced bystander C editing and minimized DNA or RNA off-targets. Additionally, we demonstrate that ABE8e-YA efficiently and precisely corrects pathogenic mutations in human cells, suggesting its high suitability for addressing 9.3% of pathogenic point mutations, higher than that of ABE8e and ABE9. Moreover, by using ABE8e-YA, we efficiently and precisely generate hypocholesterolemia and tail-loss mouse models mimicking human-associated disease, as well as performed in vivo mouse proprotein convertase subtilisin/kexin type 9 (Pcsk9) base editing for hypercholesterolemia gene therapy. Together these data indicate its great potential in broad applications for disease modeling and gene therapy.},
}
@article {pmid41093851,
year = {2025},
author = {Zhou, SK and Luo, JT and Chen, YF and Lu, ZD and Jian, QH and Jiang, SQ and Li, J and Zhang, XQ and Tan, XY and Yang, XZ and Xu, CF and Wang, J},
title = {Muscle-specific gene editing therapy via mammalian fusogen-directed virus-like particles.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9145},
pmid = {41093851},
issn = {2041-1723},
support = {32430059//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32471434//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32271442//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Muscle, Skeletal/metabolism ; Mice ; *Muscular Dystrophy, Duchenne/therapy/genetics ; CRISPR-Cas Systems ; Dystrophin/genetics/metabolism ; *Genetic Therapy/methods ; Humans ; Disease Models, Animal ; CRISPR-Associated Protein 9/genetics/metabolism ; Male ; Ribonucleoproteins/genetics/metabolism ; Exons ; Mice, Inbred C57BL ; },
abstract = {Muscle genetic defects can lead to impaired movement, respiratory failure, and other severe symptoms. The development of curative therapies is challenging due to the need for the delivery of gene-editing tools into skeletal muscle cells throughout the body. Here, we use muscular fusogens (Myomaker and Myomerger) to engineer muscle-specific virus-like particles (MuVLPs) for the systemic delivery of gene-editing tools. We demonstrate that MuVLPs can be loaded with diverse payloads, including EGFP, Cre and Cas9/sgRNA ribonucleoproteins (Cas9 RNPs), and can be delivered into skeletal muscle cells via targeted membrane fusion. Systemic administration of MuVLPs carrying Cas9 RNPs enables skeletal muscle-specific gene editing, which excised the exon containing a premature terminator codon mutation in a mouse model for Duchenne muscular dystrophy (DMD). This treatment restores dystrophin expression in various skeletal muscle tissues, including the diaphragm, quadriceps, tibialis anterior, gastrocnemius, and triceps. As a result, the treated mice exhibit a significantly increased capacity for exercise and endurance. This study established a platform for precise gene editing in skeletal muscle tissues.},
}
@article {pmid41093884,
year = {2025},
author = {Moon, J and Zhang, J and Guan, X and Yang, R and Guo, C and Schalper, KT and Avery, L and Banach, D and LaSala, R and Warrier, R and Liu, C},
title = {CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9142},
pmid = {41093884},
issn = {2041-1723},
support = {R01 AI194917/AI/NIAID NIH HHS/United States ; R33 AI154642/AI/NIAID NIH HHS/United States ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Hepacivirus/genetics/isolation &amp; purification ; *RNA, Viral/genetics/blood/analysis ; Temperature ; *CRISPR-Associated Proteins/metabolism/genetics ; HIV Infections/virology/diagnosis/blood ; Hepatitis C/diagnosis/virology ; HIV/genetics/isolation &amp; purification ; HIV-1/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.},
}
@article {pmid41096563,
year = {2025},
author = {Simoni, S and Fambrini, M and Pugliesi, C and Rogo, U},
title = {Genome Editing by Grafting.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41096563},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Genome, Plant ; *Plants, Genetically Modified/genetics ; },
abstract = {Grafting is the process of joining parts of two plants, allowing the exchange of molecules such as small RNAs (including microRNAs and small interfering RNAs), messenger RNAs, and proteins between the rootstock and the scion. Genome editing by grafting exploits RNAs, such as tRNA-like sequences (TLS motifs), to deliver the components (RNA) of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) system from transgenic rootstock to wild-type scion. The complex Cas9 protein and sgRNA-TLS produced in the scion perform the desired modification without the integration of foreign DNA in the plant genome, resulting in heritable transgene-free genome editing. In this review, we examine the current state of the art of this innovation and how it helps address regulatory problems, improves crop recovery and selection, exceeds the usage of viral vectors, and may reduce potential off-target effects. We also discuss the promise of genome editing by grafting for plants recalcitrant to in vitro culture and for agamic-propagated species that must maintain heterozygosity for plant productivity, fruit quality, and adaptation. Furthermore, we explore the limitations of this technique, including variable efficiency, graft incompatibility among genotypes, and challenges in large-scale application, while highlighting its considerable potential for further improvement and future broader applications for crop breeding.},
}
@article {pmid41096677,
year = {2025},
author = {Șerban, M and Toader, C and Covache-Busuioc, RA},
title = {CRISPR and Artificial Intelligence in Neuroregeneration: Closed-Loop Strategies for Precision Medicine, Spinal Cord Repair, and Adaptive Neuro-Oncology.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41096677},
issn = {1422-0067},
mesh = {Humans ; *Artificial Intelligence ; *Precision Medicine/methods ; Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Spinal Cord Regeneration ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Nerve Regeneration/genetics ; },
abstract = {Repairing the central nervous system (CNS) remains one of the most difficult obstacles to overcome in translational neurosciences. This is due to intrinsic growth inhibitors, extracellular matrix issues, the glial scar-form barrier, chronic neuroinflammation, and epigenetic silencing. The purpose of this review is to bring together findings from recent developments in genome editing and computational approaches, which center around the possible convergence of clustered regularly interspaced short palindromic repeats (CRISPR) platforms and artificial intelligence (AI), towards precision neuroregeneration. We wished to outline possible ways in which CRISPR-based systems, including but not limited to Cas9 and Cas12 nucleases, RNA-targeting Cas13, base and prime editors, and transcriptional regulators such as CRISPRa/i, can be applied to potentially reactivate axon-growth programs, alter inhibitory extracellular signaling, reprogram or lineage transform glia to functional neurons, and block oncogenic pathways in glioblastoma. In addition, we wanted to highlight how AI approaches, such as single-cell multi-omics, radiogenomic prediction, development of digital twins, and design of adaptive clinical trials, will increasingly be positioned to act as system-level architects that allow translation of complex datasets into predictive and actionable therapeutic approaches. We examine convergence consumers in spinal cord injury and adaptive neuro-oncology and discuss expanse consumers in ischemic stroke, Alzheimer's disease, Parkinson's disease, and rare neurogenetic syndromes. Finally, we discuss the ethical and regulatory landscape around beyond off-target editing and genomic stability of CRISPR, algorithmic bias, explainability, and equitable access to advanced neurotherapies. Our intent was not to provide a comprehensive inventory of possibilities but rather to provide a conceptual tool where CRISPR acts as a molecular manipulator and AI as a computational integrator, converging to create pathways towards precision neuroregeneration, personalized medicine, and adaptive neurotherapeutics that are ethically sound.},
}
@article {pmid41096689,
year = {2025},
author = {Haldrup, SB and McClements, ME and Cehajic-Kapetanovic, J and Corydon, TJ and MacLaren, RE},
title = {Gene Therapy Strategies for the Treatment of Bestrophinopathies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41096689},
issn = {1422-0067},
support = {NA//Aarhus University/ ; NA//Fight for Sight Denmark/ ; NA//Synoptik Fonden/ ; NA//Maskinfabrikant Jochum Jensen og hustru Mette Marie Jensen, f. Poulsens Mindelegat (fond)/ ; NA//A.P. M&#xf8;ller Foundation/ ; NA//Mette Warburgs Fond/ ; NA//APT Holding/ ; 00038189//VELUX Foundation/ ; 24OC0088426//Novo Nordisk Foundation/ ; NA//NIHR Oxford Biomedical Research Centre/ ; 304408/Z/23/Z//Wellcome Discovery Award/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Bestrophins/genetics/metabolism ; *Retinal Diseases/therapy/genetics ; *Eye Diseases, Hereditary/therapy/genetics ; Gene Editing/methods ; Mutation ; Animals ; CRISPR-Cas Systems ; Chloride Channels/genetics ; },
abstract = {The BEST1 gene encodes a transmembrane protein in the retinal pigment epithelium (RPE) in the eye, that functions as a calcium-dependent chloride channel (CaCC). Pathogenic variants in BEST1 are the underlying cause for bestrophinopathies, a group of inherited retinal disorders that vary in their pattern of inheritance, clinical appearance, and underlying molecular disease mechanisms. Currently, there are no treatments available for any of the bestrophinopathies, and gene therapy represents an attractive strategy due to the accessibility of the eye and slow disease progression. While gene augmentation may be effective for a subset of bestrophinopathies, others require allele-specific silencing or correction of the disease-causing variant to reconstitute expression of the BEST1 protein. This review aims to give an overview of the clinical diversity of bestrophinopathies and proposes the molecular disease mechanism of the pathogenic BEST1 variant as an important parameter for the choice of treatment strategy. Furthermore, we discuss the potential of different mutation-specific and mutation-independent CRISPR/Cas9-based gene editing strategies as a future treatment approach for bestrophinopathies.},
}
@article {pmid41096693,
year = {2025},
author = {He, J and Shi, N and Yao, H and Li, J and Wang, Y and Zhang, J},
title = {Genome Editing in the Chicken: From PGC-Mediated Germline Transmission to Advanced Applications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41096693},
issn = {1422-0067},
support = {2023ZD04053//the Biological Breeding-National Science and Technology Major Project/ ; 2025YFHZ0042//the Natural Science Foundation of Sichuan Province/ ; SCU2025D003//the fundamental research funds for the central universitie/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Chickens/genetics ; *Germ Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Avian genome editing has historically lagged behind mammalian research. This disparity is primarily due to a unique reproductive biology that precludes standard techniques like pronuclear injection. A pivotal breakthrough, however, came from the development of efficient in vitro culture systems for primordial germ cells (PGCs). This has established the chicken as a tractable and powerful model for genetic engineering. Our review chronicles the technological evolution this has enabled, from early untargeted methods to the precision of modern CRISPR-based systems. We then analyze the broad applications of these tools, which are now used to engineer disease resistance, enhance agricultural traits, and develop novel platforms such as surrogate hosts and oviduct bioreactors. Collectively, these advances have established PGC-based genome editing as a robust and versatile platform. Looking forward, emerging precision editors and the expansion of these techniques to other avian species are poised to drive the next wave of innovation in poultry science and biotechnology.},
}
@article {pmid41096720,
year = {2025},
author = {Peng, H and Li, J and Sun, K and Tang, H and Huang, W and Li, X and Wang, S and Ding, K and Han, Z and Li, Z and Xu, L and Wang, K},
title = {Advances and Applications of Plant Base Editing Technologies.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41096720},
issn = {1422-0067},
support = {32472182//National Natural Science Foundation of China/ ; 2024ZD0407704//Biological Breeding-National Science and Technology Major Project/ ; 2022BBF02039//Science and Technology Department of Ningxia in China/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; Genome, Plant ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; },
abstract = {Base editing represents a major breakthrough in the field of genome editing in recent years. By fusing deaminases with the CRISPR/Cas system, it enables precise single-base modifications of DNA. This review systematically summarizes the development of base editing technologies, including cytosine base editors (CBEs), adenine base editors (ABEs), and glycosylase base editors (GBEs), with a particular focus on their applications in crop improvement as well as future trends and prospects. We highlight advances in the creation of novel germplasm with enhanced stress resistance and desirable agronomic traits through base editing in rice, wheat, maize, potato, and other crops, particularly for improving herbicide resistance, disease resistance, and grain quality. Furthermore, we analyze factors that influence base editing efficiency, noting that challenges remain, such as PAM sequence constraints, limited base conversion types, off-target effects, narrow editing windows, and efficiency variation. Future efforts should aim to optimize deaminase activity, expand PAM compatibility, and develop versatile tools to facilitate the broad application of base editing in molecular breeding. This review provides a timely reference for researchers and breeders, offering theoretical guidance and practical insights into harnessing base editing for crop genetic improvement.},
}
@article {pmid41096782,
year = {2025},
author = {Huang, C and Liu, M and Kok, J},
title = {Chromosomal and Plasmid-Based CRISPRi Platforms for Conditional Gene Silencing in Lactococcus lactis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41096782},
issn = {1422-0067},
support = {201706350298//China Scholarship Council/ ; 201505990303//China Scholarship Council/ ; },
mesh = {*Lactococcus lactis/genetics ; *Plasmids/genetics ; *CRISPR-Cas Systems ; *Gene Silencing ; Streptococcus pyogenes/genetics ; Bacterial Proteins/genetics ; Gene Expression Regulation, Bacterial ; *Chromosomes, Bacterial/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Promoter Regions, Genetic ; },
abstract = {Inducible CRISPR interference (CRISPRi) systems were established in Lactococcus lactis using both plasmid and chromosomal approaches. Expression of nuclease-deficient Cas9 (dCas9) from Streptococcus pyogenes was placed under the control of the nisin-inducible promoter PnisA, while sgRNAs were transcribed from the constitutive Pusp45 promoter. To monitor expression, dCas9 was fused with superfolder GFP. Plasmid-based constructs successfully repressed a luciferase reporter gene and silenced the gene of the major autolysin, AcmA, leading to the expected morphological phenotype. However, plasmid systems showed leaky expression, producing mutant phenotypes even without induction. Chromosomal integration of dCas9 reduced its expression level by approximately 20-fold compared with plasmid-based expression, thereby preventing leaky activity and ensuring tight regulation. This chromosome-based (cbCRISPRi) platform enabled controlled repression of the essential gene ybeY, which resulted in severe growth defects. Restoration of wild-type phenotypes was achieved by introducing a synonymous codon substitution in the sgRNA target region. Transcriptome analysis of ybeY-silenced cells revealed downregulation of ribosomal protein genes and widespread effects on membrane-associated proteins, ATP synthase subunits, and various transporters. These inducible CRISPRi platforms provide robust and tunable tools for functional genomics in L. lactis, particularly for studying essential genes that cannot be deleted.},
}
@article {pmid41096922,
year = {2025},
author = {Xu, J and Pan, M and Zhu, Y and Wang, P and Jiang, L and Xu, D and Wang, X and Chen, L and Guo, W and Yang, H and Cao, D},
title = {CRISPR/Cas9-Mediated Targeted Mutagenesis of GmAS1/2 Genes Alters Leaf Shape in Soybean.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41096922},
issn = {1422-0067},
support = {YBXM2529//National Nanfan Research Institute of CAAS/ ; },
mesh = {*Glycine max/genetics/anatomy &amp; histology/growth &amp; development ; *CRISPR-Cas Systems ; *Plant Leaves/genetics/anatomy &amp; histology/growth &amp; development ; Gene Expression Regulation, Plant ; Phenotype ; *Plant Proteins/genetics/metabolism ; *Mutagenesis ; *Genes, Plant ; Gene Editing ; Transcription Factors/genetics ; Gene Expression Profiling ; Mutation ; },
abstract = {ASYMMETRIC LEAVES1 (AS1) and AS2 play essential roles in regulating leaf development in plants. However, their functional roles in soybean remain poorly understood. Here, we identified two members of the soybean AS1 gene family, GmAS1a and GmAS1c, which exhibit high expression levels in stem and leaf tissues. Using the CRISPR/Cas9 system, we targeted four GmAS1 and three GmAS2 genes, generating mutant lines with distinct leaf development phenotypes, including wrinkling (refers to fine lines and creases on the leaf surface, like aged skin texture), curling (describes the inward or outward rolling of leaf edges, deviating from the typical flat shape), and narrow. We found that functional redundancy exists among the four GmAS1 genes in soybean. GmAS1 and GmAS2 cooperatively regulate leaf curling, leaf crinkling phenotypes, and leaf width in soybean, with functional redundancy also observed between these two genes. Transcriptome sequencing analysis of w3 mutant (as1b as1c as1d as2a as2b as2c) identified 1801 differentially expressed genes (DEGs), including 192 transcription factors (TFs). Gene ontology enrichment analysis revealed significant enrichment of DEGs in pathways associated with plant hormone biosynthesis and signal transduction. A detailed examination of the DEGs showed several genes involved in the development of leaf lateral organs, such as KNOX (SHOOT MERISTEMLESS (STM), KNAT1, KNAT2, and KNAT6), LOB (LBD25, LBD30), and ARP5, were down-regulated in w3/WT (wild-type) comparison. CRISPR/Cas9-mediated targeted mutagenesis of the GmAS1/2 genes significantly impairs leaf development and polarity establishment in soybean, providing valuable germplasm resources and a theoretical framework for future studies on leaf morphogenesis.},
}
@article {pmid41097019,
year = {2025},
author = {Kapitonova, MA and Shabalina, AV and Dedkov, VG and Dolgova, AS},
title = {CRISPR-Cas12a-Based Isothermal Detection of Mammarenavirus machupoense Virus: Optimization and Evaluation of Multiplex Capability.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41097019},
issn = {1422-0067},
support = {Ensuring chemical and biological safety of the Russian Federation//state program/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Endodeoxyribonucleases/genetics ; *CRISPR-Associated Proteins/genetics ; *Molecular Diagnostic Techniques/methods ; *Arenaviridae/genetics/isolation &amp; purification ; Animals ; Bacterial Proteins ; },
abstract = {Bolivian hemorrhagic fever (BHF) is a zoonotic disease caused by Mammarenavirus machupoense (MACV) featuring severe neurological and hemorrhagic symptoms and a high mortality rate. BHF is usually diagnosed by serological tests or real-time polymerase chain reaction (RT-PCR); these methods are often inaccessible in endemic regions due to a lack of laboratory infrastructure, creating a demand for sensitive and rapid equipment-free alternatives. Here, we present an isothermal method for MACV nucleic acid detection based on the Cas12a-based DETECTR system combined with recombinase polymerase amplification (RPA) in a single tube: the RT-RPA/DETECTR assay. We demonstrate the possibility of using more than one primer set for the simultaneous detection of MACV genetic variants containing multiple point mutations. The method was optimized and tested using specially developed virus-like armored particles containing the target sequence. The multiplex RT-RPA/DETECTR method achieved a limit of detection of approximately 5 × 10[4] copies/ mL (80 aM) of armored particles. The method was validated using clinical samples spiked with virus-like particles. The assay proved to be selective and reliable in detecting certain nucleotide substitutions simultaneously.},
}
@article {pmid41097026,
year = {2025},
author = {Jeong, SK and Park, JR and Kim, EG and Kim, KM},
title = {Development of Resistance to Damping-Off in Rice, Oryza sativa L., Using CRISPR/Cas9.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41097026},
issn = {1422-0067},
mesh = {*Oryza/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; Rhizoctonia/pathogenicity ; Plant Proteins/genetics ; },
abstract = {Damping-off disease hinders rice seedling growth and reduces yield. Current control methods, such as seed or soil sterilization, rely on chemicals that cause environmental pollution and promote pathogen resistance. As a sustainable alternative, we targeted the damping-off resistance-related gene OsDGTq1 using CRISPR/Cas9. Field experiments first verified OsDGTq1's significance in resistance. The CRISPR/Cas9 system, delivered via Agrobacterium-mediated transformation, was used to edit OsDGTq1 in rice cultivar Ilmi. Lesions from major damping-off pathogens, Rhizoctonia solani and Pythium graminicola, were observed on G0 plants. All 37 regenerated plants contained T-DNA insertions. Among them, edits generated by sgRNA1-1, sgRNA1-2, and sgRNA1-3 resulted in the insertion of two thymine bases as target mutations. Edited lines were assigned names and evaluated for agronomic traits, seed-setting rates, and pathogen responses. Several lines with edited target genes showed distinct disease responses and altered gene expression compared to Ilmi, likely due to CRISPR/Cas9-induced sequence changes. Further studies in subsequent generations are needed to confirm the stability of these edits and their association with resistance. These results confirm that genome editing of OsDGTq1 alters resistance to damping-off. The approach demonstrates that gene-editing technology can accelerate rice breeding, offering an environmentally friendly strategy to develop resistant varieties. Such varieties can reduce chemical inputs, prevent pollution, and minimize seedling loss, ultimately enhancing food self-sufficiency and stabilizing rice supply.},
}
@article {pmid41097079,
year = {2025},
author = {Wu, KC and Chang, YH and Chiang, RY and Ding, DC},
title = {CAP-LAMP2b-Modified Stem Cells' Extracellular Vesicles Hybrid with CRISPR-Cas9 Targeting ADAMTS4 to Reverse IL-1β-Induced Aggrecan Loss in Chondrocytes.},
journal = {International journal of molecular sciences},
volume = {26},
number = {19},
pages = {},
pmid = {41097079},
issn = {1422-0067},
support = {TCRD 113-065//Hualien Tzu Chi Hospital/ ; },
mesh = {*Extracellular Vesicles/metabolism ; Humans ; *Chondrocytes/metabolism/cytology ; *ADAMTS4 Protein/genetics/metabolism ; *CRISPR-Cas Systems ; *Mesenchymal Stem Cells/metabolism/cytology ; *Aggrecans/metabolism ; *Interleukin-1beta/metabolism ; Cell Differentiation ; Cells, Cultured ; Gene Editing ; },
abstract = {Extracellular vesicles (EVs) from mesenchymal stem cells hold therapeutic promise for inflammatory and degenerative diseases; however, limited delivery and targeting capabilities hinder their clinical use. In this study, we sought to enhance the anti-inflammatory and chondroprotective effects of EVs through CAP-LAMP2b (chondrocyte affinity peptide fused to an EV membrane protein) engineering and ADAMTS4 gene editing hybrid vesicle formation. Human umbilical cord MSCs (hUCMSCs) were characterized via morphology, immunophenotyping, and trilineage differentiation. EVs from control and CAP-LAMP2b-transfected hUCMSCs were fused with liposomes carrying CRISPR-Cas9 ADAMTS4 gRNA. DiI-labeled EV uptake was assessed via fluorescence imaging. CAP-LAMP2b was expressed in hUCMSCs and their EVs. EVs exhibited the expected size (~120 nm), morphology, and exosomal markers (CD9, CD63, CD81, HSP70). CAP-modified hybrid EVs significantly enhanced chondrocyte uptake compared to control EVs and liposomes. IL-1β increased ADAMTS4 expression, whereas CAP-LAMP2b-ADAMTS4 EVs, particularly clone SG3, reversed these effects by reducing ADAMTS4 and restoring aggrecan. Western blotting confirmed suppressed ADAMTS4 and elevated aggrecan protein. CAP-LAMP2b-ADAMTS4 EVs, therefore, showed superior uptake and therapeutic efficacy in inflamed chondrocytes, attenuating inflammatory gene expression and preserving matrix integrity. These results support engineered EVs as a promising cell-free approach for cartilage repair and osteoarthritis treatment.},
}
@article {pmid41097540,
year = {2025},
author = {Chen, C and Wu, T and Liu, J and Gao, J},
title = {Threat and Control of tet(X)-Mediated Tigecycline-Resistant Acinetobacter sp. Bacteria.},
journal = {Foods (Basel, Switzerland)},
volume = {14},
number = {19},
pages = {},
pmid = {41097540},
issn = {2304-8158},
support = {32402890//National Natural Science Foundation of China/ ; 2023M732993//China Postdoctoral Science Foundation/ ; },
abstract = {Tigecycline is regarded as one of the last-resort antibiotics against multidrug-resistant (MDR) Acinetobacter sp. bacteria. Recently, the tigecycline-resistant Acinetobacter sp. isolates mediated by tet(X) genes have emerged as a class of global pathogens for humans and food-producing animals. However, the genetic diversities and treatment options were not systematically discussed in the era of One Health. In this review, we provide a detailed illustration of the evolution route, distribution characteristics, horizontal transmission, and rapid detection of tet(X) genes in diverse Acinetobacter species. We also detail the application of chemical drugs, plant extracts, phages, antimicrobial peptides (AMPs), and CRISPR-Cas technologies for controlling tet(X)-positive Acinetobacter sp. pathogens. Despite excellent activities, the antibacterial spectrum and application safety need further evaluation and resolution. It is noted that deep learning is a promising approach to identify more potent antimicrobial compounds.},
}
@article {pmid41097718,
year = {2025},
author = {Lye, SH and Polycarp, N and Durojaye, TJ and Tollefsbol, TO},
title = {Functional Heterogeneity and Context-Dependent Roles of LncRNAs in Breast Cancer.},
journal = {Cancers},
volume = {17},
number = {19},
pages = {},
pmid = {41097718},
issn = {2072-6694},
support = {R01 CA178441/CA/NCI NIH HHS/United States ; R01CA178441/CA/NCI NIH HHS/United States ; },
abstract = {As with other non-coding RNAs (ncRNAs), the aberrant expression of long non-coding RNAs (lncRNAs) can be associated with different forms of cancers, including breast cancer (BC). Various lncRNAs may either promote or suppress cell proliferation, metastasis, and other related cancer signaling pathways by interacting with other cellular machinery, thus affecting the expression of BC-related genes. However, lncRNAs are characterized by features that are unlike protein-coding genes, which pose unique challenges when it comes to their study and utility. They are highly diverse and may display contradictory functions depending on factors like the BC subtype, isoform diversity, epigenetic regulation, subcellular localization, interactions with various molecular partners, and the tumor microenvironment (TME), which contributes to the intratumoral heterogeneity and phenotypic plasticity. While lncRNAs have potential clinical utility, their functional heterogeneity coupled with a current paucity of knowledge of their functions present challenges for clinical translation. Strategies to address this heterogeneity include improving classification systems, employing CRISPR/Cas tools for functional studies, utilizing single-cell and spatial sequencing technologies, and prioritizing robust targets for therapeutic development. A comprehensive understanding of the lncRNA functional heterogeneity and context-dependent behavior is crucial for advancing BC research and precision medicine. This review discusses the sources of lncRNA heterogeneity, their implications in BC biology, and approaches to resolve knowledge gaps in order to harness lncRNAs for clinical applications.},
}
@article {pmid41099664,
year = {2025},
author = {Spavieri, JM and Inacio, TG and Seguchi, G and de Souza, BC and Pereira, GAG and de Mello, F},
title = {Impact of CRISPRi-Mediated Titration of GPD Genes on the Fermentative Performance of S. cerevisiae.},
journal = {ACS synthetic biology},
volume = {14},
number = {11},
pages = {4412-4423},
pmid = {41099664},
issn = {2161-5063},
mesh = {Fermentation ; *CRISPR-Cas Systems/genetics ; Glycerol-3-Phosphate Dehydrogenase (NAD+)/genetics/metabolism ; Gene Editing/methods ; Saccharomyces cerevisiae Proteins ; },
abstract = {Glycerol is one of the main byproducts in ethanol fermentation due to its importance in redox balance and response to osmotic stress in Saccharomyces cerevisiae. Since its production diverts carbon from alcohol production, traditional gene-editing methods have been applied to the glycerol synthesis pathway. However, such approaches generate undesirable phenotypes for industrial applications. In the present study, we employed the CRISPR-dCas9 system to moderately downregulate the expression of GPD1 and GPD2, the two main genes involved in this metabolism. GPD2 gene expression downregulation and a graded reduction in glycerol production after repression of four different target sites in each paralogue were achieved. Employment of the CRISPRi approach for GPD gene modulation resulted in higher specific ethanol productivity (SEP) than that of single knockout cells. Targeted modulation in a region -140 basepairs upstream of the transcription start site (TSS) of GPD1 resulted in a 3% increase in ethanol production compared to the wild type and gpd Δ strains. Such regulation, combined with GPD2 deletion, revealed the higher SEP among all tested strains. Furthermore, a GPD1-modulated strain maintained tolerance to high osmolarity in very high-gravity (VHG) fermentation while maintaining its ethanol production levels above those observed in the control strain.},
}
@article {pmid41099996,
year = {2025},
author = {Shafi, Z and Shahid, M and Ilyas, T and Singh, K},
title = {Next-generation perspectives on microbially synthesized siderophores: molecular engineering, multi-omics insights, and applications for smart climate-resilient crops.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {10},
pages = {393},
pmid = {41099996},
issn = {1573-0972},
mesh = {*Siderophores/biosynthesis/genetics/metabolism ; *Crops, Agricultural/growth &amp; development/microbiology ; Iron/metabolism ; *Bacteria/metabolism/genetics ; Metabolomics ; Gene Editing ; Genomics ; Proteomics ; CRISPR-Cas Systems ; Multiomics ; },
abstract = {Siderophores, low-molecular-weight iron-chelating compounds synthesized by microbes, play a crucial role in iron (Fe) acquisition under Fe-limited conditions. In recent years, their significance in sustainable agriculture has gained increasing attention due to their multifaceted roles in plant growth promotion, stress alleviation, and disease suppression. This review presents next-generation insights into the biosynthesis, regulation, and applications of microbial siderophores, with a focus on advanced molecular and omics-based approaches. Innovations in synthetic biology and CRISPR/Cas-mediated genome editing have enabled precise manipulation of siderophore biosynthetic gene clusters, enhancing their production and functionality. Multi-omics platforms-genomics, transcriptomics, proteomics, and metabolomics-have revealed complex regulatory networks, unveiling cryptic pathways and inter-microbial variability in siderophore synthesis. Furthermore, the use of siderophore-producing plant growth-promoting rhizobacteria (PGPR) has shown promise in improving nutrient uptake, inducing systemic resistance, and mitigating abiotic stresses in crops. The integration of nano-formulations and encapsulation technologies has enhanced the stability and field efficacy of siderophore-based bioinoculants. This review also explores emerging strategies for developing microbial consortia and smart delivery systems to meet the challenges of climate-resilient agriculture. By bridging molecular insights with field-level applications, this article underscores the potential of siderophores as eco-friendly tools for next-generation sustainable farming practices.},
}
@article {pmid41100703,
year = {2025},
author = {Liao, X and Li, Y and Wu, Y and Wen, L and Jing, M and Chen, B and Li, X and Shang, X},
title = {TEMC-Cas: Accurate Cas Protein Classification via Combined Contrastive Learning and Protein Language Models.},
journal = {ACS synthetic biology},
volume = {14},
number = {11},
pages = {4586-4596},
doi = {10.1021/acssynbio.5c00631},
pmid = {41100703},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Deep Learning ; *CRISPR-Associated Proteins/genetics/classification/metabolism ; Software ; },
abstract = {The accurate classification of Cas proteins is crucial for understanding CRISPR-Cas systems and developing genome-editing tools. Here, we present TEMC-Cas, a deep learning framework for accurate classification of Cas proteins that combines a finely tuned ESM protein language model with contrastive learning. Unlike traditional methods that rely on sequence similarity (e.g., BLAST, HMMs) or structural prediction, TEMC-Cas leverages evolutionary-scale modeling to capture distant homology while employing contrastive learning to distinguish closely related subtypes. The framework incorporates LoRA for efficient parameter adaptation and addresses class imbalance through weighted loss functions. TEMC-Cas achieves superior performance in classifying the Cas1-Cas13 families and 17 Cas12 subtypes, demonstrating particular strength in identifying remote homology. This approach provides a robust tool for the discovery of the CRISPR system and expands the toolbox for genome engineering applications. TEMC-Cas is now freely accessible at https://github.com/Xingyu-Liao/TEMC-Cas.},
}
@article {pmid41100915,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Old and new tactics of CRISPR-centric competition between bacteria and bacteriophages.},
journal = {Current opinion in structural biology},
volume = {95},
number = {},
pages = {103168},
doi = {10.1016/j.sbi.2025.103168},
pmid = {41100915},
issn = {1879-033X},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems ; *Bacteria/genetics/virology/metabolism/immunology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR-Cas system provides adaptive immunity for prokaryotes against mobile genetic elements (MGEs) such as bacteriophages and plasmids. As a countermeasure, MGEs have evolved various anti-CRISPR (Acr) mechanisms that neutralize the CRISPR-mediated immunity. Canonical Acr proteins block target binding of Cas proteins in a stoichiometric or enzymatic manner. New findings reveal that Acr also disintegrates functional Cas complexes, induces promiscuous target binding, and mimics Cas proteins and crRNA with defective mutations. Here, we summarize a broad repertoire of structural and functional mechanisms underlying CRISPR-centric competition, highlighting recent discoveries of molecular machinery that modulates CRISPR function.},
}
@article {pmid41101079,
year = {2026},
author = {Ma, Q and Rong, Z and Shen, J and Yang, M and Han, Y},
title = {Integrated one-pot RPA-CRISPR/Cas13a platform enables ultrasensitive and field-deployable JAK2 V617F detection for myeloproliferative neoplasm diagnosis.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117197},
doi = {10.1016/j.jpba.2025.117197},
pmid = {41101079},
issn = {1873-264X},
mesh = {Humans ; *Janus Kinase 2/genetics ; *CRISPR-Cas Systems/genetics ; *Myeloproliferative Disorders/genetics/diagnosis ; Mutation ; Sensitivity and Specificity ; Recombinases ; Real-Time Polymerase Chain Reaction/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Myeloproliferative neoplasms (MPNs) are clonal hematopoietic malignancies driven by mutations like JAK2 V617F, a constitutively activating variant diagnostic for polycythemia vera and subsets of essential thrombocythemia and primary myelofibrosis. Sanger sequencing is the gold standard for detecting JAK2 mutations, but it faces limitations in terms of cost, sensitivity, and portability. CRISPR diagnostics generally requires preamplification, which increases the risk of contamination. There remains an unmet need for field-deployable, rapid MPNs diagnostics in resource-limited settings. We developed ONE-CASPR, a one-pot RPA-CRISPR/Cas13a system enabling ultrasensitive (0.1 % mutant allele frequency), rapid (30 min), portable JAK2 V617F detection. The platform synergizes recombinase polymerase amplification (RPA) with Cas13a trans-cleavage activity in a single-tube reaction at 37 °C, executed via a miniaturized wireless analysis device (CPod) to eliminate cross-contamination while preserving accuracy. Clinical validation across 36 patient samples demonstrated 100 % concordance with real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) in both sensitivity and specificity. ONE-CASPR provides a rapid, sensitive, field-deployable, and user-friendly solution for MPNs point-of-care diagnosis in resource-limited settings, with broad molecular screening application potential.},
}
@article {pmid41101316,
year = {2025},
author = {Chitboonthavisuk, C and Martin, C and Huss, P and Peters, JM and Anantharaman, K and Raman, S},
title = {Systematic genome-wide mapping of host determinants of bacteriophage infectivity.},
journal = {Cell systems},
volume = {16},
number = {11},
pages = {101427},
doi = {10.1016/j.cels.2025.101427},
pmid = {41101316},
issn = {2405-4720},
support = {R21 AI156785/AI/NIAID NIH HHS/United States ; R35 GM143024/GM/NIGMS NIH HHS/United States ; R35 GM150487/GM/NIGMS NIH HHS/United States ; },
mesh = {*Escherichia coli/virology/genetics ; *Bacteriophages/genetics/pathogenicity ; *Host-Pathogen Interactions/genetics ; CRISPR-Cas Systems/genetics ; Genome, Viral/genetics ; Bacteriophage T7/genetics/pathogenicity ; Host Microbial Interactions/genetics ; },
abstract = {Bacterial host factors regulate the infection cycle of bacteriophages. Except for some well-studied host factors (e.g., receptors or restriction-modification systems), the contribution of the rest of the host genome on phage infection remains poorly understood. We developed phage-host analysis using genome-wide CRISPR interference and phage packaging ("PHAGEPACK"), a pooled assay that systematically and comprehensively measures each host gene's impact on phage fitness. PHAGEPACK combines CRISPR interference with phage packaging to link host perturbation to phage fitness during active infection. Using PHAGEPACK, we constructed a genome-wide map of genes impacting T7 phage fitness in permissive E. coli, revealing pathways that affect phage packaging. When applied to the non-permissive E. coli O121, PHAGEPACK identified pathways leading to host resistance; their removal increased phage susceptibility up to a billion-fold. Bioinformatic analysis indicates that phage genomes carry homologs or truncations of key host factors, potentially for fitness advantage. In summary, PHAGEPACK offers insights into phage-host interactions, phage evolution, and bacterial resistance.},
}
@article {pmid41101942,
year = {2025},
author = {Lim, SL and Chin, CH and Chiou, YJ and Hsu, MT and Chiang, PW and Chen, HJ and Tu, YC and Tzou, WS and Tang, SL},
title = {Unveiling Unusual Ecofunctional Traits of Endozoicomonas Species Through Comprehensive Comparative Genomics.},
journal = {Environmental microbiology},
volume = {27},
number = {10},
pages = {e70191},
doi = {10.1111/1462-2920.70191},
pmid = {41101942},
issn = {1462-2920},
support = {AS-IA-109-L05//Academia Sinica/ ; },
mesh = {*Genome, Bacterial ; Genomics ; Phylogeny ; Prophages/genetics ; Bacterial Proteins/genetics ; *Gammaproteobacteria/genetics ; Quorum Sensing ; },
abstract = {Endozoicomonas is an omnipresent marine bacterial genus, associated with various marine organisms, that contributes to host health, nutrient cycling and disease resistance. Nonetheless, its genomic features remain poorly characterised due to a paucity of high-quality genomes. In this study, we sequenced 5 novel Endozoicomonas strains and re-sequenced 1 known strain to improve genomic resolution. By integrating these 6 high-quality genomes with 31 qualified published genomes, our pan-genomic analysis revealed variation in genetic traits among clades. Notably, Endozoicomonas lacks quorum-sensing capabilities, suggesting resistance to quorum quenching mechanisms. It also lacks the capacity to synthesise and transport vitamin B12, indicating that it does not supply this nutrient to holobionts. Remarkably, Endozoicomonas genomes encode 92 identified giant proteins (15-65 kbp). These proteins cluster into three major groups associated with antimicrobial peptide synthesis, exotoxin production and cell adhesion. Additionally, we found that Endozoicomonas has acquired prophages from diverse sources via infection or other types of gene transfer. Notably, CRISPR-Cas sequences suggest evolutionary trajectories independent of both prophage acquisition and phylogenetic lineage, implying potential geographic influences or environmental pressures. This study provides new insights into the genomic diversity of Endozoicomonas and its genetic adaptations to diverse hosts.},
}
@article {pmid41101973,
year = {2025},
author = {Ishizuka, T and Tsuboyama, K and Tomari, Y},
title = {Comprehensive identification and functional analysis of fully disordered proteins essential for cell survival.},
journal = {RNA (New York, N.Y.)},
volume = {32},
number = {1},
pages = {61-70},
pmid = {41101973},
issn = {1469-9001},
mesh = {*Intrinsically Disordered Proteins/genetics/metabolism/chemistry ; Cell Survival/genetics ; Humans ; Genes, Essential ; Introns ; CRISPR-Cas Systems ; },
abstract = {Proteins have traditionally been understood through their tertiary structures, with well-defined conformations considered essential for biological function. This classical structure-function paradigm implies that proteins with high intrinsic disorder would be less critical for cellular survival. Recent discoveries have suggested that some intrinsically disordered proteins or even fully disordered proteins without any apparent tertiary structures are essential. However, the biological significance of such disordered proteins is not comprehensively understood. Here, using genome-wide CRISPR screening, we demonstrated that highly or fully disordered proteins show comparable essentiality to well-folded proteins. We found that the proportion of essential proteins is comparable across proteins of varying disorder levels, although structured proteins are more prevalent among essential genes. Focusing on FAM32A, one of the essential, fully disordered proteins identified in our screen, we show that its depletion leads to increased intron retention and downregulation of many other essential genes. These findings reshape our understanding of the structure-function paradigm, highlighting that fully disordered proteins can be essential for cellular viability.},
}
@article {pmid41102523,
year = {2025},
author = {Savage, N and Danis, E and Chokshi, CR and Custers, S and Shaikh, MV and Miletic, P and Venugopal, C and Brown, KR and Vibhakar, R and Moffat, J and Singh, SK},
title = {CRISPR screen reveals SOX2 as a critical regulator of CD133 and cellular stress response in glioblastoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {36228},
pmid = {41102523},
issn = {2045-2322},
support = {1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; 1065//Terry Fox Research Institute Program Project Grant/ ; },
mesh = {*AC133 Antigen/metabolism/genetics ; *SOXB1 Transcription Factors/genetics/metabolism ; *Glioblastoma/genetics/metabolism/pathology ; Humans ; *CRISPR-Cas Systems ; Neoplastic Stem Cells/metabolism/pathology ; Cell Line, Tumor ; *Brain Neoplasms/genetics/metabolism/pathology ; Gene Expression Regulation, Neoplastic ; *Stress, Physiological ; Animals ; Mice ; },
abstract = {Glioblastoma (GBM) remains a formidable challenge in clinical settings due to limited treatments available. The surface protein CD133 marks glioblastoma stem cells (GSCs), cells capable of overcoming therapeutic pressures and correlate with more aggressiveness tumor phenotypes. In this study, we employed a CRISPR-Cas9 functional screen to deconvolute CD133 dynamics in tumors. This led us to establish that SOX2 is a key player in controlling the PROM1 gene, which in turn influences how cells react to stress factors, including those induced by chemoradiation treatment. The discoveries in this study shed light on the complex web of mechanisms that control the survival and resistance of GSCs, offering promising new avenues for targeting and potentially overcoming therapy resistance.},
}
@article {pmid41102733,
year = {2025},
author = {Marchenko, N and Nesbitt, NM and Alexandrova, E and Reisz, JA and D'Alessandro, A and Suh, J and Uryasev, S and Pennacchia, L and Bahou, WF},
title = {Biliverdin reductase B as a new target in breast cancer.},
journal = {Breast cancer research : BCR},
volume = {27},
number = {1},
pages = {179},
pmid = {41102733},
issn = {1465-542X},
support = {R01 HL146442/HL/NHLBI NIH HHS/United States ; P30 CA046934/CA/NCI NIH HHS/United States ; R43 CA284999/CA/NCI NIH HHS/United States ; P30CA046934/CA/NCI NIH HHS/United States ; NCI CA284999/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology/metabolism/drug therapy ; Female ; Animals ; Cell Line, Tumor ; Mice ; *Oxidoreductases Acting on CH-CH Group Donors/genetics/metabolism/antagonists &amp; inhibitors ; Cell Proliferation ; Oxidation-Reduction ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Enhanced metabolic and mitochondrial activity inherent in actively proliferating cancer cells is associated with intracellular redox imbalance that impacts cellular viability. To restore redox homeostasis cancer cells evolve to activate redox protective mechanisms. This differential activation of redox defense pathways compared to normal cells provides a therapeutic window for novel targeted therapies in cancer. Although heme metabolism emerges as a crucial regulator of redox homeostasis and iron metabolism in cancer cells with frequent alteration in breast cancer, it remains largely unexplored, and no targeted translational approaches have been developed. Heme-regulated redox homeostasis is coordinately maintained through biosynthetic and degradation pathways. As a byproduct of TCA cycle, cytotoxic heme is initially derivatized by heme oxygenases and progressively metabolized to the potent antioxidant bilirubin by two non-redundant biliverdin reductases, BLVRA and BLVRB. BLVRB overexpression has been observed in breast cancers, although its function in breast cancer pathogenesis remains unknown.<br><br>METHODS: CRISPR/Cas9 deletion of BLVRB in multiple breast cancer cell lines demonstrated its profound effect on intracellular redox state and cell proliferation in vitro and in xenograft models. Integrated proteomic, metabolomic, and lipidomic studies identified and validated BLVRB-mediated adaptive metabolic responses required for breast cancer cell cytoprotection.<br><br>RESULTS: We have established BLVRB as a requisite component of the pro-survival redox defense mechanism in breast cancer cells. Targeted deletion of BLVRB induces reductive stress, leading to alterations in endoplasmic reticulum proteostasis and lipid composition. These defects impact plasma membrane functionality and endosomal recycling of multiple oncogenic receptors, such as HER2 and transferrin receptors.<br><br>CONCLUSIONS: These data collectively identify BLVRB as a novel metabolic target in breast cancer, distinct from other redox-regulating pathways. This study, along with our recent progress in developing novel specific BLVRB inhibitors, offers a unique translational opportunity for targeted therapies in personalized breast cancer medicine.},
}
@article {pmid41102841,
year = {2025},
author = {Li, X and Lv, H and Wu, C and Li, H and Yi, W and Niu, X and Peng, Q and Meng, C and Yuan, Y and Tian, S and Wang, Z and Fang, R and Zhou, Z},
title = {Corynebacterium pseudotuberculosis phospholipase D targets mitochondrial sphingomyelin and induces NLRP3-GSDMD axis-mediated pyroptosis in macrophages to promote infection.},
journal = {Veterinary research},
volume = {56},
number = {1},
pages = {198},
pmid = {41102841},
issn = {1297-9716},
support = {2021YFD1800800//National Key Research and Development Program of China/ ; CSTC2021JCYJ-MSXMX0884//Natural Science Foundation of Chongqing/ ; CSTB2024NSCQ-MSX1262//Natural Science Foundation of Chongqing/ ; XDJK2018C054//Fundamental Research Funds for the Central Universities/ ; XDJK2020B016//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Phospholipase D/metabolism ; *Sphingomyelins/metabolism ; *Corynebacterium Infections/enzymology/pathology/veterinary ; Mitochondria/physiology ; Pyroptosis/physiology ; *Macrophages/microbiology/physiology ; Bacterial Zoonoses/enzymology/pathology ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism ; Gasdermins/metabolism ; Phosphate-Binding Proteins/metabolism ; Mice, Inbred C57BL ; Animals ; Mice/microbiology ; CRISPR-Cas Systems ; *Corynebacterium pseudotuberculosis/enzymology/physiology ; Animals, Outbred Strains ; },
abstract = {Infection by Corynebacterium pseudotuberculosis (Cp), a facultative intracellular bacterium, causes mainly purulent inflammation and chronic granulomas in animals and humans. Pyroptosis is a proinflammatory form of programmed cell death that is important in response to pathogen infection. The role of pyroptosis in Cp infection and the related mechanisms are still unclear. Here, we reveal that Cp infection induces pyroptosis in macrophages. The inhibition of pyroptosis by disulfiram (DSF) or dimethyl fumarate (DMF) decreases the pathogenicity of Cp in mice and prevents the escape of this pathogen from infected macrophages. In contrast, LPS treatment achieves the opposite results in mice and macrophages infected with Cp. In addition, we revealed that phospholipase D (PLD) is a key virulence factor that induces pyroptosis and subsequently promotes Cp escape and spread in macrophages and confirmed that mutation of the enzyme active site at D66S, G80I, K114N, and G242P weakens PLD-induced pyroptosis. Furthermore, the indispensable role of the NLRP3-GSDMD axis in PLD-induced pyroptosis was confirmed using Nlrp3 knockout (Nlrp3[-/-]) and Gsdmd knockout (Gsdmd[-/-]) macrophages. Mechanistically, the PLD of Cp targets mitochondrial sphingomyelins within macrophages, induces cardiolipin externalization to the outer mitochondrial membrane, and releases mtROS, leading to pyroptosis. In conclusion, our data indicate that Cp infection induces mitochondrial dysfunction in macrophages through the secretion of PLD, which targets mitochondrial sphingomyelins and induces NLRP3-GSDMD axis-dependent pyroptosis. Inducing pyroptosis is one of the mechanisms underlying the pathogenesis and transmission of Cp, and inhibiting pyroptosis may be an important strategy for controlling this pathogen.},
}
@article {pmid41103153,
year = {2025},
author = {Chen, L and Yun, M and Chen, B and Xie, S and Liu, W and Wang, M and Yan, J and Cai, J and Yang, S and Peng, Q and Xie, D and Lin, Y and Jiang, B},
title = {Loss of CsCLV2 function causes dwarfism and determinates growth in cucumber.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {1},
pages = {e70525},
doi = {10.1111/tpj.70525},
pmid = {41103153},
issn = {1365-313X},
support = {32202477//National Natural Science Foundation of China/ ; },
mesh = {*Cucumis sativus/genetics/growth &amp; development/metabolism ; *Plant Proteins/genetics/metabolism/physiology ; *Dwarfism/genetics ; Phenotype ; Meristem/growth &amp; development/genetics ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Cucumber (Cucumis sativus L.) is a globally important vegetable crop. Ideal plant architecture optimizes spatial utilization, enhances economic coefficient, and facilitates mechanized cultivation. In this study, we identified a dwarf mutant, csdw3, exhibiting reduced plant height, shortened internodes, and fewer internodes. Genetic analysis showed that this dwarf phenotype is controlled by a single recessive gene. Fine-mapping localized the causal locus to an 80 kb region on chromosome 1, where we discovered a 102 bp deletion in CsCLV2, a gene encoding a leucine-rich repeat receptor-like protein homologous to Arabidopsis CLAVATA2. CRISPR-Cas9-generated loss-of-function mutants recapitulated the dwarf phenotype, confirming the role of CsCLV2 in plant height regulation. Histological examination revealed that CsCLV2 disruption causes premature termination of shoot apical meristem (SAM) development, reducing both internode number and length. Protein interaction assays further demonstrated that CsCLV2 associates with receptor-like kinase CsCIK1 (CLAVATA3 INSENSITIVE RECEPTOR KINASES 1), indicating their cooperative function in the CLV-WUS signaling pathway to maintain meristem activity. Our findings uncover a regulator of plant height in cucumber and provide valuable genetic resources for breeding ideotypes optimized for yield and cultivation efficiency.},
}
@article {pmid41104129,
year = {2025},
author = {Pan, L and Wei, L and Luo, S and Ren, B and Li, M and Liang, L and Li, X and Wei, G},
title = {Klebsiella pneumoniae detection by a light-controlled one-pot RPA-CRISPR/Cas12a method.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1669860},
pmid = {41104129},
issn = {2235-2988},
mesh = {*Klebsiella pneumoniae/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Humans ; *Klebsiella Infections/diagnosis/microbiology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Ultraviolet Rays ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: Klebsiella pneumoniae (KP) is a significant pathogenic bacterium responsible for severe infections in hospitals. However, existing traditional detection techniques, such as culture and PCR, are relatively inefficient. Therefore, this study aims to establish a rapid and convenient method for detecting KP.<br><br>METHODS: This study developed a single-tube detection method combining recombinant polymerase amplification (RPA) and light-controlled CRISPR/Cas12a. RPA primers were designed and screened for the rcsA gene of KP to effectively amplify the target. A light-controlled CRISPR/Cas12a system was created using crRNA modified with a photocleavable group (NPOM). The two systems were integrated into a single tube. Following RPA amplification, UV light-controlled release of crRNA inhibition activates CRISPR-mediated target recognition and Cas12a trans-cleavage, detecting fluorescent signals (FD) in conjunction with UV analysis.<br><br>RESULTS: The light-controlled RPA-CRISPR/Cas12a detection platform developed in this study uses a 15 &#x3bc;L reaction system. By optimizing key parameters such as RPA amplification time (20&#xa0;min), primer concentration (400 nM), UV light activation time (30 s), and crRNA/Cas12a concentration (300 nM), the platform achieves optimal detection efficiency. The platform has a fluorescence detection limit of 4.072&#xd7;10[2] copies/reaction and can specifically identify KP in seven common clinical strains. Clinical sample validation demonstrated that the method yields results fully consistent with PCR detection (30/30 agreement rate of 100%), showcasing excellent detection performance and clinical application potential.<br><br>CONCLUSION: We have successfully developed a light-controlled RPA-CRISPR/Cas12a detection system capable of rapidly and highly sensitively detecting KP. This system demonstrates significant advantages in terms of detection speed (completed in as little as 50 minutes), sensitivity (as low as 4.072&#xd7;10[2] copies/reaction), and ease of use, providing an efficient and reliable solution for clinical pathogen detection.},
}
@article {pmid41104521,
year = {2025},
author = {Braunreiter, K and Kempton, A and Mejia-Guerra, MK and Murray, A and Baine, S and Adegboye, K and Haile, A and Kumar Ahuja, SJ and Fedoce, A and Liu, C and Burch, P and Kabadi, AM},
title = {Characterization of a humanized mouse model of Duchenne muscular dystrophy to support the development of genetic medicines.},
journal = {Disease models &amp; mechanisms},
volume = {18},
number = {10},
pages = {},
pmid = {41104521},
issn = {1754-8411},
support = {//Sarepta Therapeutics/ ; },
mesh = {Animals ; *Muscular Dystrophy, Duchenne/therapy/genetics/physiopathology/pathology/blood ; Disease Models, Animal ; Humans ; Dystrophin/metabolism/genetics ; Muscle, Skeletal/pathology/physiopathology ; Mice, Inbred mdx ; *Genetic Therapy ; Mice ; Male ; Diaphragm/pathology ; Biomarkers/blood/metabolism ; Gene Editing ; Muscle Fibers, Skeletal/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Myocardium/pathology/metabolism ; Fibrosis ; },
abstract = {Duchenne muscular dystrophy (DMD) is a rare, progressive neuromuscular disease resulting from DMD variants, leading to loss of functional dystrophin. To evaluate human-targeted genetic medicines for functional dystrophin restoration, humanized genetic models containing the full human locus are required. This study characterized the hDMDΔ52/mdx mouse model previously reported by Pickar-Oliver and colleagues. Genomic characterization confirmed complete DMD duplication with identical exon 52 deletion junctions on both copies. Histological analysis showed increased diaphragm fibrosis and skeletal muscle central nuclei in hDMDΔ52/mdx mice versus hDMD/mdx controls. hDMDΔ52/mdx mice demonstrated reduced tibialis anterior specific force, decreased skeletal muscle fiber diameter, decreased resistance to eccentric contraction-induced damage and cardiac defects. Multiple serum biomarkers of disease were identified. Using a CRISPR/Cas9 gene-editing strategy to restore human functional dystrophin protein expression, detectable dystrophin expression in the heart and skeletal muscle and increased resistance to injury in the tibialis anterior muscle were observed. In summary, hDMDΔ52/mdx mice display multiple physiological and functional deficits associated with DMD pathology, which can be restored by human-targeted therapy, confirming the suitability of this model for developing human-targeted genetic medicines.},
}
@article {pmid41106357,
year = {2025},
author = {Gehrke, F and Puchta, H},
title = {CRISPR meets AI-based robotics: Advancing sustainable agriculture.},
journal = {Cell},
volume = {188},
number = {21},
pages = {5785-5787},
doi = {10.1016/j.cell.2025.09.011},
pmid = {41106357},
issn = {1097-4172},
mesh = {*Artificial Intelligence ; *Robotics/methods ; Gene Editing/methods ; *Crops, Agricultural/genetics ; *Agriculture/methods ; *CRISPR-Cas Systems ; },
abstract = {In this issue of Cell, Xu and colleagues develop an approach integrating genome editing, artificial intelligence, and robotics to enhance crop improvement. By reconfiguring reproductive traits for automated pollination in crops such as tomatoes and soybeans, their approach accelerates hybrid seed production and yields crops with better stress tolerance, flavor, and resilience, supporting sustainable agriculture and crop diversity.},
}
@article {pmid41106392,
year = {2026},
author = {Yang, M and Liu, S and Chen, G and Liu, X and Sun, D and Zhang, J and Wang, Y and Chen, S and Tian, R and Hu, Z},
title = {Structural and functional bases of F. rodentium Cas9 provide insights into CRISPR-Cas protein engineering.},
journal = {Cell genomics},
volume = {6},
number = {1},
pages = {101039},
pmid = {41106392},
issn = {2666-979X},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism/chemistry ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; *Protein Engineering/methods ; Cryoelectron Microscopy ; },
abstract = {The Faecalibaculum rodentium (Fr) CRISPR-Cas9 system exhibits enhanced gene-editing precision and efficiency compared to SpCas9, with distinctive advantages in targeting the TATA box in eukaryotic promoters. However, the underlying molecular mechanisms remained unexplored. Here, we present cryo-electron microscopy structures of the FrCas9-single guide RNA (sgRNA)-DNA complex in both the R-loop expansion and pre-catalytic states, shedding light on its specialized recognition of the 5'-NRTA-3' protospacer adjacent motif (PAM) and the unusual overwinding of the sgRNA-DNA heteroduplex. Our investigations into the structure and extensive mutational analyses reveal that the phosphate lock loop plays a pivotal role in finely adjusting FrCas9's off-target sensitivity and catalytic efficiency. Remarkably, targeted residue substitutions in the phosphate lock loop and the PAM-distal region were found to synergistically enhance both the editing precision and efficiency of FrCas9. These findings advance our understanding of Cas9's accuracy and potency mechanisms while providing a molecular foundation for the rational design and development of next-generation CRISPR technologies.},
}
@article {pmid41106454,
year = {2025},
author = {Xie, D and Wang, J and Huang, L and Zhao, Z and Wang, R and Long, H and Ni, Q and Liu, H and Hu, M and Huang, J},
title = {A novel X-linked retinitis pigmentosa beagle dog model for ophthalmic research established rapidly by editing the RPGR gene in zygotes.},
journal = {Experimental eye research},
volume = {261},
number = {},
pages = {110697},
doi = {10.1016/j.exer.2025.110697},
pmid = {41106454},
issn = {1096-0007},
mesh = {Animals ; *Retinitis Pigmentosa/genetics/metabolism/physiopathology ; Dogs ; *Disease Models, Animal ; *Eye Proteins/genetics ; *Gene Editing/methods ; *Genetic Diseases, X-Linked/genetics ; Female ; *Zygote/metabolism ; Electroretinography ; CRISPR-Cas Systems ; Male ; Mutation ; },
abstract = {Inherited retinal diseases (IRDs) constitute several ocular disorders leading to progressive and severe visual impairment. While significant progress has been made in understanding the genetic basis of IRDs, preclinical animal models are crucial for advancing therapeutic development. Although well-established mouse models exist, the scarcity of large animal models represents a significant limitation. Dogs (Canis familiaris) exhibit numerous physiological and anatomical similarities to humans, rendering them as potential models. X-linked retinitis pigmentosa (XLRP) is a severe, early-onset form of IRDs, primarily caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene. Here, we efficiently generated a stably inherited RPGR-knockout beagle model of XLRP in one-step via CRISPR/Cas9 gene editing tool with multiple gRNAs and zygote microinjection technology. Two F0 generation beagle dogs were successfully obtained with high knockout efficiency in the RPGR gene and the F0 female exhibited characteristic XLRP phenotypes, including progressive deterioration of both rod and cone photoreceptor function, a gradual reduction in the outer nuclear layer thickness from 7 to 35 months, and attenuated retinal arteries with minimal pigmentation. Notably, the F1 male of hemizygous RPGR-knockout beagle dogs displayed more pronounced disease phenotypes by 7 months of age. In conclusion, we successfully generated a stably inherited beagle dog model of XLRP exhibiting an intermediate characteristic disease onset, which is highly suitable for therapeutic research in gene therapy and stem cell transplantation for determining optimal intervention timing and therapeutic dosing.},
}
@article {pmid41107544,
year = {2025},
author = {Whyms, C and Zhao, Y and Addo-Yobo, D and He, H and Whittington, AC and Trasanidou, D and Salazar, CRP and Staals, RHJ and Li, H},
title = {The twist-and-squeeze activation of CARF-fused adenosine deaminase by cyclic oligoadenylates.},
journal = {The EMBO journal},
volume = {44},
number = {23},
pages = {6919-6943},
pmid = {41107544},
issn = {1460-2075},
support = {S10 RR025080/RR/NCRR NIH HHS/United States ; R24 GM145964/GM/NIGMS NIH HHS/United States ; U24 GM116788/GM/NIGMS NIH HHS/United States ; S10 RR024564/RR/NCRR NIH HHS/United States ; R35 GM152081/GM/NIGMS NIH HHS/United States ; },
mesh = {*Adenosine Deaminase/metabolism/chemistry/genetics ; *Adenine Nucleotides/metabolism ; Adenosine Triphosphate/metabolism ; Models, Molecular ; Substrate Specificity ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; *Oligoribonucleotides/metabolism ; *Nucleotides, Cyclic/metabolism ; },
abstract = {The recently identified CARF (CRISPR-associated Rossman-fold) family of proteins play a critical role in prokaryotic defense, mediating cOA (cyclic oligoadenylate)-stimulated ancillary immune responses in the type III CRISPR-Cas systems. Whereas most previously characterized CARF proteins contain nucleic acids or protein degradation effectors, a subset of the family, including the CARF-fused adenosine deaminase (ADA) (Cad1), has recently been shown to convert ATP to ITP. The enzymatic mechanism and the activation process of Cad1, however, remain incompletely understood. Here we present biochemical and structural analyses of a ring nuclease Cad1, revealing its substrate binding specificity and a sequential activation process by cOAs. Despite an overall structural similarity to canonical ADA enzymes, the ADA domain of Cad1 possesses unique structural features that confer a specificity for ATP. Supported by mutational analysis, our structural work demonstrates an allosteric link between the cOA-binding CARF and the ADA domain through a protein network within the hexameric enzyme assembly. Binding of a cA4 molecule to paired CARF domains induces a twisting of the linked ADA domains around one another, which remodels their active sites and alters interactions with neighboring ADA domains, thereby driving a sequential conformational activation mechanism.},
}
@article {pmid41108054,
year = {2025},
author = {Mariki, A and Kohlmeier, KA and Mousavi, SM and Shabani, M},
title = {A systematic review of CRISPR applications in demyelinating peripheral nervous system disorders.},
journal = {Regenerative medicine},
volume = {20},
number = {11},
pages = {653-662},
pmid = {41108054},
issn = {1746-076X},
mesh = {Humans ; Animals ; *Demyelinating Diseases/therapy/genetics ; *CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; *Peripheral Nervous System Diseases/therapy/genetics ; Charcot-Marie-Tooth Disease/therapy/genetics ; Schwann Cells/metabolism/pathology ; Gene Editing ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {AIM: This review evaluates CRISPR-based strategies for myelin regeneration in peripheral demyelinating disorders, with a focus on Guillain - Barré syndrome (GBS) and Charcot - Marie - Tooth disease type 1A (CMT1A). It aims to identify current therapeutic approaches, delivery systems, and gaps in the literature.<br><br>MATERIALS &amp; METHODS: A systematic literature search was conducted across PubMed, Scopus, Web of Science, and Google Scholar for studies published from 2010 onward, following PRISMA guidelines. Study quality was assessed using OHAT and SYRCLE tools, and 14 articles met the inclusion criteria.<br><br>RESULTS: In GBS, CRISPR interventions primarily targeted antiviral immune regulation (AXL, IFI6, IFNL2), inhibition of viral entry mechanisms (Integrin &#x3b1;v&#x3b2;5, SPCS1), and Schwann cell repair. In CMT1A, therapeutic approaches focused on correcting PMP22 overexpression. Lentiviral transduction was the most frequently used delivery method, with no major adverse effects reported. However, most studies were in vitro, and only two were in vivo, highlighting the need for further validation in animal models.<br><br>CONCLUSIONS: CRISPR technology shows considerable potential for addressing peripheral nerve demyelination through precise genetic modifications that may enhance Schwann cell function and support myelin repair. Nevertheless, the field remains at an early discovery stage, with no near-term clinical applicability demonstrated.},
}
@article {pmid41108328,
year = {2025},
author = {Liu, C and Yao, D and Jiang, Y and Huo, C and Zhang, J and Yi, S and Yang, W and Han, Y and Liu, Y and Wang, X},
title = {Electroporation-based CRISPR/Cas9 Gene Editing in Haliotis Discus Hannai.},
journal = {Marine biotechnology (New York, N.Y.)},
volume = {27},
number = {6},
pages = {148},
pmid = {41108328},
issn = {1436-2236},
support = {ZR2023QC107//Natural Science Foundation of Shandong Province/ ; ZR2022QC233//Natural Science Foundation of Shandong Province/ ; 2022LZGC015//Key Research and Development Program of Shandong Province/ ; SDAIT-14-01//Modern Agricultural Industry Technology System of Shandong Province/ ; tstp20240518//Special Funds for the Taishan Scholar Project of Shandong Province/ ; 202203//Special Supporting Funds for Leading Talents above Provincial Level in Yantai City/ ; 2023HD//Aquaculture Seed Industry Promotion Project of Yantai City/ ; },
mesh = {Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; *Gastropoda/embryology/genetics/physiology ; Gene Editing/veterinary ; Aquaculture/methods ; CRISPR-Cas Systems ; Myostatin/genetics ; Male ; Female ; },
abstract = {Abalone, a marine mollusk with significant economic and ecological value, plays a crucial role in sustainable aquaculture. The development and application of CRISPR-Cas9 gene-editing technology have opened up a new path for improving breeding efficiency. CRISPR/Cas9-mediated gene editing has been achieved in abalones via microinjection. In this study, a gene encoding myostatin MSTN in H. discus hannai; was selected as target for conducting the CRISPR-Cas9 gene editing experiment in combination with an electroporation delivery system. Our results showed that all three sgRNAs effectively targeted and cleaved the target segment, with sgRNA1 and sgRNA2 exhibiting high in vitro activity. After electroporation, the effects of transfection on embryonic development of fertilized eggs were observed and statistically analyzed. 12.7 ± 5.4% of the fertilized eggs were damaged and deformed after electroporation. Twenty-four hours after electroporation, surviving larvae were collected for DNA extraction and sequencing. Two potential mutations within the target region of MSTN were identified by sequencing. These results provide a reference for the improvement and development of CRISPR-mediated gene editing methods in marine mollusks such as abalones.},
}
@article {pmid41108536,
year = {2025},
author = {Siddiqui, S and Siddiqui, H and Riguene, E and Nomikos, M},
title = {Zebrafish: A Versatile and Powerful Model for Biomedical Research.},
journal = {BioEssays : news and reviews in molecular, cellular and developmental biology},
volume = {47},
number = {12},
pages = {e70080},
pmid = {41108536},
issn = {1521-1878},
support = {//NPRP-Blue Skies Research Award/ ; NPRP-BSRA1-0504-210082//Qatar Research Development and Innovation Council (QRDI), Doha, Qatar/ ; },
mesh = {*Zebrafish/genetics ; Animals ; Humans ; *Biomedical Research/methods ; *Disease Models, Animal ; Precision Medicine/methods ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Zebrafish (Danio rerio) have become a versatile model in precision medicine, bridging fundamental biology with translational applications. Their optical transparency, rapid development, and high genetic conservation with humans enable real-time imaging and cost-efficient high-throughput screening. Advances in CRISPR/Cas9, prime editing, and morpholino approaches have expanded their utility for modeling diverse human diseases. In addition to well-established roles in cardiovascular, neurological, metabolic, oncological, and infectious disease research, emerging applications include non-invasive larval urine assays, functional validation of rare human variants, host-microbiome interactions, and automated behavioral profiling for neuropsychiatric conditions. Limitations such as species-specific lipid metabolism and limited antibody availability remain, yet recent integration of single-cell transcriptomics, computational modeling, and machine learning is enhancing translational relevance. Collectively, these innovations position zebrafish as a scalable and powerful platform for disease modeling and personalized therapeutic strategies, underscoring their growing impact in the evolving landscape of precision medicine.},
}
@article {pmid41108596,
year = {2026},
author = {Liu, Y and Merino, I and Gutensohn, M and Johansson, AI and Johansson, K and Andersson, M and Hofvander, P and Sitbon, F},
title = {Glycoalkaloid-Free Starch Potatoes Generated by CRISPR/Cas9-Mediated Mutations of Genes in the Glycoalkaloid Biosynthesis Pathway Enable More Sustainable Uses of By-Products From Starch Production.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1344-1358},
pmid = {41108596},
issn = {1467-7652},
support = {//SLU Grogrund/ ; },
mesh = {*Solanum tuberosum/genetics/metabolism ; *Starch/metabolism/biosynthesis ; *CRISPR-Cas Systems/genetics ; Plant Tubers/metabolism/genetics ; Mutation ; Gene Editing ; Plants, Genetically Modified ; Biosynthetic Pathways/genetics ; },
abstract = {Steroidal glycoalkaloids (SGAs) are toxic cholesterol-derived secondary metabolites present in several Solanaceae species. In potato, tuber SGA levels are for reasons of toxicity of concern in both table and starch cultivars. In the latter, SGAs bind to proteins and fibres in starch production side-streams and prevent their further uses as food and feed. To enable more sustainable uses of starch by-products, we have here reduced SGA biosynthesis in a starch potato cultivar using DNA-free CRISPR/Cas9. Six SGA genes were targeted, encoding enzymes acting either before cholesterol (SMO1-L, DWF1-L, DWF7-L), or after (16DOX, CYP88B1, TAMiso2). Editing efficiencies varied between 20% and 49%, and generated mutants were investigated under greenhouse and field conditions. Target mass-spectrometric analyses confirmed reduced SGA levels and alterations of sterol metabolism in mutated events. Plant height and tuber yield were reduced in several events, although this was not correlated to low SGA levels. Several knockout mutants had almost SGA-free leaves and tubers, the latter also under two SGA-inducing conditions. Similarly, both fibre and protein fractions isolated from side-streams in the starch production process from mutant tubers had very low SGA levels. By contrast, the corresponding wild-type SGA levels were almost 10-fold and, respectively, 40-fold higher than the recommended upper safe limit. The results demonstrate that glycoalkaloid-free mutants can be generated and grown with moderate yield reductions under both greenhouse and field conditions. This suggests a potential for sustainable production of high-value products, e.g., food-grade protein and fibre, from starch production side-streams of SGA knockout tubers.},
}
@article {pmid41108631,
year = {2025},
author = {Harzli, I and Mostafa, K and Mutlu, VA and Shamsi, IH and Kavas, M},
title = {Exploring Novel Microbial Approaches for Enhancing Crop Resilience to Abiotic Stress: Mechanisms and Applications.},
journal = {Physiologia plantarum},
volume = {177},
number = {5},
pages = {e70582},
doi = {10.1111/ppl.70582},
pmid = {41108631},
issn = {1399-3054},
support = {121O463//T&#xfc;bitak/ ; },
mesh = {*Crops, Agricultural/microbiology/physiology ; *Stress, Physiological ; Mycorrhizae/physiology ; Gene Editing ; Plant Breeding ; },
abstract = {The current state of agriculture heavily relies on chemical fertilizers and pesticides, which can negatively impact plant nutritional quality, plant health, and productivity. Additionally, abiotic stresses pose significant challenges to global agricultural productivity, threatening food security and crop sustainability. Therefore, developing and implementing sustainable alternatives to chemical fertilizers and pesticides is crucial to enhance agricultural productivity and resilience. Recent research highlights the potential of microorganisms, such as plant growth-promoting rhizobacteria (PGPR), mycorrhizal fungi, and endophytes, as sustainable solutions to improve plant resilience under abiotic stress conditions. However, challenges including scalability, ecological impacts, and the need for standardized application methods persist. This review explores novel microbial approaches to improving crop resilience against abiotic stress, focusing on how microorganisms interact with plants to mitigate stress impacts. Key mechanisms include the production of stress-alleviating compounds, enhanced nutrient uptake, and modulation of plant stress response pathways. We also examine advanced strategies in plant breeding, emphasizing CRISPR/Cas-mediated genome editing technologies as powerful tools for elucidating plant-microbe interactions. A thorough understanding of these interactions is essential for effectively applying genome editing to enhance the functional capacities of plants or associated microbes, ultimately improving key agronomic traits. This review provides a comprehensive overview of these innovative microbial approaches and their practical applications in sustainable agriculture, offering insights into future research directions, such as developing novel microbial strains and optimizing field applications.},
}
@article {pmid41108681,
year = {2025},
author = {Espinoza, A and Ingersoll, S and Ren, X},
title = {Protocol for quantifying phase-separated condensates in living cells using HILO microscopy imaging and genetic engineering.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104151},
pmid = {41108681},
issn = {2666-1667},
mesh = {Humans ; *Genetic Engineering/methods ; CRISPR-Cas Systems/genetics ; *Biomolecular Condensates ; Microscopy, Fluorescence/methods ; *Microscopy/methods ; },
abstract = {Spatial clustering of epigenetic factors through phase separation is an emerging concept in transcriptional regulation and genome organization. Here, we present a protocol to quantify the phase separation capacity of epigenetic factors in living cells via HILO (highly inclined and laminated optical sheet) microscopy. We describe steps for using CRISPR-Cas9 to generate a cell line with two distinct fusion proteins containing fluorophores and either CRE-LoxP- or dTAG-inducible degradation systems. We then detail the procedures for imaging fusion proteins to analyze and quantify phase separation capacity. For complete details on the use and execution of this protocol, please refer to Brown et al.[1].},
}
@article {pmid41109141,
year = {2026},
author = {Song, L and Bu, S and Ding, Y and Chai, Y and Fu, Y and Zhang, P and Yuan, R},
title = {CRISPR-enhanced low-potential ECL sensor based on AuPt-functionalized porphyrin AlOG for ultrasensitive aflatoxin B1 detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {292},
number = {},
pages = {118089},
doi = {10.1016/j.bios.2025.118089},
pmid = {41109141},
issn = {1873-4235},
mesh = {*Aflatoxin B1/analysis/isolation &amp; purification/chemistry ; *Biosensing Techniques/methods ; CRISPR-Cas Systems ; Limit of Detection ; Luminescent Measurements/methods ; Electrochemical Techniques/methods ; *Porphyrins/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Aluminum/chemistry ; Food Contamination/analysis ; },
abstract = {Herein, an ultrasensitive low-potential electrochemiluminescence (ECL) sensor was developed by integrating an AuPt nanoparticle-functionalized porphyrin aluminum-organic gel (P-AlOG@AuPt) emitter with an enhanced CRISPR/Cas12a amplification module for the detection of aflatoxin B1 (AFB1) related to food safety. Impressively, the P-AlOG@AuPt emitter exhibited strong ECL emission and high signal-to-noise ratio at a low triggering potential (-0.8 V), addressing key limitations of previous high-potential aluminum-organic gels. Meanwhile, the in situ enriched AuPt as a coreaction accelerator catalyzed the reduction of coreactant S2O8[2-] to produce abundant SO4[•-] for facilitating its interactions with P-AlOG@AuPt[•-], resulting in a significant ECL enhancement. Furthermore, a synergistic catalytic hairpin assembly (CHA) pre-amplification and tetrahedral DNA nanostructure (TDN)-anchored ssDNA reporters significantly improved the cleavage efficiency of CRISPR/Cas12a, boosting the detection sensitivity of the ECL sensor for AFB1. The established ECL sensor exhibited ultrasensitive detection of AFB1 with a linear range of 0.1 pg mL[-1] - 500 ng mL[-1] and detection limit of 0.044 pg mL[-1], outperforming most reported ECL- and CRISPR-based AFB1 sensors. The practical usability of the proposed ECL sensor was verified by applying it for the quantification of AFB1 in rice samples. This work offers a promising low-potential ECL sensing platform for practical mycotoxin monitoring and broader biosensing applications.},
}
@article {pmid41109509,
year = {2026},
author = {Gujjar, RS and Sherin, S and Batra, S and Mahto, A and Goswami, SK and Kumar, R and Tiwari, RK and Singh, A and Kumari, M},
title = {Emerging trends in the development of efficient CAS nucleases for meticulous gene editing in plants.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {362},
number = {},
pages = {112820},
doi = {10.1016/j.plantsci.2025.112820},
pmid = {41109509},
issn = {1873-2259},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Plants/genetics ; *Endonucleases/genetics/metabolism ; },
abstract = {CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated nuclease) is a formidable and precise gene-editing system, invented as a natural immune system of bacterial defense. The system relies on a guide RNA/CrRNA (Crispr RNA) sequence, specifically designed to bind to the target nucleic acid, enabling the Cas nuclease to act like a molecular scissor and cleave the target sequence. Recent research has substantially advanced the development, optimization, and application of Cas nucleases, particularly Cas9, Cas12, and their engineered orthologs to enhance the fidelity, flexibility, and proficiency of specific edits at the target site. This review focuses on the latest advancements in the research on Cas nucleases such as Cas9, Cas12a, Cas12b, CasΦ, Cas13, and Cas14, along with their mode of function and the exclusive features of each nuclease. Further, we have elaborated on the emergence of redesigned Cas9 orthologs, such as dCas9 (deactivated Cas9) and nCas9 (Cas9 nickase). The modified Cas9 orthologs have empowered the CRISPR-based editing with sophisticated techniques such as base editing and prime editing, which are capable of providing preferred edits with more precision and specificity. These advanced genome editing approaches have been favorably employed to improve the desirable agronomic traits such as stress tolerance, herbicide resistance, and yield stability in a wide range of crops, including rice, maize, wheat, tomato, and cotton. KEY MESSAGE: Our manuscript offers the recent advancement in the research on Cas nucleases, and their modified orthologs to perform CRISPR-based gene edits with enhanced more precision and specificity.},
}
@article {pmid41109516,
year = {2026},
author = {Pandya, K and Kumar, D},
title = {CRISPR/cas genome editing for neurodegenerative diseases: Mechanisms, therapeutic advances, and clinical prospects.},
journal = {Ageing research reviews},
volume = {113},
number = {},
pages = {102922},
doi = {10.1016/j.arr.2025.102922},
pmid = {41109516},
issn = {1872-9649},
mesh = {Humans ; *Gene Editing/methods/trends ; *Neurodegenerative Diseases/genetics/therapy ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Therapy/methods/trends ; },
abstract = {Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Amyotrophic Lateral Sclerosis (ALS), Spinocerebral Ataxia (SCA), and Huntington's disease (HD) are major global health challenges. Current treatments are only symptomatic and do not address the underlying pathogenic genetic mechanisms. The development of the CRISPR/Cas genome editing technologies, has increased possibilities for targeted repair of pathological mutations. CRISPR/Cas9, Cas12, and Cas13 systems enable targeted editing and transcriptome modulation in various preclinical models. CRISPR/Cas9 disruption of mutant APP, Tau, and LRRK2 genes, reducing toxic protein aggregration in AD models has restored normal genetic function. While correction of CAG nucleotide repeats in HD, and reduction of alpha-synuclein expression in PD. RNA targeting systems like Cas13 offers additional therapeutics potential by selectively degrading disease assciated transcript without altering genomic DNA. Advancements in engineered Cas variants with enhanced specificity, such as SpCas9-HF1, base editors and prime editors, with innovative delivery strategies including adeno-associated virus (AAVs) and nanoparticle-based systems, have improved genome editing. However, challenges remain, including off-target effects, mosaicism, and delivery across the BBB, and long-term safety. Ethical consideration focuses on somatic versus germline editing, equitable access, and regulatory oversight. While somatic editing shows acceptance in treating neurological disorders. Germline interventions face strict regulations due to potential multigeneration impacts. Collectively, these technologies are the vanguard of precision molecular medicine, advancing from symptom management towards potentially curative gene therapies for neurological disorders.},
}
@article {pmid41109522,
year = {2025},
author = {Garcia, AFS and Farinati, S and Draga, S and Riommi, D and Gabelli, G and Vannozzi, A and Barcaccia, G and Palumbo, F},
title = {Establishing a cutting-edge protoplast technology platform for applying new genomic techniques in Cichorium spp.},
journal = {New biotechnology},
volume = {90},
number = {},
pages = {206-222},
doi = {10.1016/j.nbt.2025.10.008},
pmid = {41109522},
issn = {1876-4347},
mesh = {*Protoplasts/metabolism ; *Gene Editing/methods ; *Genomics/methods ; CRISPR-Cas Systems ; *Cichorium intybus/genetics ; },
abstract = {Genome editing technologies, especially those based on the CRISPR/Cas9 system, have revolutionized crop breeding by enabling precise genetic modifications. Specifically, delivering preassembled ribonucleoprotein (RNP) complexes-consisting of the Cas9 endonuclease coupled to specific single guide RNAs (sgRNAs)-into protoplasts offers an effective DNA-free method that prevents the integration of foreign genetic material. Despite the availability of detailed protocols, establishing a standardized and efficient in vitro regeneration procedure-from protoplast isolation to whole plant regeneration-remains challenging due to significant variability in regeneration efficiency across different varieties and biotypes. Therefore, optimizing each step is essential to maximize the recovery of successful edited plants. In this study, we developed an efficient protocol for regenerating whole plants from protoplasts isolated from 12 representative Italian varieties of chicory and endive. We focused on leaf chicory and endive biotypes with high horticultural value, including Radicchio types, which are important targets for quality improvement. Our optimized platform supports protoplast isolation, PEG-mediated transfection, and plant regeneration, demonstrating promising potential for future genome editing applications. Notably, the high responsiveness of protoplasts to PEG-mediated transfection suggests that coupling this method with our regeneration procedure could facilitate the use of advanced biotechnological strategies. The combination of high transient transformation efficiency, versatile encapsulation techniques, and successful plant regeneration establishes chicory and endive as promising candidates for DNA-free genome editing via protoplasts, providing a technically precise approach with reduced environmental and economic impacts compared to conventional breeding methods.},
}
@article {pmid41110011,
year = {2025},
author = {De La Mora, CL and Havey, MJ and Krysan, PJ},
title = {Efficient production of gene-edited onion (Allium cepa) plants using biolistic delivery of cas9 RNPs and transient expression constructs.},
journal = {Plant cell reports},
volume = {44},
number = {11},
pages = {243},
pmid = {41110011},
issn = {1432-203X},
support = {1844304//Division of Integrative Organismal Systems/ ; },
mesh = {*Onions/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Biolistics/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics/metabolism ; Plant Proteins/genetics/metabolism ; },
abstract = {Delivery of Cas9/sgRNA RNPs and DNA vectors designed to transiently express morphogenic regulatory genes or an antibiotic resistance gene enabled efficient recovery of gene edited onion lines. We developed a protocol for efficiently producing gene edited onion plants that does not depend on stable transformation. The process makes use of transient gene expression to enrich for gene editing among plants regenerated from immature embryos bombarded with ribonucleoprotein (RNP) complexes composed of CRISPR-associated protein 9 (Cas9) and single guide RNAs (sgRNAs). We used the Allium cepa Downy Mildew Resistant 6 (AcDMR6) gene as the target gene for our studies and produced a total of 47 onion plants with edited AcDMR6 alleles, including 13 homozygous plants, 12 biallelic plants, 7 heterozygous plants, and 15 chimeric plants. The most effective strategy for producing gene edited onion plants involved co-delivering plasmids encoding a hygromycin-resistance protein and plant developmental regulator genes along with the Cas9/sgRNA RNPs followed by transient Hygromycin selection for 48 h. Using this approach, up to 12% of the regenerated onion plants carried edited alleles of AcDMR6. By comparison, no editing was observed among the 146 plants regenerated from explants transfected with the Cas9/sgRNA RNPs alone. The strategy we describe here for using transient gene expression to enrich for gene editing in onion could potentially be extended to other crop species as well.},
}
@article {pmid41110509,
year = {2026},
author = {Faber, NR and Ashok, K and Venkatesan, T and Wertheim, B and Bulgarella, M},
title = {Leveraging advances in RNAi and CRISPR for improved biological pest control.},
journal = {Current opinion in insect science},
volume = {73},
number = {},
pages = {101453},
doi = {10.1016/j.cois.2025.101453},
pmid = {41110509},
issn = {2214-5753},
mesh = {*RNA Interference ; *Pest Control, Biological/methods ; Animals ; *CRISPR-Cas Systems ; *Gene Editing ; *Insecta/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The limitations of chemical pesticides and their associated risks highlight the need for more sustainable pest management strategies. Biological control using natural enemies offers an eco-friendly alternative but is sometimes constrained by efficiency and scalability. Emerging molecular tools-RNA interference (RNAi) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing-present novel opportunities to enhance existing biological control or to control pests directly. RNAi induces targeted gene knockdown via a non-heritable, transient response. CRISPR enables precise genetic modifications and could improve traits in beneficial insects or disrupt essential genes in pests, optionally including a gene drive for increased power. Although limitations remain for several species, these technologies could be valuable tools for integrated pest management. Their future implementation raises biosafety and regulatory considerations, particularly for self-propagating systems like gene drives. This review showcases developments in RNAi and CRISPR-based pest control, and calls for risk-based, adaptive governance to enable their responsible use in sustainable agriculture.},
}
@article {pmid41110569,
year = {2025},
author = {Cui, Z and Liang, W and Li, J and Bai, Z},
title = {CRISPR/Cas9 gene editing strategy for cancer therapy: non-viral nanocarrier-mediated delivery of plasmids, RNA and ribonucleoprotein complexes.},
journal = {International journal of biological macromolecules},
volume = {331},
number = {Pt 1},
pages = {148389},
doi = {10.1016/j.ijbiomac.2025.148389},
pmid = {41110569},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; *Ribonucleoproteins/genetics ; *Neoplasms/therapy/genetics ; *Plasmids/genetics ; Animals ; *RNA/genetics ; *Gene Transfer Techniques ; Genetic Therapy/methods ; Nanoparticles/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing systems are among the most prevalent tools in modern genetic research and have been extensively studied in the context of cancer therapy. CRISPR/Cas9 systems can be categorized into three main delivery forms: plasmid DNA (pDNA) containing both Cas9 and guide RNA (sgRNA), messenger RNA (mRNA) of Cas9 along with sgRNA, and ribonucleoprotein (RNP) complexes consisting of Cas9 protein bound to sgRNA. This article will review the various CRISPR/Cas9 systems, focusing on the non-viral especially nanocarrier delivery strategies and common protocols used for their delivery and the challenges encountered during this process. This review will offer guidance on the potential applications of CRISPR/Cas9 gene editing technology for the treatment of malignant tumors in the future.},
}
@article {pmid41112309,
year = {2025},
author = {Cruz-Cárdenas, JA and López-Arredondo, A and Cázares-Preciado, JA and Rodríguez-Gonzalez, M and Palomares, LA and Brunck, MEG},
title = {Development of CRISPR/Cas9-mediated CD16b[-/-] and CD32a[-/-] promyelocytic cell lines to study FcγR signaling in human neutrophils.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1633609},
pmid = {41112309},
issn = {1664-3224},
mesh = {Humans ; *Receptors, IgG/genetics/metabolism/immunology ; *Neutrophils/immunology/metabolism ; *CRISPR-Cas Systems ; *Signal Transduction/immunology ; HL-60 Cells ; Phagocytosis ; GPI-Linked Proteins/genetics ; Reactive Oxygen Species/metabolism ; Cytokines/metabolism ; },
abstract = {INTRODUCTION: Neutrophils use Fc gamma receptors (FcγRs) to recognize IgG-opsonized pathogens, triggering antimicrobial functions including phagocytosis, ROS production, and cytokine release. CD16b, the most abundant FcγR on neutrophils, plays a key role in initiating these responses, while CD32a is another abundant FcγR on neutrophils that contributes to modulating immune functions. CD16b lacks an intracellular domain and its signaling mechanisms remain unclear. The prevalence of the CD16b-deficient phenotype on donor neutrophils is estimated at <1% of the global population, which complicates its study. To address this, we employed CRISPR/Cas9 to generate HL-60-derived neutrophil-like cells deficient for CD16b or CD32a, that facilitate investigation of their respective roles in neutrophil biology.<br><br>METHODS: We disrupted the FCGR3B or FCGR2A genes using CRISPR/Cas9 in the HL-60 cell line and differentiated clones into neutrophil-like cells using 1.3% DMSO. Functional assays were performed, including phagocytosis, ROS production, SYK phosphorylation, and cytokine responses.<br><br>RESULTS AND DISCUSSION: Both CD16b[-/-] and CD32a[-/-] HL-60-derived clones maintained neutrophilic differentiation and phagocytic capacity but displayed impaired Fc&#x3b3;R-mediated ROS production and SYK phosphorylation, with more pronounced defects in CD16b[-/-] cells. Cytokine production was altered in both lines, with CD16b[-/-] cells producing less IL-6 and IL-1&#x3b2;, and CD32a[-/-] cells producing less TNF-&#x3b1; and IL-10. This model provides new insights into the distinct roles of CD16b and CD32a in neutrophil activation and immune responses.},
}
@article {pmid41113410,
year = {2025},
author = {Feng, L and Huang, Y and Zhao, R and Zhang, K and Yang, W},
title = {[Effect of Different Caenorhabditis elegans U6 Promoters on the Efficiency of CRISPR/Cas9-Mediated Gene Editing].},
journal = {Sichuan da xue xue bao. Yi xue ban = Journal of Sichuan University. Medical science edition},
volume = {56},
number = {4},
pages = {1038-1044},
pmid = {41113410},
issn = {1672-173X},
mesh = {Animals ; *Caenorhabditis elegans/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Promoter Regions, Genetic/genetics ; *RNA, Small Nuclear/genetics ; Plasmids/genetics ; Caenorhabditis elegans Proteins/genetics ; },
abstract = {OBJECTIVE: To investigate the effects of Caenorhabditis elegans (C. elegans) endogenous U6 promoters on dpy-10 gene editing efficiency.<br><br>METHODS: We screened endogenous U6 small nuclear RNA (snRNA) genes of C. elegans from the WormBase database and constructed 14 editing plasmids targeting dpy-10 by replacing the U6 r07e5.16 promoter in the pSX524 plasmid (Peft-3::cas9::tbb-2 terminator::U6 r07e5.16::dpy-10 sgRNA) through molecular cloning. Gene editing was performed in wild-type C. elegans using a standardized microinjection protocol. Gene editing efficiency and the high-efficiency gene editing index were quantified based on the screening of dpy-10 mutant phenotypes in the F1 progeny.<br><br>RESULTS: A total of 15 U6 snRNA genes (r07e5.16, f35c11.9, t20d3.13, k09b11.15, k09b11.16, w05b2.8, c28a5.7, f54c8.9, k09b11.11, k09b11.12, k09b11.14, t20d3.12, f54c8.8, f54c8.10, and k09b11.13) were identified from the WormBase database. Based on the editing efficiency and high-efficiency gene editing index, the activity of these promoters was evaluated, and 4 U6 promoters (w05b2.8, c28a5.7, f54c8.9, and k09b11.11) were found to have significantly enhanced gene editing success rates, outperforming other promoters, including U6 r07e5.16 and U6 k09b11.12 , which are commonly used in the C. elegans research community. Notably, the gRNA[F+E] scaffold did not show superior editing efficiency over the gRNA scaffold when paired with the optimal U6w05b2.8 promoter.<br><br>CONCLUSION: In this study, U6 promoters that significantly improve gene editing efficiency in C. elegans are identified and the critical role of promoter optimization in CRISPR-Cas9 systems is highlighted. These findings provide a valuable foundation for improving genome editing strategies and offer new ideas for optimizing the CRISPR technology applied in nematode research.},
}
@article {pmid41114873,
year = {2025},
author = {Su, CW and Tsai, LC and Hsu, YC and Lu, YS and Lee, JC and Linacre, A and Hsieh, HM},
title = {Saliva identification by RT-LAMP integrated with CRISPR-Cas and LFA.},
journal = {Forensic science, medicine, and pathology},
volume = {21},
number = {4},
pages = {1720-1729},
pmid = {41114873},
issn = {1556-2891},
support = {MOST 110-2320-B-015-001 and MOST 111-2320-B-015-001-MY2//National Science and Technology Council/ ; },
mesh = {Humans ; *Saliva/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Female ; *CRISPR-Cas Systems ; Reproducibility of Results ; Male ; Semen/chemistry ; Cervix Mucus/chemistry ; RNA ; Limit of Detection ; *Molecular Diagnostic Techniques ; Salivary Proteins and Peptides ; },
abstract = {Saliva is a frequently encountered body fluid at crime scenes, however currently there are no definite means to rapidly identify a body fluid as being saliva. In this study, a novel detection method for saliva using a modified Loop-mediated Isothermal Amplification (LAMP) integrated with CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein) and LFA (Lateral Flow Assay) was developed to detect the expression of a saliva-specific gene: follicular dendric cell secreted protein (FDCSP). To determine the specificity of the assay, RNA from saliva plus other commonly encountered body fluids was tested (peripheral blood, semen, vaginal fluid, and menstrual blood): positive results were only observed from RNA extracted from known saliva samples and RNA from all the other body fluids exhibited a negative result. To assess the reproducibility, triplicates were used from one saliva sample, and the assay was performed on three different days: positive results were observed from all triplicates. The limit of detection was 2[-6] (0.3906 ng RNA) or 2[-7] (0.1953 ng RNA). This preliminary study for the identification of saliva requires no complex equipment and is easy to perform, offering an alternative means for body fluid identification.},
}
@article {pmid41115973,
year = {2025},
author = {Mladenov, E and Kallies, M and Stuschke, M and Gkika, E and Iliakis, G},
title = {CRISPR/Cas9 generated DSB clusters mimic complex lesions induced by high-LET radiation and shift repair from c-NHEJ to mutagenic repair pathways.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {36480},
pmid = {41115973},
issn = {2045-2322},
mesh = {*CRISPR-Cas Systems ; Humans ; *DNA Breaks, Double-Stranded/radiation effects ; *DNA End-Joining Repair/radiation effects/genetics ; *Linear Energy Transfer ; Hypoxanthine Phosphoribosyltransferase/genetics ; Animals ; *DNA Repair ; Cell Line ; Poly (ADP-Ribose) Polymerase-1/genetics/metabolism ; Mutagenesis ; },
abstract = {DNA double-strand break (DSB) clusters are a hallmark of high-linear energy transfer (high-LET) radiation and are associated with pronounced biological effects, including reduced cell survival and elevated genomic instability. Our previous work in Chinese hamster cells, engineered with variably designed clusters of I-SceI recognition sites, integrated at multiple genomic locations, revealed that DSB clusters suppress classical non-homologous end-joining (c-NHEJ) and induce chromosomal translocations that ultimately increase cell lethality. Here, we extend this line of investigation to human cell lines and generate DSB clusters using alternative approaches that do not require prior genetic manipulation of the test cell lines. We employ CRISPR/Cas9-technology to generate DSB clusters of specific design at a selected genomic locus and examine their consequences on locus integrity. We target Exon 3 of the human HPRT (hHPRT) gene and introduce single DSBs or DSB clusters of varying numbers and inter-DSB distances. Alterations at the locus reflecting hHPRT gene inactivation, are quantified as mutations causing resistance to 6-thioguanine (6TG). Our results show that DSB clusters are markedly more potent inducers of mutations than single DSBs and that DSBs spaced within ~ 600 base pairs synergize in mutation induction. Mechanistic analyses using small-molecule inhibitors and engineered gene knockout cell lines reveal that the increased mutagenicity of clustered DSBs is primarily mediated by DNA end resection and PARP1-dependent alternative end-joining (alt-EJ) pathways. These findings reinforce the biological relevance of DSB clusters as a severe form of complex DNA damage and provide mechanistic insights into high-LET radiation-induced increased cell killing and genomic instability.},
}
@article {pmid41116722,
year = {2025},
author = {Lin, Y and Jiang, D and Dong, X and Li, Y and Wu, X and Li, R and Li, F and Sun, D and Yu, Y},
title = {A label-free orthogonal dual-channel CRISPR-Cas platform for simultaneous detection of Mycobacterium tuberculosis and respiratory syncytial virus.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {91},
pages = {17914-17917},
doi = {10.1039/d5cc05269f},
pmid = {41116722},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems ; *Mycobacterium tuberculosis/isolation &amp; purification/genetics ; Humans ; *Respiratory Syncytial Viruses/isolation &amp; purification/genetics ; Aptamers, Nucleotide/chemistry ; Biosensing Techniques ; },
abstract = {A label-free, orthogonal dual-channel CRISPR-Cas platform is developed for the simultaneous detection of diverse pathogens. Cas12a is reported by a G4-PPIX complex, and Cas13 by a split Broccoli-DFHBI aptamer, which ensure exclusive channel specificity and eliminate optical cross-talk. Clinical tests on 50 samples show 100% concordance with PCR.},
}
@article {pmid41117290,
year = {2025},
author = {Ye, T and Xue, M and Xu, Y and Fan, M and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Inverted Tetrahedral DNA Reporters Enable Label-Free Ratiometric CRISPR Electrochemical Aptasensing of Kanamycin.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {43},
pages = {27694-27702},
doi = {10.1021/acs.jafc.5c06928},
pmid = {41117290},
issn = {1520-5118},
mesh = {*Kanamycin/analysis ; *Anti-Bacterial Agents/analysis ; CRISPR-Cas Systems ; *Electrochemical Techniques/instrumentation/methods ; Biosensing Techniques/instrumentation/methods ; Food Contamination/analysis ; Food Analysis/methods ; Limit of Detection ; Milk/chemistry ; *DNA Nanostructures/chemistry ; },
abstract = {Integrating CRISPR technology with electrochemical sensing has promising potential in point-of-care testing applications. However, inappropriate immobilization of the reporter on the heterogeneous surface leads to a poor trans-cleavage efficiency. Additionally, the accuracy and reliability of electrochemical sensing still face challenges. Herein, an inverted tetrahedral DNA reporter was developed for electrochemical CRISPR aptasensing. Thiol-modified single-strand oligonucleotides were self-assembled on the edge of tetrahedral DNA nanostructures (TDNs) as a scaffold, enabling an inverted immobilization of DNA tetrahedra via the Au-S bond. The trans-cleavage activity of CRISPR/Cas12a on the single-stranded oligonucleotides resulted in the TDNs dissociating from the electrode surface. The recovery of electron transfer of potassium ferricyanide on the electrode enhances the electrochemical response, while the signal of adsorbed methylene blue on the skeleton of TDNs decreases, enabling a ratiometric signal output. As a proof of concept, the proposed inverted tetrahedral DNA reporters were employed to develop a label-free ratiometric electrochemical aptasensing method for kanamycin detection. Under the optimal conditions, as low as 0.35 pM kanamycin was detected in 50 min with a 4 orders of magnitude dynamic range from 1 pM to 10 nM. Furthermore, the practical application ability of the proposed method for kanamycin detection in a spiked milk sample was also demonstrated. This work offers a new perspective for electrochemical CRISPR sensing development.},
}
@article {pmid41117602,
year = {2025},
author = {Keshry, SS and Nayak, U and Mamidi, P and Mohanty, S and Ghorai, U and Swain, RK and Chattopadhyay, S},
title = {CRISPR-Induced Mutations of mk2b and mk3 Host Proteins Enhance Chikungunya Virus Susceptibility and Modulate Host Immune Responses in Zebrafish.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {20},
pages = {e71112},
doi = {10.1096/fj.202501236RR},
pmid = {41117602},
issn = {1530-6860},
support = {113-2334-6704/2K23/1//CSIR | Human Resource Development Centre, Council of Scientific And Industrial Research (HRDC)/ ; IF180156//Department of Science and Technology, Ministry of Science and Technology, India (DST)/ ; BT/PR42322/TRM/120/525/2021//Department of Biotechnology, Ministry of Science and Technology, India (DBT)/ ; },
mesh = {Animals ; *Zebrafish/genetics/virology/immunology ; *Chikungunya virus/immunology ; *Chikungunya Fever/immunology/genetics/virology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Intracellular Signaling Peptides and Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Mutation ; Clustered Regularly Interspaced Short Palindromic Repeats ; MAP-Kinase-Activated Kinase 2 ; },
abstract = {Host factors are essential at every stage of the viral life cycle and therefore represent attractive and potentially effective targets for the development of antiviral therapeutics. This study highlights the crucial roles of host factors, specifically mitogen-activated protein kinase 2 (mk2) and mitogen-activated protein kinase 3 (mk3), both of which are stress-stimulated serine/threonine kinases. The roles of mk2 and mk3 were investigated by generating single (mk2b-/- and mk3-/-) and double knockouts (mk2b-/-mk3-/-) in a zebrafish model using the CRISPR-Cas9 technique, followed by chikungunya virus (CHIKV) infection. All knockout lines exhibited significantly higher CHIKV titers and severe phenotypes compared to the WT control, with mk3[-]/[-] showing the greatest susceptibility. After CHIKV infection, expression levels of TNF-α changed across all knockout models. Notably, mk2b[-]/[-] and mk2b[-]/[-]mk3[-]/[-] double knockout larvae exhibited reduced TNF-α expression, suggesting that higher levels of TNF-α may be associated with viral clearance via the p38-MK2-TNF-α signaling axis. In contrast, mk3-/- zebrafish exhibited increased vulnerability to CHIKV through alternative, yet unidentified, pathways. Furthermore, an increase in viral titer corresponded with an enhanced host immune response, as indicated by significantly higher expression levels of ifnɸ1 and rsad2 in all knockout groups. In conclusion, this study confirms that the mk2b and mk3 host proteins are essential in controlling CHIKV infection at the organism level. These findings might have implications towards designing strategies for future antiviral therapeutics. Furthermore, the knockout model of mk2b and mk3 in zebrafish could serve as a valuable tool for studying their roles in other viral infections.},
}
@article {pmid41117937,
year = {2025},
author = {Menge, S and Segura, I and Hartmann, M and Decker, L and Kiran, S and Danzer, KM and Iben, S and Harbauer, AB and Oeckl, P and Freischmidt, A},
title = {Comparing loss of individual fragile X proteins suggests strong links to cellular senescence and aging.},
journal = {Cellular and molecular life sciences : CMLS},
volume = {82},
number = {1},
pages = {358},
pmid = {41117937},
issn = {1420-9071},
mesh = {Humans ; *Cellular Senescence/genetics ; *Fragile X Messenger Ribonucleoprotein 1/genetics/metabolism ; *Aging/genetics/metabolism ; *RNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Autophagy ; Cell Line, Tumor ; Proteomics/methods ; Mitochondria/metabolism ; Fragile X Syndrome/metabolism/genetics/pathology ; Neurodegenerative Diseases/metabolism/genetics/pathology ; },
abstract = {Members of the fragile X protein (FXP) family (FMR1, FXR1 and FXR2) are differentially expressed in most types of cancer and major neurodegenerative diseases. While increased expression of FXR1 in cancer has been linked to senescence evasion and consequently tumor initiation and progression, decreased expression of FXPs in neurodegeneration may contribute to pathogenic protein aggregation and death of vulnerable neurons. However, due the causal role in fragile x syndrome, most data are available about loss of FMR1 in neurons while functions of FXR1 and especially FXR2 remain largely unexplored. To address this knowledge gap, and to directly compare functions of the FXPs, we used proteomics of CRISPR/Cas9 edited HAP1 cells carrying knockouts of the individual FXPs for identification of cellular mechanisms associated with these proteins. Further exploration of proteomic findings suggests roles of the FXPs in ribosome biogenesis, autophagy and mitochondrial health linked to organismal aging, and cellular senescence. Validation of FXP induced defects relevant for neurodegenerative diseases in neuroblastoma cell line SH-SY5Y upon FXP knockdown revealed high cell type specificity of individual FXP functions. Overall, we provide a comprehensive overview and comparison of cellular mechanisms related to the individual FXPs, as well as starting points for further studying this protein family in respective cell types of FXP associated diseases, and in aging in general.},
}
@article {pmid41118255,
year = {2025},
author = {Ong, CJN and Elesho, OE and Bramwell, BB and Cabuhat, KSP and Bacalzo, GD and Nuevo, JJM and Fortaleza, JAG},
title = {Staphylococcus aureus: Antimicrobial resistance, quorum sensing, and antibiofilm approaches.},
journal = {European journal of microbiology &amp; immunology},
volume = {15},
number = {4},
pages = {195-209},
pmid = {41118255},
issn = {2062-509X},
abstract = {Staphylococcus aureus is a clinically important bacterial pathogen causing infections from superficial skin lesions to life-threatening systemic diseases. The emergence of methicillin-resistant S. aureus (MRSA) has compounded the global health burden, particularly in low- and middle-income countries, as its quorum-sensing (QS) mediated mechanisms contribute to its persistence, resistance, and evasion from host immune responses and antimicrobial treatments. Thus, these features compromise the effectiveness of conventional antibiotics, urging the need for alternative therapeutic approaches. To resolve these issues, several non-antibiotic antibiofilm approaches have been developed. Bacteriophages and phage-derived enzymes show promising specificity in lysing bacterial cells and disrupting biofilms. Antimicrobial peptides (AMPs), with their broad-spectrum activity, destabilize bacterial membranes and modulate immune responses. Monoclonal antibodies can neutralize toxins or inhibit adhesion molecules within biofilms. Phytochemicals have demonstrated activity against QS pathways and efflux pumps. Metal ion chelators like deferiprone interfere with iron acquisition, which is essential for biofilm stability. Nanoparticles (NPs), ranging from metallic and polymeric to lipid-based and cyclodextrin-based systems, enhance drug delivery and biofilm penetration. CRISPR-Cas systems provide precise genome editing to target resistance genes and virulence factors. Rhamnolipids disrupt biofilm matrix integrity, while enzymes such as dispersin B degrade extracellular polymeric substances. Photodynamic and laser therapies offer localized disruption of biofilm structures through oxidative stress. Collectively, this review offers a transformative complementary approach to traditional antibiotics, enhancing treatment efficacy while potentially reducing the emergence of resistance. Continued research on delivery systems, safety profiles, and synergistic combinations will be pivotal for their clinical translation against S. aureus infections.},
}
@article {pmid41118394,
year = {2025},
author = {Gouin, Y and Wilcockson, A and Chan, AM and Suttle, CA and Zhong, KX},
title = {The genome of Gallaecimonas pentaromativorans strain 10A, isolated from a Pacific oyster, sheds light on an environmentally widespread genus with remarkable metabolic potential.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334406},
pmid = {41118394},
issn = {1932-6203},
mesh = {Animals ; *Genome, Bacterial ; Phylogeny ; *Ostreidae/microbiology ; },
abstract = {Bacteria in the genus Gallaecimonas are known for their ability to breakdown complex hydrocarbons, making them of particular ecological and biotechnological significance. However, few species have been isolated to date, and their ecological distribution has yet to be examined. Here, we report a novel strain of G. pentaromativorans, designated as strain 10A, which was isolated from a Pacific oyster (Magallana gigas, a.k.a. Crassostrea gigas) collected from a farm experiencing a mass mortality event in British Columbia (BC), Canada. Gallaecimonas pentaromativorans strain 10A is a rod-shaped, motile bacterium and has a circular genome of 4,322,156 bp encoding 3,928 protein-coding sequences (CDS). Phylogenetic analysis showed that strain 10A is closely related to members of G. pentaromativorans. Like other Gallaecimonas members, strain 10A is predicted to harbor specific pathways involved in degrading xenobiotic compounds including polycyclic aromatic hydrocarbons (PAHs), producing biosurfactants, and assimilating nitrate and sulfate; however, it is uniquely equipped with an additional 166 genes belonging to 147 protein families, including a putative higB-higA that likely contributes to enhanced stress response. Strain 10A also possesses Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated (Cas) system (CRISPR-Cas), prevalent in Gallaecimonas (detected in three out of four species), implying a potential defense mechanism against exogenous mobile genetic elements such as plasmids and viruses. We also mined publicly available databases to establish the widespread distribution of bacteria in the genus Gallaecimonas in seawater, sediments, and freshwater across latitude, suggesting its versatility and importance to environmental processes. Ultimately, this study demonstrates that the genome of G. pentaromativorans strain 10A, isolated from a Pacific oyster, may encode a suite of putative functions, including xenobiotic breakdown, biosurfactant production, and CRISPR-Cas defense. This plasticity and breadth in metabolic function help to explain the cosmopolitan distribution of members of this genus.},
}
@article {pmid41118570,
year = {2025},
author = {Braithwaite, J and Cannon, C and Chalmers, R and Edwards, H},
title = {Single-colony resolution of CRISPR-Cas adaptation in E. coli reveals altered spacer-source bias during solid-phase growth.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
pmid = {41118570},
issn = {1362-4962},
support = {RPG-2020-079//Leverhulme Trust/ ; //University of Nottingham/ ; },
mesh = {*Escherichia coli/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Plasmids/genetics ; Escherichia coli Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Lac Operon ; Adaptation, Physiological/genetics ; *DNA, Intergenic/genetics ; },
abstract = {CRISPR-Cas systems provide adaptive immunity by integrating short DNA fragments from mobile genetic elements into host arrays. While the core biochemical mechanism of adaptation is well defined, its modulation by physiological contexts is less well understood. Here, we present a visual papillation assay that enables single-colony detection of CRISPR-Cas adaptation in Escherichia coli. Spacer acquisition restores the reading frame of a disrupted lacZ gene, forming blue papillae on lactose X-gal plates. The assay is semi-quantitative, highly sensitive, capable of detecting single events among 109 cells, and responds predictably to Cas1-Cas2 expression levels. Spacer mapping revealed a major shift in source bias: in liquid culture, 64% of spacers were plasmid-derived, but on solid medium this dropped to ∼9%. Adjusting inducer concentration to match liquid conditions did not restore plasmid bias, indicating a physiological basis linked to colony growth. Accounting for the molar excess of chromosomal DNA, the 9% plasmid share reflects near-neutral DNA source sampling rather than plasmid overrepresentation. These findings suggest that the spatial and metabolic structure of colonies strongly shapes the adaptation landscape. The assay provides a scalable platform for dissecting condition-specific features of CRISPR-Cas adaptation, including spacer origin, sequence features, and growth context.},
}
@article {pmid41118576,
year = {2025},
author = {Shu, WJ and Ma, Z and Jia, L and Guo, B and Tian, X and He, C and Wang, F},
title = {MiR-ON-CRISPR: a microRNA-activated CRISPR-dCas9 system for precise gene therapy in living cells and mouse models of sepsis.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
pmid = {41118576},
issn = {1362-4962},
support = {32271512//National Natural Science Foundation of China/ ; 82572281//National Natural Science Foundation of China/ ; 32201199//National Natural Science Foundation of China/ ; 2023-JC-ZD-43//Natural Science Basic Research Program of Shaanxi/ ; 2025JC-YBQN-1129//Natural Science Basic Research Program of Shaanxi/ ; 2022JC-56//Natural Science Basic Research Program of Shaanxi/ ; 2024SF-YBXM-138//Key Research and Development Program of Shaanxi/ ; 0959202513035//Young Talent Fund of Xi'an Association for Science and Technology/ ; 24YXYJ0031//Xi'an Science and Technology Research Program/ ; },
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; *Sepsis/therapy/genetics/pathology ; *CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; *Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Endoplasmic Reticulum Stress/genetics ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; NF-E2-Related Factor 2/genetics/metabolism ; },
abstract = {The CRISPR-dCas9 technology is a powerful tool for manipulating the expression of target genes in a variety of biomedical applications. Nevertheless, it is imperative that the activity of the CRISPR-dCas9 system be tightly controlled to improve its safety and applicability. In this study, we successfully designed a microRNA-activated CRISPR-dCas9 system, termed miR-ON-CRISPR, in which the core components (dCas9 and sgRNA) are both regulated by endogenous miRNA. Our findings demonstrated that the miR-ON-CRISPR system can regulate firefly luciferase reporter gene expression to faithfully visualize miRNA activity and image the differentiation status of neural cells. Moreover, the miR-ON-CRISPR was designed as an AND/OR gate system, thereby enabling the simultaneous detection of two distinct miRNAs. Furthermore, the system was adapted to achieve cell type-specific killing by activating the exogenous DTA genes or endogenous BAX genes. Finally, in mouse models of sepsis, the miR-ON-CRISPR system was shown to alleviate the sepsis-induced liver injury as well as the associated oxidative stress damage and endoplasmic reticulum stress via activating the nuclear erythroid 2-related factor 2 gene. In conclusion, this proof-of-concept study demonstrates the feasibility of the miR-ON-CRISPR system for cell type-specific control of CRISPR-dCas9 activity and its therapeutic applications in the treatment of genetic diseases.},
}
@article {pmid41118579,
year = {2025},
author = {Hanai, Y and Hilario, PLL and Shiraishi, Y and Yoshida, N and Murakami, S and Shimizu, Y and Kano, N and Kojima, M and Murai, K and Kawai, T and Okamura, K},
title = {The knock-in atlas: a web resource for targeted protein trap by CRISPR/Cas9 in human and mouse cell lines.},
journal = {Nucleic acids research},
volume = {53},
number = {19},
pages = {},
pmid = {41118579},
issn = {1362-4962},
support = {//NAIST for the Creation of Innovation in Science and Technology/ ; 24KJ1692//JSPS Research Fellowship/ ; 25KJ1828//NAIST Granite Program/ ; 17K20145//JSPS Found for the Promotion of Joint International Research/ ; //Academic Assistant Grant by Office for Gender Equality at NAIST/ ; //Takeda Science Foundation/ ; 20H03468//KAKENHI/ ; 23K14546//JSPS/ ; },
mesh = {Humans ; Animals ; *CRISPR-Cas Systems/genetics ; Mice ; *Gene Knock-In Techniques/methods ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Internet ; Cell Line ; RNA-Binding Proteins/genetics ; HeLa Cells ; *Databases, Genetic ; },
abstract = {Various cell engineering techniques have been developed by leveraging the CRISPR-Cas9 technology, but large-scale resources for targeted gene knock-in are still limited. Here we introduce the Knock-in Atlas, a web resource for gene tagging by fluorescent proteins by inserting artificial exons in target gene introns. To produce knock-in cells efficiently and reproducibly, we carefully chose and catalogued guide RNAs (gRNAs) for targeting genes in the human and mouse genomes by taking the gRNA efficacy scores and protein structures around the insertion sites into account. As of August 2025, we have characterized knock-in cell lines for 350 proteins, with a focus on RNA binding proteins, by flow cytometry and confocal microscopy. The transfection and flow cytometry protocols were optimized for several cell lines including HEK293T, eHAP1, HeLa, THP-1, Neuro2a, mouse embryonic fibroblast (MEF) and mouse embryonic stem cell (mESC). A website has been launched to organize the results of initial characterization including flow cytometry data after transfection, confocal microscopy, and western blot results for the genes for which knock-in HEK293T cell lines were already made. The site also provides a database to organize the information of pre-designed gRNAs for the human and mouse genomes. <https://rnabio.naist.jp/atlas/>.},
}
@article {pmid41120304,
year = {2025},
author = {Wang, Z and Liu, F and Chen, N and Wu, J and Li, X and Fang, M and Yan, M and Zhang, J and Deng, B and Wang, L and Wang, X and Liu, M and Zeng, D and Zou, Z and Wang, B and Songyang, Z and He, B and Liu, Q},
title = {Chromatin looping-based CRISPR screen identifies TLK2 as chromatin loop formation regulator in cancer stemness plasticity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9288},
pmid = {41120304},
issn = {2041-1723},
support = {82321003//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82341020//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82473123//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82173367//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82403653//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82302929//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82303009//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2022A1515010915//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; },
mesh = {Humans ; *Chromatin/metabolism/genetics ; Animals ; *Neoplastic Stem Cells/metabolism/pathology ; Female ; Mice ; Kruppel-Like Factor 4 ; *Breast Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/metabolism/genetics ; CCCTC-Binding Factor/metabolism/genetics ; Cell Plasticity/genetics ; Kruppel-Like Transcription Factors/genetics/metabolism ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Cell Cycle Proteins/metabolism ; Chromosomal Proteins, Non-Histone/metabolism ; Lung Neoplasms/secondary/genetics ; },
abstract = {Targeting cancer cell plasticity through chromatin organization is an emerging research area, yet the molecular mechanisms that govern chromatin loop formation remain unclear. Here, we develop a CRISPR screen based on our engineered live-cell CTCF-cohesin contact reporters to identify regulators of chromatin loops. Our findings reveal that tousled-like kinase 2 (TLK2) functions as a key regulator of chromatin loop formation during the cancer stemness transition. Mechanistically, TLK2 phosphorylates DYNLL1, enhancing its interaction with CTCF to promote CTCF-cohesin hub formation at the KLF4 locus. Suppressing TLK2 impairs cancer stemness plasticity, sensitizes cancer cells to cytotoxic stress in vitro, and reduces lung metastases and enhances immunotherapy response in breast cancer mouse models. Clinically, elevated TLK2 expression correlates with poor prognosis in breast cancer patients. Collectively, these findings identify TLK2 as a potential therapeutic target for mitigating cancer stemness plasticity, highlighting chromatin loop-targeting therapy as a promising strategy to eradicate cancer stem cells.},
}
@article {pmid41121296,
year = {2025},
author = {Shi, X and Lu, S and Tang, Q and Zhao, X},
title = {Targeted modification of cis-elements in the CUL3 gene to restore exon 9 inclusion for treating Gordon syndrome.},
journal = {Human genomics},
volume = {19},
number = {1},
pages = {119},
pmid = {41121296},
issn = {1479-7364},
support = {ZR2022MH300//the Shandong Province Natural Science Foundation/ ; 82271540//the National Natural Science Foundation of China/ ; QDFY+X2024131//the Natural Science Foundation of the Affiliated Hospital of Qingdao University/ ; },
mesh = {*Cullin Proteins/genetics ; Humans ; *Exons/genetics ; RNA Splice Sites/genetics ; Introns/genetics ; RNA Splicing/genetics ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {BACKGROUND: The weak splice acceptor site (AS) of exon 9 underlies almost all pathogenic variants of Cullin3 (CUL3) causing exon 9 skipping in Gordon syndrome, emphasizing the need for splicing-targeted therapeutic strategies. This study explored universal therapeutic targets to modulate AS and investigated their potential and mechanisms for restoring normal splicing.<br><br>RESULTS: Through bioinformatic prediction, minigene assays, EMSA, CRISPR/Cas9-mediated construction of mutant cell lines and RIP, three rescue sites in the polypyrimidine (Py) tract of intron 8 were identified, including A(-9)T, A(-10)T and AA(-9, -10)TT, with AA(-9, -10)TT most effectively promoting exon inclusion by extending the Py-tract to increase U2AF2 binding. Additionally, previous candidate target A18G was confirmed to rescue exon 9 skipping by weakening hnRNP A1 splicing inhibition in endogenous cell models.<br><br>CONCLUSIONS: Our findings highlight the therapeutic potential of AA(-9, -10)TT and A18G in CUL3-related Gordon syndrome, suggesting the targeted modification of cis-elements could be an ideal and universal strategy to develop treatments for splicing-related diseases.},
}
@article {pmid41121307,
year = {2025},
author = {Li, C and Peng, W and Zhong, Z and Zhang, C and Wang, X and Qin, R and Lei, Q and Lv, J and Liu, F and Zhao, Y and Lv, Z and Li, C and Yang, S and Zhang, H and Tao, Z and Sun, C},
title = {CRISPR/Cas9 library screening reveals that STK19 has synergistic antitumor effects when combined with cisplatin on tongue squamous cell carcinoma.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1142},
pmid = {41121307},
issn = {1479-5876},
support = {82360568, 81960543//Natural Science Foundation of China/ ; 82160452//Natural Science Foundation of China/ ; 202401AY070001-137//Applied Basic Research Foundation of Yunnan Province/ ; 202301AY070001-247//Yunnan Fundamental Research Projects/ ; 2023YJKTB01//Basic Research Program of the First People's Hospital of Qujing/ ; 202305AF150091//Yunnan Province Academician Expert Workstation/ ; },
mesh = {*Cisplatin/pharmacology/therapeutic use ; *Tongue Neoplasms/drug therapy/genetics/pathology ; Humans ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Drug Synergism ; *Carcinoma, Squamous Cell/drug therapy/genetics/pathology ; *Protein Serine-Threonine Kinases/metabolism/genetics ; Xenograft Model Antitumor Assays ; *Antineoplastic Agents/pharmacology/therapeutic use ; DNA Damage/drug effects ; Mice ; Mice, Nude ; Cell Proliferation/drug effects ; },
abstract = {BACKGROUND: Tongue squamous cell carcinoma (TSCC) is a common oral cancer that has a high propensity for recurrence and metastasis. Therefore, TSCC has a 50% 5-year survival rate. Platinum-based chemotherapy is an effective treatment for squamous cell carcinoma, however, chemotherapy resistance remains a major issue. Therefore, innovative and effective drug combinations are needed to improve TSCC patient prognosis.<br><br>METHODS: In this study, we conducted an in vitro CRISPR/Cas9 library screen using two TSCC cell lines (Tscca and Cal27) to identify specific genes that, when inhibited, synergize with cisplatin to effectively suppress tumor growth.<br><br>RESULTS: We identified STK19 as a potential drug target. Inhibition of STK19 enhances the response of TSCC to cisplatin. Through genetic and pharmacological methods, it has been demonstrated that reducing STK19 activity enhances cisplatin-induced DNA damage. The mechanism involves the depletion of MGMT with STK19 inhibition, leading to conditional lethality and synergistic reduction of tumors in vivo when combined with cisplatin. Overall, in this study, unbiased genetic testing was used to successfully identify synthetic lethal drug combinations for TSCC.<br><br>CONCLUSION: STK19 was identified as a promising target that could enhance the killing effects of cisplatin on tongue squamous carcinoma cells, offering a novel therapeutic option for individuals who are insensitive to conventional treatment methods.},
}
@article {pmid41121349,
year = {2025},
author = {Safarzadeh Kozani, P and Safarzadeh Kozani, P},
title = {Preventing secondary primary malignancies (SPMs) in CAR-T cell therapy through site-specific transgene integration into genomic safe harbors (GSHs).},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1155},
pmid = {41121349},
issn = {1479-5876},
mesh = {Humans ; *Transgenes/genetics ; *Neoplasms, Second Primary/prevention &amp; control/genetics ; *Receptors, Chimeric Antigen/metabolism ; Animals ; *Immunotherapy, Adoptive/adverse effects ; Gene Editing ; *Genomics ; CRISPR-Cas Systems ; },
abstract = {Chimeric antigen receptor (CAR)-T cell therapy has revolutionized oncology by achieving durable remissions in refractory hematologic malignancies. However, emerging reports link this therapy to second primary malignancies, including CAR+ lymphomas and leukemias, driven by insertional mutagenesis from semi-random viral vector integration near oncogenes or tumor suppressor loci. These rare but serious complications underscore the dual challenge of eradicating primary tumors while mitigating delayed genotoxic risks. Conventional CAR-T cell manufacturing, reliant on gamma-retroviral or lentiviral vectors, introduces genomic instability through integration into fragile sites or transcriptionally active regions. CRISPR/Cas9-mediated genome editing further amplifies risks via off-target double-strand breaks and chromosomal rearrangements. This review evaluates genomic safe harbors (GSHs)-such as AAVS1, TRAC, CCR5, ROSA26 and CLYBL-as loci validated for stable, high-level CAR transgene expression without oncogenic disruption. GSHs meet stringent criteria: distal from cancer-related genes, resistant to epigenetic silencing, and transcriptionally permissive. Preclinical studies demonstrate that site-directed CAR integration into GSHs preserves antitumor efficacy while eliminating malignant transformation risks. Challenges persist in optimizing homology-directed repair efficiency, mitigating residual dsDNA toxicity, and standardizing regulatory frameworks for long-term genomic surveillance. Emerging technologies-base/prime editing, hybrid nucleases, and rigorous monitoring-promise enhanced precision and safety. By reconciling therapeutic innovation with genomic integrity, GSH-engineered CAR-T cells herald a paradigm shift toward precision immunotherapies, offering curative potential while preempting secondary oncogenesis. Collaborative efforts to refine manufacturing, harmonize global standards, and prioritize patient-specific risk stratification will be critical to advancing this transformative approach.},
}
@article {pmid41121375,
year = {2025},
author = {Amieva, R and Rico-San Román, L and Pastor-Fernández, I and Hemphill, A and Boubaker, G and Collantes-Fernández, E and Ortega-Mora, LM and Horcajo, P},
title = {Loss of NcBPK1 impairs bradyzoite differentiation and enhances virulence in Neospora caninum.},
journal = {Parasites &amp; vectors},
volume = {18},
number = {1},
pages = {422},
pmid = {41121375},
issn = {1756-3305},
support = {PRE2020-092101//Spanish Ministry of Science, Innovation and Universities/ ; },
mesh = {*Neospora/pathogenicity/genetics/growth &amp; development ; Animals ; Virulence ; Mice ; *Coccidiosis/parasitology/veterinary ; *Protozoan Proteins/genetics/metabolism ; Female ; Cattle ; Macrophages/parasitology ; Pregnancy ; Virulence Factors/genetics ; Cattle Diseases/parasitology ; CRISPR-Cas Systems ; Host-Parasite Interactions ; Disease Models, Animal ; Gene Deletion ; },
abstract = {BACKGROUND: Neospora caninum is an apicomplexan parasite responsible for bovine neosporosis, a disease that leads to substantial economic losses in cattle due to abortion and reduced productivity. The pathogenesis of N. caninum is shaped by complex host-parasite interactions, and virulence is known to vary between strains. BPK1 (Bradyzoite pseudokinase 1), a pseudokinase previously identified as a potential virulence factor in Toxoplasma gondii, has not yet been functionally characterized in N. caninum.<br><br>METHODS: To investigate the role of NcBPK1 in parasite virulence, a knockout strain (Nc&#x394;BPK1) was generated using CRISPR/Cas9 genome editing. The virulence of the mutant was evaluated in a pregnant mouse model by assessing neonatal survival and parasite burden in dam tissues. In vitro assays were conducted to examine parasite replication in bovine macrophages and to analyze the expression of stage-specific genes.<br><br>RESULTS: Deletion of NcBpk1 resulted in enhanced parasite virulence in vivo, as shown by a decrease in neonatal survival and higher parasite loads in maternal brain tissue. The Nc&#x394;BPK1 mutant also displayed enhanced replication in bovine macrophages and reduced expression of bradyzoite-specific genes, suggesting a defect in stage conversion.<br><br>CONCLUSIONS: These findings indicate that NcBPK1 is crucial for regulating the balance between acute replication and chronic persistence. Its absence promotes rapid tachyzoite proliferation and worsens disease outcomes. This study sheds light on the molecular mechanisms underlying N. caninum virulence. Further research is needed to elucidate the signaling pathways and protein interactions involving NcBPK1.},
}
@article {pmid41121528,
year = {2026},
author = {Horsley, N and Sythoff, AVB and Delgado, M and Liu, S and Cabernard, C},
title = {CrisprBuildr: an open-source application for CRISPR-mediated genome engineering in Drosophila melanogaster.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {1},
pages = {},
pmid = {41121528},
issn = {2160-1836},
support = {P40 OD018537/OD/NIH HHS/United States ; R35 GM148160/GM/NIGMS NIH HHS/United States ; R35GM148160/NH/NIH HHS/United States ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; *Genome, Insect ; *Genetic Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR/Cas9 is a powerful tool for targeted genome editing experiments. Using CRISPR/Cas9, genes can be deleted or modified by inserting specific DNA sequences, encoding for fluorescent proteins, small peptide tags, or other modifications. Such experiments are essential for detailed gene and protein characterization. However, designing and cloning the corresponding constructs can be repetitive, time-consuming, and laborious. To assist users in CRISPR/Cas9-based genome engineering, we developed CrisprBuildr, an open-source, web-based application for designing modifications to their target genes. CrisprBuildr guides users through creating guide RNAs and repair template vectors to generate cloning maps. The application is designed for the Drosophila melanogaster genome but can serve as a template for other available genomes. We also created new tagging vectors using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments.},
}
@article {pmid41122064,
year = {2025},
author = {Balobaid, A and Waterworth, WM and Vila Nova, SF and Causier, B and Sharma, V and Park, MR and Pandey, MK and West, CE},
title = {Arabidopsis thaliana FANCONI ANAEMIA I (FANCI) has roles in the repair of interstrand crosslinks and CRISPR-Cas9 induced DNA double strand breaks.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {2},
pages = {e70533},
pmid = {41122064},
issn = {1365-313X},
support = {/WT_/Wellcome Trust/United Kingdom ; //King Saud University/ ; BB/S002081/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; BB/Y010426/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/metabolism/genetics ; *DNA Repair/genetics ; *DNA Breaks, Double-Stranded ; CRISPR-Cas Systems/genetics ; Mitomycin/pharmacology ; Cross-Linking Reagents ; Mutation ; },
abstract = {DNA repair is crucial for genome stability, in particular for plants which are exposed to high levels of damage arising from UV irradiation, soil pollutants and reactive oxygen species. Damage that affects both strands of the DNA duplex is harder to repair due to both the lack of a template strand and the potential for physical separation of fragmented chromosomes. As such, DNA double-strand breaks (DSBs) and interstrand DNA crosslinks (ICL) are particularly cytotoxic forms of damage. Here we report the functions of FANCONI ANAEMIA I (FANCI), an Arabidopsis thaliana homologue of the mammalian ICL repair protein. We show that in plant cells, as in mammals, FANCI forms a nuclear localised complex with FANCD2. Genetic analysis of plants lacking FANCI displays significant hypersensitivity to the DNA crosslinking reagent mitomycin C. Furthermore, mutation of FANCI in combination with mutations in a second ICL repair factor, METHYL METHANESULFONATE AND UV-SENSITIVE PROTEIN 81 (MUS81), results in increased levels of programmed cell death compared to the corresponding single mutants, revealing roles in maintaining plant genome stability. Sequence analysis of mutational repair of CRISPR-Cas9-induced DSBs revealed that FANCI promotes single nucleotide insertions and reduces longer deletions. This pattern of mutations may reflect roles for FA proteins in replication-coupled repair of a subset of DSBs. Taken together, this analysis finds evidence for multiple roles for FANCI in the maintenance of plant genome stability.},
}
@article {pmid41122778,
year = {2025},
author = {Hasselbeck, S and Wang, J and Bai, Z and Hüfner, T and Hummer, G and Grote, P and Cheng, X},
title = {A Chemically Induced CRISPR/dCas13[FCPF] Platform for Precise and Programmable RNA Regulation.},
journal = {Journal of medicinal chemistry},
volume = {68},
number = {21},
pages = {22633-22649},
pmid = {41122778},
issn = {1520-4804},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *RNA/genetics/metabolism ; Alternative Splicing/drug effects ; Survival of Motor Neuron 2 Protein/genetics ; HEK293 Cells ; Exons ; },
abstract = {Alternative splicing enhances proteomic diversity, yet its dysregulation drives cancer, neurodegeneration, and inherited disease. Small-molecule splicing modulators, while clinically validated, like risdiplam, often lack locus specificity, producing off-target effects. CRISPR/Cas13 enables programmable transcript-level targeting, but dCas13 fusion effectors are bulky and can hamper delivery and RNA homeostasis. Building on our previous Chem-CRISPR/dCas9[FCPF] system for epigenome editing, we now introduce Chem-CRISPR/dCas13[FCPF], a modular platform that covalently tethers a perfluorobiphenyl-tagged small molecule to dCas13 via a four-residue FCPF π-clamp tag. Guided by crRNAs, dRfxCas13d[FCPF] recruits a risdiplam-derived conjugate to the SMN2 exon 7 splice region, inducing exon inclusion at ligand doses ∼500-fold lower than those of free risdiplam and with no detectable effects at known risdiplam-sensitive transcripts in our assays. The approach generalizes to additional transcripts by crRNA redesign. By coupling CRISPR addressability with dose-sparing chemical action, Chem-CRISPR/dCas13[FCPF] establishes a proximity-induced, chemically controllable route to precise RNA modulation suitable for therapeutic exploration.},
}
@article {pmid41123029,
year = {2026},
author = {Burnase, N and Kumar, B and Kumar, PS and Dharshini, RS and Das, R and Barapatre, A},
title = {Advances in Antiviral Strategies for Oral Herpes Infections in Immunocompromised Patients.},
journal = {Oral diseases},
volume = {32},
number = {2},
pages = {300-321},
doi = {10.1111/odi.70090},
pmid = {41123029},
issn = {1601-0825},
mesh = {Humans ; *Immunocompromised Host ; *Antiviral Agents/therapeutic use ; *Herpesviridae Infections/drug therapy/immunology ; Genetic Therapy ; *Stomatitis, Herpetic/drug therapy/immunology ; Drug Resistance, Viral ; RNA Interference ; CRISPR-Cas Systems ; *Herpes Simplex/drug therapy ; },
abstract = {OBJECTIVE: This review explores conventional antiviral therapies and innovative treatment strategies for managing oral herpes simplex virus (HSV) or human herpesvirus (HHV) infections, particularly in immunocompromised patients. It aims to evaluate clinical outcomes, address challenges such as drug resistance, and assess the sustainability of current and emerging therapeutic approaches.<br><br>SUBJECT: A descriptive review of current treatment strategies for oral HSV infections in immunosuppressed individuals is presented. Relevant studies are analyzed for their contributions, highlighting therapeutic advancements, limitations of existing drugs, and novel approaches in development.<br><br>RESULTS: Oral herpesvirus infections can lead to considerable morbidity and mortality in immunocompromised patients. Resistance to standard antivirals often develops due to prolonged or inconsistent use and impaired cell-mediated immunity. To overcome these issues, innovative strategies like gene therapy, RNA interference (RNAi), and CRISPR-Cas9 have emerged. These approaches aim to suppress viral gene expression, strengthen innate immunity, and genetically modify host cells to resist viral entry and replication.<br><br>CONCLUSION: Emerging antiviral therapies show promise in addressing the limitations of conventional treatments, particularly drug resistance and non-specific targeting. These novel approaches offer more effective, precise, and safer options for treating oral viral infections in immunosuppressed populations at heightened risk for severe outcomes.},
}
@article {pmid41123065,
year = {2025},
author = {Sohail, M and Ma, S and Mushtaq, B and Haider, MU and Li, B and Zhang, X and Huang, H},
title = {Fabricating Four-Element Doped Carbon Dots-Based Fluorescent Ratiometric Reporter Platform for CRISPR/Cas-Driven Precise Sensing of Nucleic Acids.},
journal = {Analytical chemistry},
volume = {97},
number = {43},
pages = {23868-23878},
doi = {10.1021/acs.analchem.5c03228},
pmid = {41123065},
issn = {1520-6882},
mesh = {*Carbon/chemistry ; *CRISPR-Cas Systems ; *Quantum Dots/chemistry ; *Biosensing Techniques/methods ; *Fluorescent Dyes/chemistry ; Humans ; *Nucleic Acids/analysis ; },
abstract = {Conventional CRISPR/Cas sensing platforms exhibit poor efficiency concerning reporter-based demerits, including their interference-labile nature, photobleaching, low robustness due to a single output signal, and probe-concentration dependence. Herein, a carbon dots (CDs)-based dual-emissive fluorescent ratiometric CRISPR/Cas reporter platform was fabricated for biosensing and other analytical applications to bottleneck the demerits of conventional reporters, integrating the benefits of a ratiometric strategy and four-element doped carbon dots (4D CDs) as a transducer. Briefly, doping enhances the optical and physicochemical traits of CDs and minimizes the effect of the interfering species. A series of state-of-the-art N, P, S, and Cu codoped CDs (4D CDs) were synthesized using the hydrothermal approach and statistical tools, such as Box-Behnken design, analysis of variance, and others, enhancing photophysical traits, surface features, and sensitivity of CDs. The red-emissive CDs were prepared by using the same procedure but different precursors. The optimum 4D CDs (blue-emissive) and red-emissive CDs were used to unleash the principle of the fluorescent ratiometric CRISPR/Cas reporter system for diverse applications. Finally, the designed 4D CDs-based CRISPR/Cas biosensor was applied for nucleic acid monitoring, such as the COVID-19 nucleic acid. This project disclosed the controlled-doping principle to synthesize 4D CDs and unleashed the mechanism of ratiometric dual-emissive CRISPR/Cas-powered reporters for precise sensing applications. We anticipate the implementation of this technology in commercial analytical, biosensing, point-of-care, and other applications.},
}
@article {pmid41123354,
year = {2026},
author = {Choi, NY and Kim, MH and Jang, HA and Pyo, SW and Park, KY and Lee, H and Bae, EK and Ko, JH},
title = {Elucidating the Genetic Basis of Columnar Upright Architecture in Populus Through CRISPR Disruption of TILLER ANGLE CONTROL1.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1377-1397},
pmid = {41123354},
issn = {1467-7652},
support = {RS-2023-NR076519//National Research Foundation of Korea/ ; FG0702-2023-01-2025//Korea Forest Service/ ; 2023489B10-2325-AA01//R&amp;D Program for Forest Science Technology/ ; },
mesh = {*Populus/genetics/growth &amp; development/metabolism ; Plants, Genetically Modified ; *Genes, Plant ; *Trees/genetics/growth &amp; development/metabolism ; CRISPR-Cas Systems ; Codon, Nonsense ; Gene Expression Regulation, Plant ; Gravitropism ; Plant Leaves/growth &amp; development ; *Meristem/metabolism ; Plant Growth Regulators/metabolism ; },
abstract = {Narrow or upright branch angles in shoots and leaves lead to columnar, upright-growing tree architectures, as observed in various tree species such as Lombardy poplar (Populus nigra var. italica). However, the genetic mechanism underlying this unique growth habit in Lombardy poplar has not yet been elucidated. In this study, we identified a nonsense mutation in the PnTAC1-1 gene of Lombardy poplar, an ortholog of the rice TILLER ANGLE CONTROL 1 (TAC1) gene known to regulate branch angles. To confirm the functional role of TAC1 in regulating tree architecture, we generated transgenic hybrid poplar (Populus alba × Populus glandulosa, clone BH) with targeted mutations in TAC1 homologues using CRISPR/Cas9 gene editing. The resulting TAC1-CRISPR hybrid poplars exhibited a stable upright branching phenotype closely resembling that of Lombardy poplar, as confirmed by two consecutive years of living modified organism (LMO) field trials. Anatomical analysis revealed increased cell elongation specifically in the lower petiole region and significantly enhanced gravitropic responses in TAC1-CRISPR hybrid poplars compared to wild-type BH clones. RNA sequencing analysis further demonstrated that TAC1 disruption triggered extensive transcriptomic reprogramming of axillary meristem, notably altering hormonal and photomorphogenic signalling pathways, which redirected auxin accumulation toward the abaxial region and increased gibberellin biosynthesis, ultimately promoting upright growth. This research uncovers the genetic and molecular mechanisms behind columnar growth in poplar and provides a promising approach for engineering tree architecture to enhance planting density, harvest efficiency and woody biomass productivity.},
}
@article {pmid41123407,
year = {2025},
author = {Xu, Z and Huang, Y and Dong, Y and An, X and Tong, Y and Li, M},
title = {Engineering broad-spectrum phage-resistant Escherichia coli via adaptive and programmable defense strategies.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {11},
pages = {e0159625},
pmid = {41123407},
issn = {1098-5336},
support = {2022FY101100//Science &amp; Technology Fundamental Resources Investigation Program/ ; 202423m10050004//Anhui Province Scientific and Technological Research Project/ ; 22322908D//S&amp;T Program of Hebei/ ; 82341119//National Natural Science Foundation of China/ ; XK2025-05//Interdisciplinary Research Center of Beijing University of Chemical Technology/ ; },
mesh = {*Escherichia coli/virology/genetics ; *Coliphages/genetics/physiology/isolation &amp; purification ; CRISPR-Cas Systems ; Genome, Viral ; Fermentation ; },
abstract = {UNLABELLED: Phage contamination, which impacts product quality and production efficiency, remains a major challenge in industrial fermentation. Although bacteria have evolved various defense systems to combat phage infection, these systems often suffer from narrow host specificity and limited efficacy. In this study, we isolated and characterized a novel lytic Escherichia coli phage, TR2, from a contaminated fermentation substrate. Its strong environmental stability, short latency period, and high lytic activity render it a significant threat to fermentation processes. Genomic sequencing revealed that phage TR2 has a linear, double-stranded DNA genome of 45,171 bp with a G+C content of 44% and 74 coding sequences. On the basis of the physiological characteristics and genomic features of this phage, we developed two strategies to generate phage-resistant E. coli strains: (i) selection of spontaneous mutations in bacterial surface receptors to prevent phage adsorption and infection and (ii) integration of an exogenous CRISPR/Cas9 system to confer sequence-specific immunity. Spontaneous mutation provides broad-spectrum resistance but at the cost of fitness and evolutionary stability, whereas CRISPR/Cas9 ensures long-term, programmable immunity with minimal growth defects. Importantly, both strategies successfully protected bacterial cultures from phage infection without compromising recombinant protein production, highlighting their potential for industrial application. Our findings provide a practical approach for mitigating phage contamination in industrial fermentation processes. This study also highlights the advantages and limitations of spontaneous mutations and natural phage defense systems, offering valuable insights for the design of more effective phage-resistant microbial platforms.<br><br>IMPORTANCE: Phage contamination is a significant challenge in industrial fermentation and severely impacts product quality and production efficiency. We systematically compared spontaneous mutation and CRISPR/Cas9-mediated immunity as two strategies for engineering phage-resistant E. coli strains. Both approaches effectively protected bacterial cultures from phage infection without compromising recombinant protein production, underscoring their potential for industrial applications. Notably, spontaneous mutation conferred broad-spectrum resistance but was associated with fitness costs and limited evolutionary stability. In contrast, CRISPR/Cas9-based immunity offered long-term, programmable protection with minimal growth impairment. By delineating the trade-offs between these two strategies, our work provides a framework for selecting tailored phage resistance solutions suited to diverse biomanufacturing scenarios.},
}
@article {pmid41123679,
year = {2025},
author = {Rocha, DC and Omoregbee, MO and Contiliani, DF and Mandlik, R and Li, G and Mascoveto, J and Coleman, G and Culver, JN and Leal, DR and de Souza, AA and Qi, Y},
title = {Transgene-free genome editing in citrus and poplar trees using positive and negative selection markers.},
journal = {Plant cell reports},
volume = {44},
number = {11},
pages = {244},
pmid = {41123679},
issn = {1432-203X},
support = {IOS-2132693//Division of Integrative Organismal Systems/ ; 2224203//Division of Integrative Organismal Systems/ ; IOS-2428015//Division of Integrative Organismal Systems/ ; 2021-67013-34554//National Institute of Food and Agriculture/ ; 2020-33522-32274//National Institute of Food and Agriculture/ ; 2024-33522-42755//National Institute of Food and Agriculture/ ; 2020-70029- 33161//National Institute of Food and Agriculture/ ; DE-SC0023011//Department of Energy/ ; 2023/09068-9//FAPESP/ ; },
mesh = {*Populus/genetics ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Citrus/genetics ; Transgenes/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant ; Genetic Markers ; Herbicide Resistance/genetics ; },
abstract = {Transgene-free genome editing of the gene of interest in citrus and poplar has been achieved by co-editing the ALS gene via transient transgene expression of an efficient cytosine base editor. CRISPR-Cas genome editing systems have been widely used in plants. However, such genome-edited plants are nearly always transgenic in the first generation when Agrobacterium-mediated transformation is used. Transgene-free genome-edited plants are valuable for genetic analysis and breeding as well as simplifying regulatory approval. It can be challenging to generate transgene-free genome-edited plants in vegetatively propagated or perennial plants. To advance transgene-free genome editing in citrus and poplar, we investigated a co-editing strategy using an efficient cytosine base editor (CBE) to edit the ALS gene to confer herbicide resistance combined with transient transgene expression and potential mobile RNA-based movement of CBE transcripts to neighboring, non-transgenic cells. An FCY-UPP based cytotoxin system was used to select non-transgenic plants that survive after culturing on 5-FC containing medium. While the editing efficiency is higher in poplar than in citrus, our results show that the CBE-based co-editing strategy works in both citrus and poplar, albeit with low efficiency for biallelic edits. Unexpectedly, the addition of the TLS mobile RNA sequence reduced genome editing efficiency in both transgenic and non-transgenic plants. Although a small fraction of escaping plants is detected in both positive and negative selection processes, our data demonstrate a promising approach for generating transgene-free base-edited plants.},
}
@article {pmid41123685,
year = {2025},
author = {Yang, S and Liu, Y and Zhang, J and Xu, J and Li, T and Huang, H and Zhu, Z and Li, M and Wang, H and Yang, C},
title = {An integrated lab-in-a-tube platform for point-of-care detection of blaKPC in urinary tract infections.},
journal = {Mikrochimica acta},
volume = {192},
number = {11},
pages = {748},
pmid = {41123685},
issn = {1436-5073},
support = {82472374//National Natural Science Foundation of China/ ; YG2024LC02//Cross-Research Fund of Biomedical Engineering of Shanghai Jiaotong University/ ; 2024ZY008//The Clinical and Excellence Program at the Institute of Molecular Medicine, Shanghai Jiao Tong University School of Medicine/ ; },
mesh = {Humans ; *Urinary Tract Infections/microbiology/diagnosis/urine ; *Klebsiella pneumoniae/enzymology/genetics/drug effects/isolation &amp; purification ; *beta-Lactamases/genetics/urine ; *Bacterial Proteins/genetics/urine ; *Point-of-Care Systems ; Klebsiella Infections/urine/diagnosis/microbiology ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; },
abstract = {Carbapenem-resistant Klebsiella pneumoniae (CRKP), predominantly mediated by the blaKPC carbapenemase gene, poses a critical therapeutic challenge for urinary tract infections (UTIs). To enable rapid identification at the point of need, we engineered TubeCARE (tube-integrated platform for carbapenem antimicrobial resistance evaluation), an integrated, disposable lab-in-a-tube system for direct blaKPC detection from urine samples. This self-contained system uniquely combines urine processing, nucleic acid extraction, recombinase polymerase amplification (RPA), and CRISPR/Cas12a reaction within a single sealed unit, offering dual-modality outputs: smartphone-based real-time fluorescence or naked-eye lateral flow strip interpretation. The full workflow delivers "sample-in, result-out" in 40 min, eliminating external multi-step nucleic acid extraction and reducing contamination risks. Both detection modes demonstrated laboratory-grade sensitivity (1 CFU/mL) with 100% specificity against non-target carbapenemase genes. Clinical validation using 24 urine samples (20 blaKPC-positive, 4 negative) showed 100% concordance with quantitative PCR. In a representative UTI case, TubeCARE enabled 40-min bedside blaKPC detection; early implementation would circumvent 72-h diagnostic delays, facilitating timely carbapenem avoidance and preventing bloodstream complications. Featuring integrated lab-in-a-tube operation, cost-effective manufacturing, user-friendly workflow, and laboratory-grade accuracy, TubeCARE provides actionable antimicrobial resistance surveillance in diverse clinical and resource-limited settings.},
}
@article {pmid41123840,
year = {2025},
author = {Remiszewski, P and Siedlecki, E and Wełniak-Kamińska, M and Mikula, M and Czarnecka, AM},
title = {Genetically Modified Mouse Models for Sarcoma Research: A Comprehensive Review.},
journal = {Current oncology reports},
volume = {27},
number = {11},
pages = {1221-1248},
pmid = {41123840},
issn = {1534-6269},
support = {2019/35/O/NZ2/03761 (AMC)//Narodowe Centrum Nauki/ ; },
mesh = {*Sarcoma/genetics/pathology ; Animals ; Humans ; *Disease Models, Animal ; Mice ; CRISPR-Cas Systems ; Animals, Genetically Modified ; Gene Editing ; },
abstract = {PURPOSE OF REVIEW: Sarcomas are a heterogeneous group of over 170 malignant tumours of mesenchymal origin. The poor prognosis highlights the need for novel therapeutic strategies. Preclinical modelling is essential, yet challenging, given that sarcomas differ substantially from carcinomas and resources are very limited.<br><br>RECENT FINDINGS: GEMMs allow for the precise modelling of recurrent sarcoma genetics. The Cre-loxP system offer spatial and temporal control over the activation of oncogenes or the loss of tumour suppressors, while the CRISPR-Cas9 system enables the rapid, simultaneous editing of key drivers such as Trp53, Nf1, Kras and Pten. These models reproduce key features of human sarcomas, including their histopathology, the initiation of tumours in specific lineages and sites, and tumour-immune interactions within immune-competent hosts. GEMMs have been used to investigate hypotheses about the cells of origin, to test radiotherapy and immunotherapy, and to compare fusion-driven sarcomas with those with a complex karyotype. Despite variability, GEMMs remain essential tools for investigating the mechanisms of initiation, progression, and response to therapy. GEMMs offer mechanistic fidelity, but their use is limited by factors such as breeding burden, variability in recombination, off-target effects of CRISPR, underrepresentation of genomic complexity and inconsistent metastasis. These weaknesses reduce their predictive value, particularly with regard to advanced disease and immunotherapy. Progress will require the integration of Cre-loxP with CRISPR-Cas9, the standardisation of induction and reporting, and a closer alignment with distinct sarcoma subtypes, in order to enhance translational relevance.},
}
@article {pmid41123979,
year = {2025},
author = {Wang, S and Wei, Z and Feng, Y and Gan, Z and Yu, L and Cheng, H and Xiao, Y},
title = {Rationally Trapped Polycrystalline Perovskite in Lanthanide MOF Cages for Ammonia-Mediated Nucleic Acid Intelligent Visualization.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {49},
pages = {e07214},
doi = {10.1002/smll.202507214},
pmid = {41123979},
issn = {1613-6829},
support = {82273895//National Natural Science Foundation of China/ ; 82304440//National Natural Science Foundation of China/ ; 82073811//National Natural Science Foundation of China/ ; 2024M763727//China Postdoctoral Science Foundation/ ; },
mesh = {*Calcium Compounds/chemistry ; *Oxides/chemistry ; *Titanium/chemistry ; *Lanthanoid Series Elements/chemistry ; *Ammonia/chemistry ; *Nucleic Acids/chemistry/analysis ; *Metal-Organic Frameworks/chemistry ; },
abstract = {Perovskites nanoparticles (PNPs), promising materials in fluorescence biosensing, have has their practical applications stymied by poor stability in polar solvents. Integrating PNPs into metal-organic frameworks (MOFs) offers a solution by enhancing their compatibility with various environments. Lanthanide MOFs (Ln-MOFs) are particularly advantageous due to their customizable structure, enhanced stability, and intrinsic fluorescence. Herein, the mechanism by which hybrid materials achieve a balance between physical and fluorescence properties is elucidated. Through theoretical calculations, 2,2'-bipyridine-5,5'-dicarboxylic acid is selected as the ligand to sensitize Eu[3+] and facilitate Pb[2+] chemisorption. In addition, 4-bromobutyric acid is not only employed to create hierarchical Eu-MOFs and optimize the framework for in situ growth of polycrystalline perovskites but also utilized to generate zwitterionic ligands through an SN2 reaction with MOF cages restricted n-octylamine, ensuring the stable dispersion of hybrid materials in ethyl acetate (polar solvent). The prepared PNPs@Ln-MOF exhibits significantly enhanced fluorescence lifetime (50-fold) and stability in polar solvents. Besides, the highly sensitive fluorescence color shift of PNPs@Ln-MOF in response to ammonia offers a generalizable strategy for ammonia-mediated biosensing device. Supported by CRISPR/Cas technology, this device allows for precise on-site nucleic acid assay (LOD = 200 fM), pioneering advanced applications of perovskite-based hybrid materials in biosensing.},
}
@article {pmid41124025,
year = {2025},
author = {Su, F and Zhang, H and Ren, C and Jiang, Y and Qiao, Y and Zhang, S and Liu, J and Li, S and Li, Y},
title = {MnO2@Mn3O4 Heterojunction as a Coreactant Catalyst Collaborated with T-Shaped DNA Cycling-CRISPR/Cas12a Cascade Amplification for Locus-Specific N6-Methyladenosine RNA Modification Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {43},
pages = {24165-24175},
doi = {10.1021/acs.analchem.5c04791},
pmid = {41124025},
issn = {1520-6882},
mesh = {*Adenosine/analogs &amp; derivatives/analysis ; *Manganese Compounds/chemistry ; *Oxides/chemistry ; *Biosensing Techniques/methods ; Humans ; *RNA/analysis/chemistry ; Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics ; Metal Nanoparticles/chemistry ; Catalysis ; Gold/chemistry ; Nucleic Acid Amplification Techniques ; CRISPR-Associated Proteins/genetics ; Limit of Detection ; Luminescent Measurements ; HeLa Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {As the predominant RNA modification, N6-methyladenosine (m[6]A) is recognized to play pivotal regulatory roles in fundamental cellular functions and oncogenic processes. However, the precise analysis of site-specific m[6]A modifications continues to present significant challenges. In this work, a unique electrochemiluminescence (ECL) biosensor for the locus-specific detection of m[6]A in RNA was developed in the first utilization of a MnO2@Mn3O4 heterojunction as a coreaction catalyst in collaboration with a T-shaped DNA cycling-CRISPR/Cas12a cascade amplification strategy. The MnO2@Mn3O4 heterojunction was observed to significantly enhance coreactant catalytic activity, yielding a 7.3-fold increase in the ECL intensity of the gold nanoparticles (AuNPs)/MnO2@Mn3O4/(2,2'-bipyridine) dichlororuthenium(II) (Ru(bpy)3[2+])/Nafion/GCE compared to the AuNPs/Ru(bpy)3[2+]/Nafion/GCE. Sequentially, the T-shaped DNA cycling amplification strategy effectively converted the target m[6]A RNA into an amplified biosignal, further enhanced by a CRISPR/Cas12a signal amplification system mediated by framework nucleic acid (FNA)-based photocontrollable nucleic acid protection, ensuring the sensitivity and specificity of m[6]A RNA. The integration of the triple signal amplification strategy achieved detection limits as low as 0.05 pM (S/N = 3) for a linearity spanning from 100 fM to 100 nM. The proposed ECL biosensor has been applied in detecting site-specific m[6]A modifications in total real RNA samples extracted from HeLa cells, demonstrating its promising applications for clinical diagnosis.},
}
@article {pmid41124242,
year = {2025},
author = {Lu, X and Zhu, Y and Wei, C and Cheng, L and Goodier, KD and Kong, J and Gao, X and Yu, D and Liu, X and Long, Y and Lin, J and Ma, J and Su, Y and Mao, HQ},
title = {A multistep platform identifies spleen-tropic lipid nanoparticles for in vivo T cell-targeted delivery of gene-editing proteins.},
journal = {Science advances},
volume = {11},
number = {43},
pages = {eady5579},
pmid = {41124242},
issn = {2375-2548},
support = {P41 EB028239/EB/NIBIB NIH HHS/United States ; R01 CA293906/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; *Gene Editing/methods ; *Spleen/metabolism/cytology ; Mice ; *T-Lymphocytes/metabolism/immunology ; *Nanoparticles/chemistry ; *Lipids/chemistry ; *Gene Transfer Techniques ; Humans ; Receptors, CCR5/genetics ; CRISPR-Cas Systems ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are a promising nonviral delivery system for gene-editing proteins, but optimal formulations remain underexplored. Unlike messenger RNA-based approaches, ribonucleoprotein delivery enables immediate genome editing without relying on endogenous translation. However, intracellular delivery remains a major challenge due to protein size, charge variability, and susceptibility to denaturation and degradation. Here, we present a multistep screening platform to optimize LNP formulations for gene-editing protein delivery, focusing on in vivo T cell targeting. Through in vitro screening of a composition library, we identified top-performing candidates. In vivo screening in Ai9 mice revealed a spleen-tropic LNP formulation that preferentially targets T cells, enabling efficient gene editing in vivo. Using this LNP formulation, we achieved targeted knockout of CCR5 and PD-1 in splenic T cells, supporting potential applications in HIV resistance and cancer immunotherapy. Furthermore, a machine learning-guided mechanistic study revealed key design principles for LNP-based protein delivery, highlighting unexplored opportunities for in vivo genome-editing therapies.},
}
@article {pmid41125159,
year = {2026},
author = {Zhou, C and Zhu, S and Luo, C and Wang, W and Fan, H and Gao, Y and Xu, X and Wang, Q and You, Y and Xie, T},
title = {From IscB to Cas9: Engineering and advances in the next generation of miniature gene editing tools.},
journal = {Biotechnology advances},
volume = {86},
number = {},
pages = {108743},
doi = {10.1016/j.biotechadv.2025.108743},
pmid = {41125159},
issn = {1873-1899},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; DNA Transposable Elements/genetics ; Animals ; *CRISPR-Associated Protein 9/genetics ; },
abstract = {The CRISPR-Cas system, distinguished by its inherent modularity and broad programmability, has catalyzed a paradigm shift in genome engineering due to its unprecedented accuracy, specificity, and on-target efficiency, now serving as the cornerstone of modern genome manipulation. The efficient delivery of gene editing tools remains a major technical hurdle to clinical application, primarily due to the lack of compact editors. The recent identification of the transposon-associated nuclease IscB as an evolutionary ancestor of Cas9 has provided important insights into the molecular evolution of the CRISPR-Cas9 system. Notably, IscB is a highly compact nuclease, approximately one-third the size of Cas9, capable of precise nucleic acid cleavage in eukaryotic cells under the guidance of ωRNA. These features make it a promising candidate for the development of next-generation miniaturized genome editors. However, natural IscB exhibits limited editing performance in eukaryotic systems. This review first outlines the biochemical function of the transposon IscB and briefly traces the evolutionary origin of the Cas9 system. It then describes and compares the structural characteristics and cleavage mechanisms of OgeuIscB and Cas9. Subsequent sections summarize various engineering strategies for current IscB systems, including the development of base editors and recent advances in their application. Finally, the limitations of existing systems are discussed, and potential directions for future optimization are proposed, aiming to provide new insights and facilitate the advancement of IscB-based miniaturized editors.},
}
@article {pmid41125583,
year = {2025},
author = {Yang, J and Li, H and Li, M and Song, R and Shen, T and Wang, G and Xu, D and Hao, M and Jia, A and Rehman, SU and Hua, L and Liang, Y and Chi, C and Lan, C and Deng, XW and Dubcovsky, J and Song, B and Wang, X and Chen, S},
title = {Genome-assisted identification of wheat leaf rust resistance gene Lr.ace-4A/Lr30.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9339},
pmid = {41125583},
issn = {2041-1723},
mesh = {*Triticum/genetics/microbiology/immunology ; *Plant Diseases/microbiology/genetics/immunology ; *Disease Resistance/genetics ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; Plant Leaves/microbiology/genetics ; Basidiomycota ; Genome, Plant ; *Genes, Plant ; CRISPR-Cas Systems ; Puccinia ; },
abstract = {Leaf rust is a devastating disease of wheat. Growing rust-resistant wheat varieties is the best strategy to mitigate this threat. Here, we generate a 10.51-gigabase chromosome-scale assembly of the durum wheat landrace PI 192051. Using mutagenesis and transcriptome sequencing, we identify the leaf rust resistance gene Lr.ace-4A within a recombination-sparse region of PI 192051 and demonstrate that Lr.ace-4A is identical to the previously designated Lr30 gene in hexaploid wheat. Lr.ace-4A/Lr30 encodes a non-canonical coiled-coil nucleotide-binding leucine-rich repeat receptor, featuring tandem nucleotide-binding domains. This gene is both necessary and sufficient to confer resistance to leaf rust, as demonstrated by CRISPR/Cas9-induced mutations and transgenic complementation. Lr.ace-4A provides near-immunity resistance in durum wheat, though its effectiveness is diminished in hexaploid wheat. Two amino acid polymorphisms differentiate the resistant and susceptible Lr.ace-4A haplotypes, with transgenic plants carrying either susceptible variant showing susceptibility. The cloning of Lr.ace-4A will accelerate its deployment in wheat breeding programs.},
}
@article {pmid41128695,
year = {2026},
author = {Plugge, SF and Ma, H and van der Vaart, JY and Sprangers, J and Morsink, FHM and Xanthakis, D and Jamieson, C and Stroot, KBW and Keijzer, AR and Margaritis, T and Candelli, T and Straver, R and de Ridder, J and Holstege, FCP and de Leng, WWJ and Offerhaus, GJA and Merenda, A and Maurice, MM},
title = {Intestinal LKB1 Loss Drives a Premalignant Program Along the Serrated Cancer Pathway.},
journal = {Gastroenterology},
volume = {170},
number = {2},
pages = {298-314},
doi = {10.1053/j.gastro.2025.07.041},
pmid = {41128695},
issn = {1528-0012},
mesh = {Humans ; *Protein Serine-Threonine Kinases/genetics/deficiency/metabolism ; Animals ; AMP-Activated Protein Kinase Kinases ; *Peutz-Jeghers Syndrome/genetics/pathology/enzymology ; Organoids/pathology ; *Colorectal Neoplasms/genetics/pathology/enzymology ; Mice ; *Precancerous Conditions/genetics/pathology/enzymology ; Signal Transduction ; ErbB Receptors/metabolism ; Mice, Knockout ; Colon/pathology/enzymology ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Disease Models, Animal ; Proto-Oncogene Proteins p21(ras)/genetics/metabolism ; *Cell Transformation, Neoplastic/genetics/pathology ; Intestinal Mucosa/pathology ; CRISPR-Cas Systems ; AMP-Activated Protein Kinases ; },
abstract = {BACKGROUND &amp; AIMS: Heterozygous inactivating mutations of Serine Threonine Kinase 11/Liver Kinase B1 (LKB1) are causative to the Peutz-Jeghers syndrome (PJS), a hereditary disease characterized by gastrointestinal hamartomatous polyposis and increased cancer susceptibility. Although LKB1 loss-induced polyp formation has been ascribed to nonepithelial tissues, how LKB1 deficiency increases cancer risk of patients by altering the phenotypical landscape and hierarchical organization of epithelial tissues remains poorly understood.<br><br>METHODS: Using CRISPR/Cas9, we generated heterozygous and homozygous Lkb1-deficient mouse small intestinal and human colon organoids. These organoids were characterized by an integrated approach that combines imaging, bulk and single-cell RNA sequencing, and growth factor dependency assays. Our findings were validated in human PJS-derived tissues using immunohistochemistry and linked to colorectal cancer profiles using the Cancer Genome Atlas (TCGA) cancer database.<br><br>RESULTS: Our results reveal that heterozygous Lkb1 loss is sufficient to push intestinal cells into a premalignant transcriptional program associated with serrated colorectal cancer, which is further amplified by loss of heterozygosity. This altered epithelial growth state associates with persistent features of regeneration and enhanced EGFR ligand and receptor expression, conferring niche-independent growth properties to Lkb1-deficient organoids. Moreover, our newly generated LKB1-mutant signature is enriched in sporadic serrated colorectal cancer, and synergistic cooperation of Lkb1 deficiency with mutant Kras was experimentally confirmed by assessing organoid growth properties and transcriptomes.<br><br>CONCLUSIONS: Heterozygous loss of LKB1 pushes intestinal cells into a chronic regenerative state, which is amplified on loss of heterozygosity. Lkb1 deficiency thereby generates fertile ground for serrated colorectal cancer formation in the intestine, potentially explaining the increased cancer risk observed in PJS.},
}
@article {pmid41129856,
year = {2026},
author = {Zhou, J and Ren, XM and Gao, J and Wu, RP and Chen, L and Li, Z},
title = {Amplification-free detection of mycoplasma pneumoniae via CRISPR-Cas12a and deep learning-optimized crRNAs on a lateral flow platform.},
journal = {Journal of pharmaceutical and biomedical analysis},
volume = {268},
number = {},
pages = {117196},
doi = {10.1016/j.jpba.2025.117196},
pmid = {41129856},
issn = {1873-264X},
mesh = {*Mycoplasma pneumoniae/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; *Deep Learning ; Limit of Detection ; Point-of-Care Testing ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; Sensitivity and Specificity ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Accurate and rapid diagnosis of Mycoplasma pneumoniae infection is essential for reducing its significant health burden. An amplification-free CRISPR-Cas12a-mediated detection platform has been developed, incorporating a deep learning-optimized crRNA library (CCDLCL) targeting conserved regions of the MP P1 gene. The system enables visual readout via lateral flow strips, supporting its potential as a point-of-care testing (POCT) nucleic acid testing strategy. Through computational design and screening, 16 highly active crRNAs were identified from an initial set of over 50 candidates. Combinatorial use of these crRNAs demonstrated synergistic enhancement of fluorescence signal intensity and reaction kinetics. Compared to single-crRNA assays, the multiplexed crRNA library improved sensitivity by 16.8-fold, achieving a limit of detection (LOD) of 0.15 pM, and reduced time to signal saturation by 30 %. When deployed on lateral flow strips, the assay exhibited a tenfold increase in visual detection sensitivity, with a LOD of 100 pM. Clinical evaluations confirmed high specificity-showing no cross-reactivity with SARS-CoV-2, hepatitis B virus (HBV), or human genomic DNA-and over 95 % agreement with standard clinical results without target pre-amplification, delivering outcomes within 45 min. This study establishes a deep learning-facilitated crRNA design framework and a novel crRNA library-based detection system, offering a feasible approach for POCT nucleic acid testing in resource-limited settings and paving the way for streamlined clinical translation of CRISPR-Cas diagnostics.},
}
@article {pmid41130353,
year = {2026},
author = {Lee, SY and Kim, HS and Kim, YG and Shin, S and Kweon, S and Lee, JJ and Lee, GM},
title = {Genome-wide CRISPR screening identifies genes in recombinant human embryonic kidney 293 cells for increased ammonia resistance.},
journal = {Metabolic engineering},
volume = {93},
number = {},
pages = {184-193},
doi = {10.1016/j.ymben.2025.10.008},
pmid = {41130353},
issn = {1096-7184},
mesh = {Humans ; *Ammonia/pharmacology/metabolism ; HEK293 Cells ; *CRISPR-Cas Systems ; *Antibodies, Monoclonal/genetics/biosynthesis ; Gene Knockout Techniques ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Genome, Human ; },
abstract = {Ammonia, a byproduct of glutamine metabolism, inhibits cell growth and reduces product yield and quality in mammalian cell culture. To identify novel genes associated with ammonia resistance, a genome-wide CRISPR knockout screening was conducted in monoclonal antibody (mAb)-producing human embryonic kidney 293 (HEK-mAb) cells using a virus-free, recombinase-mediated cassette exchange-based gRNA interrogation method. The knockout cell library was subcultured for five consecutive passages under 20 mM NH4Cl, enriching cells with a sgRNA that conferred a proliferation advantage under high-ammonia conditions. Next-generation sequencing analysis of the enriched population identified three target genes -WNT3, TSPAN1, and CYHR1-among 19,114 genes. Knockout of these genes in HEK-mAb cells resulted in a 1.33- to 1.56-fold increase in maximum viable cell concentration and a 1.28- to 1.58-fold increase in maximum mAb concentration under 20 mM NH4Cl. Notably, WNT3 knockout maintained N-glycan galactosylation proportions of mAb despite ammonia stress. These findings highlight the effectiveness of genome-wide CRISPR knockout screening in identifying novel gene targets for ammonia-resistant HEK293 cell, offering a promising strategy for improving mAb production.},
}
@article {pmid41130365,
year = {2026},
author = {Zou, M and Tao, Y and Shi, B and Xu, R and Zhu, D and Li, Y and Han, R and Wang, R},
title = {CRISPR/Cas9-based gene deletion and targeted metabolomics reveal ectoine flux reprogramming in Halomonas campaniensis.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {67-76},
doi = {10.1016/j.jbiotec.2025.10.006},
pmid = {41130365},
issn = {1873-4863},
mesh = {*Halomonas/genetics/metabolism ; *Amino Acids, Diamino/metabolism/biosynthesis/genetics ; Metabolomics/methods ; Gene Deletion ; *CRISPR-Cas Systems/genetics ; Betaine/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Ectoine and betaine are widely used compatible solutes. In Halomonas campaniensis XH26, the hom gene is involved in betaine biosynthesis, and the doeA gene participates in ectoine degradation. Deletion of hom and doeA may lead to poorly understood changes in metabolic flux within the ectoine biosynthesis pathway. The metabolically deficient XH26/Δhom and XH26/Δhom/ΔdoeA strains were constructed using a CRISPR/Cas9 approach. Comparative analyses of colony morphology, growth characteristics, and intracellular ectoine yield were conducted to evaluate the regulatory roles of the hom and doeA genes. RT-qPCR and targeted metabolomics were used to assess changes in gene expression related to ectoine biosynthesis and shifts in central carbon metabolic flux. The metabolically deficient strains XH26/Δhom and XH26/Δhom/ΔdoeA were constructed. Compared to the strain XH26, both mutant strains exhibited smaller colony diameters and shorter, broader cells. Intracellular ectoine yield increased by 13.3 % and 33.3 %, respectively, while betaine yield significantly decreased by 73.08 % and 76.92 %. RT-qPCR analysis revealed the significant upregulation of asd, lysC, ectA, ectB, and ectC, suggesting an enhanced metabolic flux toward ectoine biosynthesis. Targeted metabolomics indicated that the differentially abundant metabolites were mainly involved in four key energy metabolism pathways. These results indicate that knocking out the key genes hom and doeA in the ectoine biosynthesis pathway led to the restructuring of carbon metabolic flux in H. campaniensis. More carbon entered the ectoine biosynthesis pathway, resulting in the enhanced production of ectoine and a concomitant reduction in its degradation. These findings offer theoretical support for engineering high-yield ectoine-producing strains.},
}
@article {pmid41130838,
year = {2026},
author = {Crooke, H and Schwindt, S and Fletcher, SL and Isken, O and Harding, S and Berkley, N and Tait-Burkard, C and Warren, C and Whitelaw, CBA and Tautz, N and Lillico, SG},
title = {DNAJC14 gene-edited pigs are resistant to classical pestiviruses.},
journal = {Trends in biotechnology},
volume = {44},
number = {2},
pages = {570-586},
doi = {10.1016/j.tibtech.2025.09.008},
pmid = {41130838},
issn = {1879-3096},
mesh = {Animals ; Swine/genetics ; *Gene Editing/methods ; *Classical Swine Fever Virus/genetics/physiology/pathogenicity ; CRISPR-Cas Systems ; *Classical Swine Fever/genetics/virology ; Diarrhea Viruses, Bovine Viral ; *Disease Resistance/genetics ; },
abstract = {Infectious diseases remain a major impediment to livestock production, negatively impacting both productivity and welfare. Where key interactions between viruses and host proteins have been identified, it is possible to rationally devise intervention strategies. In vitro studies have identified the host protein DNAJC14 as a core component of the replicative cycle of classical pestiviruses. Outbreaks caused by this group of viruses cause enormous losses in stock farming due to culling and export restrictions. Using CRISPR/Cas9 gene editing, we produced a cohort of pigs with altered DNAJC14. Primary cells from these animals did not support replication of either classical swine fever virus (CSFV) or bovine viral diarrhoea virus (BVDV) in vitro. In vivo challenge with CSFV revealed that the edited pigs displayed complete resistance to infection. This establishes gene editing as an additional strategy that can contribute to the control of classical pestiviruses.},
}
@article {pmid41131871,
year = {2025},
author = {Campbell, KB and Ouye, RB and Wong, BL and Jiang, A and Okada, K and McKenney, RJ and Fisher, AJ and Beal, PA},
title = {Control of ADAR2 Dimerization and RNA Editing Efficiency by Site-Specific 2'-Fluoro Modification of Guide RNAs.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2637-2648},
doi = {10.1021/acschembio.5c00493},
pmid = {41131871},
issn = {1554-8937},
support = {R35 GM124889/GM/NIGMS NIH HHS/United States ; },
mesh = {*Adenosine Deaminase/metabolism/chemistry/genetics ; *RNA Editing ; *RNA-Binding Proteins/metabolism/chemistry/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; RNA, Double-Stranded/metabolism/chemistry ; Protein Multimerization ; Binding Sites ; },
abstract = {Adenosine Deaminases Acting on RNA (ADARs) are an important class of RNA editing enzymes that catalyze the deamination of adenosine (A) to inosine (I) in double-stranded RNA (dsRNA). Since inosine is typically read as guanosine (G) during translation, ADARs can produce A to G transitions in dsRNA. Site-directed RNA editing (SDRE) is a promising therapeutic tool wherein guide RNAs can be used to direct endogenous human ADARs to reverse disease-causing mutations in specific RNA transcripts. Guide RNA (gRNA) modifications at locations that contact the ADAR active site are often used to improve editing efficiency. However, little is known about rate-enhancing chemical modifications in the gRNA at the dsRNA binding domain (dsRBD)-RNA interface. Analysis of published crystal structures of ADAR2 bound to dsRNA suggested positions at this interface would be sensitive to gRNA modification. In this work, gRNAs bearing 2'-modifications in the dsRBD binding site were synthesized and subsequently tested to determine their effects on the editing rate of therapeutically relevant ADAR targets. We found that replacing a single 2'-OH at specific positions on the gRNA with a 2'-F substantially increased the rate of in vitro ADAR2-catalyzed adenosine deamination for two different sequences, whereas 2'-OMe at these positions was inhibitory. This effect was also validated in cellulo. The rate of ADAR1-catalyzed deamination is not stimulated by these 2'-F modifications. A crystal structure of an ADAR2 fragment bound to duplex RNA bearing a single 2'-F at guide position +13 suggested a favorable interaction between the side chain of N241 of the auxiliary ADAR2 monomer and the 2'-F modification. Furthermore, electrophoretic mobility shift assays and mass photometry indicate 2'-F at position +13 facilitates ADAR2 dimerization on the RNA substrate. This work advances our understanding of the RNA features that define superior ADAR substrates and inform the design of gRNAs for therapeutic RNA editing.},
}
@article {pmid41132368,
year = {2025},
author = {Morais, C and Costa, SS and Hanke, D and Santos, A and Krüger-Haker, H and Pomba, C and Feßler, AT and Schwarz, S and Couto, I},
title = {Genomic analysis of the Staphylococcus pseudintermedius mobilome associated with antimicrobial resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1640322},
pmid = {41132368},
issn = {1664-302X},
abstract = {The increasing antimicrobial resistance (AMR) in Staphylococcus pseudintermedius causing skin and soft-tissue infections (SSTIs) in companion animals is a public health concern. The aim of this study was to verify if mobile genetic elements (MGEs), in particular plasmids, are related to the carriage of AMR genes among circulating and clinically relevant S. pseudintermedius. In total, 56 S. pseudintermedius, representing predominant and emerging clonal lineages associated with SSTIs in dogs and cats collected in Lisbon (Portugal), were subjected to plasmid DNA extraction and digestion with EcoRI and XbaI. Each unique restriction pattern was assigned to a plasmid profile. A subset of 17 strains was further selected for hybrid whole genome sequencing (WGS) on Oxford Nanopore MinION and Illumina MiSeq platforms. Thirty-one of the 56 S. pseudintermedius strains carried one or more plasmid(s), mostly of small or medium sizes, corresponding to eight plasmid profiles. Two of the identified plasmids carried AMR determinants; plasmid pSP-G3C4, isolated from ST71 strains, carried the tetracycline resistance gene tet(K) and plasmid pSP5912, isolated from a ST2061 strain, harbored the qacG biocide resistance gene. Other AMR determinants were detected as part of MGEs integrated into the bacterial chromosomal DNA, namely Tn552, Tn552-like, Tn553, Tn916, Tn5405-like, Tn5801, Tn5801-like GI6287 and pRE25-like elements. In addition, a new chromosomal cassette, carrying fusC, was identified in a ST1183 strain. The 12 methicillin-resistant S. pseudintermedius studied carried staphylococcal cassette chromosome mec (SCCmec) type III (n = 5), SCCmec type IVg (n = 3), SCCmec NA45 (n = 1), ΨSCCmec 57395 (n = 1), the recently described cassettes SCCmec 7017-61515 (n = 1), or SCCmec type V(T)SL/154 (n = 1). Most strains carried intact prophages without AMR determinants. Intact restriction-modification systems were detected in 12 out of the 17 strains and CRISPR/Cas in five strains, four of which were methicillin-susceptible. The results of this study suggest that the AMR content in S. pseudintermedius is mainly related to MGEs integrated into the chromosomal DNA rather than located on plasmids. These results provide important insights that may lead to a better understanding of multidrug resistance in S. pseudintermedius towards improved SSTIs treatment in companion animals.},
}
@article {pmid41133737,
year = {2025},
author = {Devine, R and Noble, K and Wilkinson, B and Hutchings, M},
title = {Microbe Profile: Streptomyces formicae KY5: an ANT-ibiotic factory.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {10},
pages = {},
pmid = {41133737},
issn = {1465-2080},
mesh = {*Streptomyces/genetics/metabolism/isolation &amp; purification/classification ; *Ants/microbiology ; Multigene Family ; Anti-Bacterial Agents/biosynthesis ; Animals ; Antifungal Agents/metabolism/pharmacology ; Genome, Bacterial ; Secondary Metabolism/genetics ; Biosynthetic Pathways/genetics ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {Streptomyces formicae KY5 was isolated from a Tetraponera penzigi plant-ant nest. It is primarily known for its production of the formicamycins, antibiotics with potent activity against Gram-positive pathogens including methicillin-resistant Staphylococcus aureus, and additionally produces an antifungal compound that inhibits multi-drug-resistant fungal pathogens including Lomentospora prolificans. S. formicae is genetically tractable using CRISPR-Cas9 gene editing, allowing for detailed analysis of the formicamycin biosynthetic gene cluster. AntiSMASH analysis predicts the genome to encode at least 45 secondary metabolite biosynthetic gene clusters, many of which appear to encode novel compounds. Current research efforts are focussing on characterising the regulation of secondary metabolism at a global level in order to switch on pathways that are not typically expressed under standard laboratory conditions with the aim of identifying novel antimicrobials.},
}
@article {pmid41134772,
year = {2025},
author = {Xu, X and Zhu, L and Xu, X and Chen, J and Du, X and Zhu, L and Yu, S and Qian, L and Jiang, X and Zhou, L and Dong, Y and Wang, Y and Huang, Y and Wang, Y},
title = {TSSKL is essential for sperm mitochondrial morphogenesis and male fertility in moths.},
journal = {PLoS genetics},
volume = {21},
number = {10},
pages = {e1011914},
pmid = {41134772},
issn = {1553-7404},
mesh = {Animals ; Male ; *Spermatozoa/metabolism ; *Moths/genetics ; *Mitochondria/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Fertility/genetics ; Bombyx/genetics ; Female ; Infertility, Male/genetics ; Morphogenesis/genetics ; Testis/metabolism ; CRISPR-Cas Systems ; },
abstract = {Sperm deliver male genomic DNA to the ovum, playing a pivotal role in sexual reproduction across the animal kingdom. The molecular regulation of sperm morphogenesis has consequently become a focal point of genetic research, with dual implications for both reproductive medicine and sustainable agriculture. Here, we characterize the functional role of the testis-specific serine/threonine protein kinase-like (TSSKL) gene in the model lepidopteran insect Bombyx mori and the globally destructive crop pest Plutella xylostella. RNA-seq and qPCR analyses revealed TSSKL's testis-specific expression pattern. Using CRISPR/Cas9-mediated mutagenesis, we demonstrate that TSSKL knockout induces complete male sterility, while female fertility remains unaffected compared to wild-type. Fluorescence microscopy and ultrastructural analyses revealed that TSSKL deletion leads to severe morphological defects in both eupyrene and apyrene sperm, accompanied by impaired mitochondrial dynamics and aberrant autophagy. Comparative transcriptome and functional analyses linked these phenotypes to dysregulated energy metabolism pathways. Crucially, this sterility phenotype is conserved in P. xylostella, recapitulating the findings in B. mori. Our study demonstrates that TSSKL is crucial for male fertility, coordinating both structural and metabolic aspects of sperm development. Beyond advancing fundamental knowledge of insect reproductive biology, this work also identifies TSSKL as an ideal target for lepidopteran pest control through precision sterility induction.},
}
@article {pmid41134871,
year = {2025},
author = {Charlier, J and Sherkatghanad, Z and Makarenkov, V},
title = {Similarity-based transfer learning with deep learning networks for accurate CRISPR-Cas9 off-target prediction.},
journal = {PLoS computational biology},
volume = {21},
number = {10},
pages = {e1013606},
pmid = {41134871},
issn = {1553-7358},
mesh = {*Deep Learning ; *CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Computational Biology/methods ; *Gene Editing/methods ; Humans ; Machine Learning ; },
abstract = {Transfer learning has emerged as a powerful tool for enhancing predictive accuracy in complex tasks, particularly in scenarios where data is limited or imbalanced. This study explores the use of similarity-based pre-evaluation as a methodology to identify optimal source datasets for transfer learning, addressing the dual challenge of efficient source-target dataset pairing and off-target prediction in CRISPR-Cas9, while existing transfer learning applications in the field of gene editing often lack a principled method for source dataset selection. We use cosine, Euclidean, and Manhattan distances to evaluate similarity between the source and target datasets used in our transfer learning experiments. Four deep learning network architectures, i.e. Multilayer Perceptron (MLP), Convolutional Neural Networks (CNNs), Feedforward Neural Networks (FNNs), and Recurrent Neural Networks (RNNs), and two traditional machine learning models, i.e. Logistic Regression (LR) and Random Forest (RF), were tested and compared in our simulations. The results suggest that similarity scores are reliable indicators for pre-selecting source datasets in CRISPR-Cas9 transfer learning experiments, with cosine distance proving to be a more effective dataset comparison metric than either Euclidean or Manhattan distances. An RNN-GRU, a 5-layer FNN, and two MLP variants provided the best overall prediction results in our simulations. By integrating similarity-based source pre-selection with machine learning outcomes, we propose a dual-layered framework that not only streamlines the transfer learning process but also significantly improves off-target prediction accuracy. The code and data used in this study are freely available at: https://github.com/dagrate/transferlearning_offtargets.},
}
@article {pmid41135303,
year = {2026},
author = {Lin, Y and Zhou, Y and Yuan, X and Wang, X and Xu, H and Lei, F and Wang, L and Lan, Q and Zhu, B},
title = {RPA-CRISPR-Cas12a/RPA-CRISPR-Cas12a-LFD: Two detection platforms for the rapid identification of five forensic body fluids.},
journal = {Forensic science international. Genetics},
volume = {81},
number = {},
pages = {103376},
doi = {10.1016/j.fsigen.2025.103376},
pmid = {41135303},
issn = {1878-0326},
mesh = {Humans ; Female ; Semen/chemistry ; Menstruation ; RNA, Messenger/genetics/metabolism ; Saliva/chemistry ; *CRISPR-Cas Systems ; *Forensic Genetics/methods ; Male ; Vagina/metabolism ; Genetic Markers ; Cervix Mucus/chemistry ; beta-Globins/genetics ; Real-Time Polymerase Chain Reaction ; },
abstract = {Identifying tissue source types of human peripheral blood, menstrual blood, vaginal secretion, semen, and saliva left at a crime scene can offer crucial clues for case investigation. Although messenger RNA (mRNA) profiling has become a robust tool for body fluid identification, conventional methods remain constrained by time-consuming workflows and dependence on laboratory infrastructure, hindering rapid on-site forensic analysis. To address these limitations, this study designed the RPA-CRISPR-Cas12 experiment and independently developed an RPA-CRISPR-Cas12a-LFD detection system, which achieved simple, rapid (<40 min), and accurate forensic identification of five kinds of critical body fluids. Initially, we mined the Genotype-Tissue Expression database and related literatures to screen candidate mRNA markers, subsequently employed RT-qPCR to evaluate their expression levels, and applied the Boruta algorithm to optimize the number of mRNA markers, ultimately identifying five mRNA markers: HBB (peripheral blood), MMP10 (menstrual blood), DKK4 (vaginal secretion), AKAP4 (semen), and HTN3 (saliva). The developed system exhibited exceptional specificity. Furthermore, the sensitivity values of HBB, MMP10, DKK4, AKAP4, and HTN3 markers in RPA-CRISPR-Cas12 and RPA-CRISPR-Cas12a-LFD detection systems were 0.1, 0.1, 1, 0.1, 0.1 ng; and 0.1, 0.1, 1, 1 and 1 ng, respectively. Among them, the sensitivity of HBB and DKK4 markers was superior to that of previous studies. Notably, the platform successfully resolved mixed samples (excluding AKAP4 marker), and MMP10 and DKK4 markers retained detectability in aged menstrual blood and vaginal secretion samples. The proposed method provides the groundwork for further development of tissue origin identification tools of forensic body fluids, which may eventually be suitable for on-site rapid detection environments.},
}
@article {pmid41135356,
year = {2026},
author = {Wang, W and Zheng, Y and Zhang, L and Bao, Y and Liu, J and Zang, J and Qu, Y and Zhang, K and Han, R and Ren, H and Zhu, L and Cao, Y and Wang, B and Shen, Q and Sun, W},
title = {A multiplex RPA-CRISPR/Cas12a platform for rapid and accurate toxinotyping of Clostridium perfringens.},
journal = {Talanta},
volume = {298},
number = {Pt B},
pages = {128998},
doi = {10.1016/j.talanta.2025.128998},
pmid = {41135356},
issn = {1873-3573},
mesh = {*Clostridium perfringens/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Bacterial Toxins/genetics/analysis ; *Nucleic Acid Amplification Techniques/methods ; *Recombinases/metabolism ; Humans ; Limit of Detection ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Clostridium perfringens (C. perfringens) is a leading cause of foodborne disease worldwide, requiring rapid and accurate toxinotyping for effective outbreak control and surveillance. Herein, we developed C. perfringens-multiplex RPA-CRISPR/Cas12a, an integrated detection platform combing multiplex Recombinase Polymerase Amplification (RPA) with Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 12a (CRISPR/Cas12a)-mediated detection for comprehensive toxinotyping. The system simultaneously identifies six key toxin genes (cpa, cpb, etx, iap, cpe, netB) in two reaction tubes, enabling discrimination of all seven C. perfringens toxinotypes (A-G). The C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform exhibited exceptional analytical performance, achieving a detection limit of ≤10 copies/μL for across all targets while maintaining absolute specificity against the human genomic DNA and 5 common foodborne pathogens. In validation testing with 12 naturally contaminated food samples, the C. perfringens-multiplex RPA-CRISPR/Cas12a assay platform demonstrated superior performance to commercial qPCR kits, accurately identifying eight Type A (cpa-gene-positive) and four Type F (cpa-gene and cpe-gene co-positive) strains. When coupled with a portable detection device, the platform completed the entire diagnostic workflow within 50 min while maintaining laboratory-level accuracy under field conditions. The rapid, cost-effective, and equipment-free system is particularly suited for decentralized toxin surveillance in resource-limited settings. By integrating high sensitivity, multiplex capability, and field applicability, this system significantly advances Point-of-care Testing (POCT) capabilities for food safety monitoring, supporting global food safety initiatives.},
}
@article {pmid41135510,
year = {2026},
author = {Yang, Q and Sun, Y and Sun, L and Chi, T and Chen, Z},
title = {Cryo-EM structure of the RfxCas13d-crRNA-off-target-RNA complex.},
journal = {Structure (London, England : 1993)},
volume = {34},
number = {1},
pages = {11-19.e2},
doi = {10.1016/j.str.2025.09.010},
pmid = {41135510},
issn = {1878-4186},
mesh = {Cryoelectron Microscopy ; Models, Molecular ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Protein Binding ; *RNA, Bacterial/chemistry/metabolism ; Catalytic Domain ; },
abstract = {The CRISPR-Cas system is crucial for the adaptive immune response of prokaryotes and has been widely applied for genetic engineering. Cas13d, a type VI-D CRISPR-Cas effector, functions as RNA-guided ribonuclease and has been engineered for programmable RNA editing, which is a commonly used, active, and well-characterized small type VI editor. Here, we determined cryoelectron microscopy (cryo-EM) structures of Ruminococcus flavefaciens Cas13d in a RfxCas13d-crRNA-off-target-RNA ternary complex and RfxCas13d-crRNA binary complex at 3.10 and 3.13 Å resolution. The ternary complex consists of RfxCas13d, crRNA, and a captured short off-target ssRNA at a complex state of binding proximal mismatched RNA. RfxCas13d undergoes conformational changes with or without the off-target RNA, but the catalytic sites remain unchanged. Mg[2+] aids in stabilizing the crRNA repeat region structure, which may be crucial for RNA binding. This discovery provides the foundation for developing RfxCas13d as a mature tool and offers a framework for advancing transcriptome engineering.},
}
@article {pmid41135835,
year = {2026},
author = {Chiu, CN and Tam, BM and Burns, P and Moritz, OL},
title = {RPE65 knockout Xenopus laevis have a compromised but detectable electroretinogram and altered visual responses, without retinal degeneration or altered melanophore dispersion.},
journal = {Experimental eye research},
volume = {262},
number = {},
pages = {110699},
doi = {10.1016/j.exer.2025.110699},
pmid = {41135835},
issn = {1096-0007},
mesh = {Xenopus laevis/genetics ; Animals ; Gene Knockout Techniques ; *Retinoid Isomerohydrolase/genetics ; Disease Models, Animal ; *Rhodopsin ; Retinal Rod Photoreceptor Cells/pathology ; Electroretinography ; *Photophobia/physiopathology ; *Melanophores/physiology ; Retinal Cone Photoreceptor Cells ; CRISPR-Cas Systems ; Larva ; Behavior, Animal ; },
abstract = {The isomerohydrolase RPE65 is a critical element of the visual cycle, the series of enzymatic reactions by which the chromophore of the visual pigments is regenerated following light exposure. In humans, mutations in the RPE65 gene cause a severe form of blindness called Leber's congenital amaurosis. Studies of Rpe65[-/-] mice have shown dramatic depletion of 11-cis-retinal in the retina, resulting in a slow retinal degeneration. However, a number of studies suggest that RPE65 may not be necessary for the regeneration of photopigment in all photoreceptor types. Using CRISPR/Cas9 technology, we previously generated RPE65 knockout Xenopus laevis in order to test the involvement of rhodopsin chromophore in the cell death mechanisms associated with rhodopsin mutations and rhodopsin quality control. Here we further characterize the effects of RPE65 knockout in these animals, and show their rod photoreceptors have shortened outer segments that lack detectable rhodopsin photopigment. However, there is no progressive degeneration of rods or cones. Via electroretinography we found greatly reduced but significant responses to light under scotopic and photopic conditions. We also found reduced behavioral sensitivity to light, while light-induced melanophore dispersion was unaffected. RPE65 knockout X. laevis may be a useful system for examining RPE65-independent photosensation mechanisms in vertebrates.},
}
@article {pmid41136394,
year = {2025},
author = {Chow, JT and Desjardins, A and Lee, DKC and Grigore, IA and Lee, L and Fu, NJ and Chau, S and Lee, BY and Gabra, MM and Salmena, L},
title = {A microRNA CRISPR screen reveals microRNA-483-3p as an apoptotic regulator in prostate cancer cells.},
journal = {Cell death &amp; disease},
volume = {16},
number = {1},
pages = {752},
pmid = {41136394},
issn = {2041-4889},
mesh = {*MicroRNAs/genetics/metabolism ; Humans ; *Prostatic Neoplasms/genetics/pathology/metabolism ; Male ; *Apoptosis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Cell Survival/genetics ; },
abstract = {The development of traditional protein-targeted cancer therapies is a slow and arduous process, often taking years or even decades. In contrast, RNA-based therapies targeting crucial microRNA (miRNA) offer a faster alternative due to the sequence-specific nature of miRNA inhibitor binding. This, combined with the capacity of individual miRNA to influence multiple cellular pathways, makes these small RNA attractive targets for cancer therapy. While miRNA are known to be dysregulated in prostate cancer (PCa), identifying their individual contributions to disease progression and the identification of therapeutically actionable miRNA targets in PCa has been challenging due to limited profiling and lack of screening tools. To address this need, we developed miRKOv2, a miRNA-only CRISPR knockout library enabling systematic, genome-wide loss-of-function screens to identify miRNA essential for PCa cell survival. Our screens uncovered 70 potential essential miRNA candidates, with miR-483 demonstrating the most significant impact on PCa cell viability. Functional characterization revealed that miR-483 disruption potentiated apoptosis in PCa cell lines. Mechanistically, we uncovered a novel regulatory axis wherein miR-483-3p directly modulates a BCLAF1/PUMA/BAK1 apoptotic signaling network, highlighting its critical role in maintaining PCa cell survival. Our findings provide novel insights into the complex regulatory role of miRNA in PCa progression and offer a potential therapeutic strategy for targeting miRNA-mediated pathways in metastatic disease.},
}
@article {pmid41136797,
year = {2025},
author = {Basarali, MK and Daemi, A and Tahiraga, RG and Özbolat, G and Hooshiar, MH and Shirazi, MSR and Döğüş, Y},
title = {Artificial intelligence-driven epigenetic CRISPR therapeutics: a structured multi-domain meta-analysis of therapeutic efficacy, off-target prediction, and gRNA optimization.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {223},
pmid = {41136797},
issn = {1438-7948},
mesh = {*Artificial Intelligence ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic ; *Genetic Therapy/methods ; },
abstract = {CRISPR-based epigenetic editing enables reversible regulation of gene expression without permanent DNA modification. The integration of artificial intelligence (AI) enhances guide RNA (gRNA) design, off-target prediction, and delivery optimization. We conducted a systematic review and meta-analysis (2015-2025) in accordance with PRISMA 2020 guidelines to evaluate the impact of AI on the precision, safety, and therapeutic efficacy of epigenetic CRISPR tools. From 540 screened records, 58 studies met inclusion criteria, of which 41 provided extractable quantitative data for meta-analysis and 17 contributed to qualitative synthesis. Random-effects models, subgroup analyses, and bias assessments were applied. Pooled analyses demonstrated strong positive effects across three domains: therapeutic efficacy (SMD = 1.67), gRNA optimization (SMD = 1.44), and off-target prediction (AUC = 0.79). Publication bias was minimal, and subgroup analyses indicated the strongest impact in therapeutic applications. Deep learning models were consistently associated with higher effect sizes. Qualitative synthesis revealed trends in interpretable AI, omics integration, and delivery innovations, underscoring AI's role in safer and more precise CRISPR editing. Overall, AI significantly improves the precision and therapeutic performance of CRISPR-based epigenetic tools, with the strongest effects observed in therapeutic efficacy, supporting their potential for personalized gene editing.},
}
@article {pmid41136894,
year = {2025},
author = {Liu, H and Zhu, G and Chen, L and Ye, H and Zhang, Y and Han, G},
title = {Machine learning prediction of bacterial optimal growth temperature from protein domain signatures reveals thermoadaptation mechanisms.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {957},
pmid = {41136894},
issn = {1471-2164},
support = {32172769//National Natural Science Foundation of China/ ; },
abstract = {UNLABELLED: Cultivating the vast majority of uncultured microbes requires knowledge of their physiological preferences, particularly optimal growth temperature (OGT). We present a machine learning approach that utilizes protein domain frequencies from bacterial genomes to predict OGT across a wide continuous range (1–83 °C). Our Random Forest model, trained on a dataset of 1,498 genomes, achieved high predictive accuracy (R[2]=0.853 on test data, 82.4% of predictions within a ± 10 °C error margin), substantially advancing current capabilities and offering a practical tool to guide cultivation experiments. Analysis of the model identified key protein domain signatures associated with thermal adaptation. The enrichment of domains related to polyamine metabolism, the tRNA methyltransferase family, and CRISPR-Cas systems was positively correlated with higher OGTs, providing genomic evidence for their roles in thermotolerance. Conversely, domains involved in redox homeostasis, transport, and nucleic acid binding were more abundant at lower temperatures. These findings not only facilitate targeted cultivation efforts but also deepen our understanding of the molecular strategies bacteria employ to thrive across diverse thermal niches.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12162-z.},
}
@article {pmid41137484,
year = {2025},
author = {Lee, SY and Park, HH},
title = {Investigating the molecular mechanisms underlying the anti-CRISPR function of AcrIIA13b protein.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70304},
pmid = {41137484},
issn = {1742-4658},
support = {RS-2025-02316334//NRF-Korea/ ; },
abstract = {The CRISPR-Cas systems of adaptive immunity in bacteria and archaea provide resistance against phages and other mobile genetic elements. Counteractive anti-CRISPR (Acr) proteins in phages and archaeal viruses impede these CRISPR-Cas systems. Although CRISPR-Cas systems have revolutionized genome editing, potential off-target events remain a safety concern. Hence, a thorough comprehension of the structural and molecular basis of diverse Acrs is imperative to unravel the fundamental mechanisms governing CRISPR-Cas regulation. Here, we present the structure of AcrIIA13b from Staphylococcus haemolyticus and analyze its structural and functional features to reveal the molecular basis underlying the inhibition of Cas9 by AcrIIA13b. Our structural analysis shows that AcrIIA13b eliminates the cleavage activity of Staphylococcus aureus Cas9 (SauCas9) by blocking the PAM-binding region of Cas9 so that Cas9 cannot recognize the target DNA. In addition, we demonstrate that the 15 amino acid residues at the N terminus of AcrIIA13b, which were revealed to be important for its dimerization, are critical for its inhibitory activity against Cas9. Our findings shed light on the molecular basis of AcrIIA13b-mediated CRISPR-Cas inhibition and provide valuable insights into the arms race between bacteria and phages.},
}
@article {pmid41137488,
year = {2026},
author = {Kang, YW and Park, HH},
title = {The anti-CRISPR protein AcrIE8.1 inhibits the type I-E CRISPR-Cas system by directly binding to the Cascade subunit Cas11.},
journal = {FEBS letters},
volume = {600},
number = {3},
pages = {334-341},
pmid = {41137488},
issn = {1873-3468},
support = {2024//Chung-Ang University/ ; RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Protein Binding ; *Viral Proteins/chemistry/metabolism/genetics ; Crystallography, X-Ray ; Bacteriophages/metabolism/genetics ; Models, Molecular ; },
abstract = {CRISPR-Cas systems provide adaptive immunity to bacteria by recognizing and destroying foreign genetic elements. The type I-E CRISPR-Cas system utilizes a multi-subunit Cascade complex to detect target DNA and recruit the Cas3 nuclease for degradation. To overcome this defense, bacteriophages have evolved anti-CRISPR (Acr) proteins that inhibit various steps of the CRISPR interference pathway. Here, we determined the crystal structure of AcrIE8.1, an uncharacterized Acr, revealing it binds to Cas11, a Cascade subunit, to disrupt function. AcrIE8.1 has a compact fold with a defined Cas11-binding interface, suggesting a unique inhibitory mechanism among AcrIE proteins. These findings highlight Cas11 as a critical target for Acr-mediated immune evasion. Impact statement Through a combination of structural and biochemical analyses, we demonstrate that AcrIE8.1 directly binds to the Cas11 subunit of the Cascade complex to inhibit the CRISPR-Cas system. This represents a novel inhibitory strategy not previously observed among AcrIE proteins.},
}
@article {pmid41138228,
year = {2025},
author = {Hodge, CA and Donegan, NP and Armstrong, DA and Hayden, MS and Howell, AL},
title = {Enhanced cleavage of genomic CCR5 using CASX2[Max].},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-18},
pmid = {41138228},
issn = {1555-8584},
support = {I01 BX005248/BX/BLRD VA/United States ; P30 CA023108/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Receptors, CCR5/genetics/metabolism/chemistry ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. CasX2 (PlmCas12e) belongs to the class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils and offers several advantages as a potential therapeutic CRISPR system over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9). These advantages include its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and the absence of pre-existing immunity in humans. We compared the cleavage efficiency and double-stranded break repair characteristics between CasX2 and CasX2[Max], a recently generated CasX2 variant with three amino acid substitutions, for targeting CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two single guide RNAs (sgRNAs) were designed that flank the 32 bases deleted in the natural CCR5 ∆32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2[Max] using sgRNAs with 20 nt and 23 nt spacer lengths, enabled cleavage of genomic CCR5. Structural modelling indicated that two of the CasX2[Max] amino acid substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2[Max] in cellular gene excision. In sum, CasX2[Max] consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.},
}
@article {pmid41138445,
year = {2026},
author = {Hu, M and Liu, F and Zhang, J and Yang, L and Yan, Y and Wang, Y and Liu, M and An, H and Zhu, L and Ai, Y and Jiang, X},
title = {Integrated CRISPR/Cas12a-activated liposomal SERS amplification in microfluidic chips for ultrasensitive ampicillin detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118131},
doi = {10.1016/j.bios.2025.118131},
pmid = {41138445},
issn = {1873-4235},
mesh = {*Ampicillin/analysis/isolation &amp; purification ; Liposomes/chemistry ; CRISPR-Cas Systems/genetics ; *Spectrum Analysis, Raman/methods ; *Biosensing Techniques ; Limit of Detection ; *Anti-Bacterial Agents/analysis ; Animals ; Gold/chemistry ; Lab-On-A-Chip Devices ; Milk/chemistry ; Silver/chemistry ; Humans ; Metal Nanoparticles/chemistry ; },
abstract = {The misuse of antibiotics has created a vicious cycle: from excessive use in livestock breeding to elevated residue levels in food products, ultimately leading to heightened antibiotic resistance in humans, a phenomenon with significant public health consequences. Food-based antibiotic testing is crucial for preventing antibiotic misuse and protecting human health. However, traditional analytical methods exhibit high equipment dependency, restricting on-site detection in hills, plateaus, and other regions with inconvenient transportation. This study developed an integrated CRISPR/Cas12a-activated liposomal Surface-enhanced Raman scattering (SERS) amplification system within a microfluidic chip for ultrasensitive ampicillin detection. The method achieves primary signal amplification by converting ampicillin concentration into a DNA signal through catalytic hairpin assembly (CHA), followed by secondary signal amplification using an Au@Ag substrate coupled with SERS technology for 4-MPBA@liposome marker detection. The platform demonstrated a linear detection range of 1 fM-1 nM with a limit of detection as low as 740 aM. Notably, the method exhibited excellent stability, selectivity, and sensitivity, successfully detecting ampicillin residues in environmental water and milk samples. It provides a technological approach for environmental monitoring and food safety, demonstrating significant practical application value.},
}
@article {pmid41138562,
year = {2025},
author = {Marteau, S and Duboscq-Bidot, L and Aizawa, T and Hamlin, M and Villard, E and Guicheney, P and Fontaine, V},
title = {Generation of a FAM189A2/ENTREP1 knockout human induced pluripotent stem cell line using CRISPR/Cas9 technology.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103857},
doi = {10.1016/j.scr.2025.103857},
pmid = {41138562},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; Cell Line ; *Membrane Proteins/genetics/metabolism ; *Gene Knockout Techniques ; },
abstract = {FAM189A2/ENTREP1 encodes a transmembrane protein, EREP1, ubiquitously expressed, with higher levels in thyroid gland, skeletal muscle, heart and glial cells, suggesting tissue-specific roles. It is regulated in various cancers and genetic studies suggested that it could be associated with cardiac and cardiometabolic diseases. EREP1 was reported to activate ITCH, an E3 ubiquitin ligase, involved in endosomal dynamics and protein trafficking. We generated by CRISPR/Cas9 technology a human induced pluripotent stem cells (hiPSCs) line carrying two compound heterozygous deletions leading to gene invalidation, with the aim of further exploring the poorly understood function of this protein.},
}
@article {pmid41138779,
year = {2025},
author = {Afresham, S and Khan, MK and Mughal, MAS and Mehmood, MS and Ali, S and Bashir, M and Abbas, Z and Azeem, A and Ahmed, W and Imran, M and Abbas, RZ and Sindhu, ZU and Sajid, MS},
title = {Recent advancements in the diagnosis of parasitic diseases.},
journal = {Molecular and biochemical parasitology},
volume = {264},
number = {},
pages = {111706},
doi = {10.1016/j.molbiopara.2025.111706},
pmid = {41138779},
issn = {1872-9428},
mesh = {Humans ; *Parasitic Diseases/diagnosis/parasitology ; *Molecular Diagnostic Techniques/methods ; Animals ; Parasites/genetics/isolation &amp; purification ; High-Throughput Nucleotide Sequencing ; *Diagnostic Tests, Routine/methods ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {Parasitic infections present a significant health risk to the public, affecting millions of people, particularly in underdeveloped and developing countries. In developing countries, these infections are also responsible for causing significant economic challenges due to elevated healthcare expenditure. Accurate diagnosis and effective treatment methods are essentially required to combat this global issue. For decades, traditional diagnostic methods such as microscopy, serological testing, histopathology, and culturing have been used for the diagnosis of these parasitic infections. While these methods can be effective and helpful in many ways, they often consume a lot of time, require an elevated level of expertise, and have limited applications particularly in endemic regions having issues like poor infrastructure and limited access to healthcare facilities. This review aims to highlight the urgent need for a revolution to replace these conventional techniques with more affordable, quick, and field-adjustable tools such as rapid diagnostic tests (RDTs) and molecular methods and provides a comprehensive picture of advanced diagnostic tools used in the identification of parasites. With the advancements in science and technology, molecular methods such as Polymerase chain reaction, Next generation sequencing, and isothermal loop-mediated amplification have remarkably enhanced the sensitivity and accuracy of parasite detection and identification. The range of these diagnostic methods has further extended by advanced serological methods, imaging techniques, and immunological methods. Moreover, the innovations in nanotechnology, CRISPR-Cas methods, and multi-omics techniques for identification of parasite DNA, antigens, metabolites, and host responses are invaluable for diagnostic accuracy, comprehensive understanding of parasite biology, and for the discovery of new therapeutic targets and diagnostic biomarkers. However, further research and developments are required for an effective and long-lasting impact of these advancements.},
}
@article {pmid41139369,
year = {2025},
author = {Wei, H and Li, D and Xie, K and Lou, S and Dong, G and Guo, F and Lian, G and Pan, X and Zeng, Z and Han, N and Gao, Z and Bian, H},
title = {Creation of new rice germplasm with cross-resistance to auxin herbicides picloram and dicamba by genome editing of OsAFB4.},
journal = {TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik},
volume = {138},
number = {11},
pages = {282},
pmid = {41139369},
issn = {1432-2242},
support = {LGN21C130006//Natural Science Foundation of Zhejiang Province/ ; 32201834//Innovative Research Group Project of the National Natural Science Foundation of China/ ; },
mesh = {*Oryza/genetics/drug effects/growth &amp; development ; *Herbicide Resistance/genetics ; *Gene Editing ; *Picloram/pharmacology ; *Herbicides/pharmacology ; *Dicamba/pharmacology ; Indoleacetic Acids ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Plant Breeding ; Gene Expression Regulation, Plant ; Plants, Genetically Modified ; },
abstract = {Genome Editing of OsAFB4 in rice cultivar Jiahe212 confers cross-resistance to synthetic auxin herbicides picloram and dicamba without reducing grain yield, providing a novel germplasm for herbicide-resistant rice breeding. Synthetic auxin herbicides (SAHs) are widely employed in global agriculture owing to low toxicity and high efficiency in weed management. Mutations in OsAFB4, an AUXIN SIGNALING F-BOX gene, led to specific resistance to SAH picloram in rice. However, potential application of OsAFB4 modification for resistance to other SAHs and its effect on agronomic traits in rice remain uncharacterized. In this study, we employed CRISPR/Cas9-mediated genome editing to generate OsAFB4 mutations in the elite commercial rice cultivar Jiahe212 (JH212). Hydroponic-culture experiments showed that Osafb4 mutant lines exhibited significant resistance to both SAHs: picloram and dicamba. Promoter activity assays using a pOsAFB4:GUS reporter line revealed that SAH treatments induced significant upregulation of GUS expression specifically in shoot apices. Under picloram or dicamba treatment, expressions of OsIAA1, OsIAA3, OsIAA9 and OsIAA20 were significantly upregulated in wild-type plants, while remarkably suppressed in Osafb4 mutants, revealing a critical role for OsAFB4 in regulating auxin-responsive IAA genes during OsAFB4-mediated sensitivity to SAH. Selected for field trials, the Cas9-free Osafb4-13 mutant line exhibited robust resistance to both picloram and dicamba without reducing grain yield compared to untreated controls. This study provides a new cross-herbicide-resistant rice germplasm without reducing grain yield by targeted editing of OsAFB4.},
}
@article {pmid41139756,
year = {2025},
author = {Kang, CM and Jeong, RD},
title = {Rapid and visual detection of Lentinula edodes spherical virus using CRISPR/Cas12a-based RT-RPA.},
journal = {Virus genes},
volume = {61},
number = {6},
pages = {724-732},
pmid = {41139756},
issn = {1572-994X},
mesh = {*Shiitake Mushrooms/virology/genetics ; *CRISPR-Cas Systems ; *Fungal Viruses/genetics/isolation &amp; purification ; Republic of Korea ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; },
abstract = {Lentinula edodes (shiitake) is a globally important edible mushroom, but viral infections can hinder mycelial growth, reduce yield, and degrade quality, leading to economic losses. This study examined viral infections in L. edodes cultivated in South Korea. We developed a CRISPR/Cas12a-based reverse transcription recombinase polymerase amplification (RT-RPA) assay for the rapid and accurate detection of L. edodes spherical virus (LeSV), the predominant mycovirus-infecting L. edodes in Korea. The optimized RT-RPA-CRISPR/Cas12a assay can be completed within 20 min at 34 °C and demonstrated 100 times higher sensitivity than RT-PCR. Field sample verification further confirmed its superior detection rate over RT-PCR. The RT-RPA-CRISPR/Cas12a method described herein has the potential to facilitate early diagnosis of LeSV infections and support disease management in L. edodes cultivation.},
}
@article {pmid41139833,
year = {2025},
author = {Neo, DM and Ben-Zion, I and Bagnall, J and Solomon, MY and Bond, AN and Gath, E and Zhang, S and Shoresh, N and Gomez, J and Hung, DT},
title = {A Multiplexed, Target-Based Phenotypic Screening Platform Using CRISPR Interference in Mycobacterium abscessus.},
journal = {ACS infectious diseases},
volume = {11},
number = {11},
pages = {3263-3275},
pmid = {41139833},
issn = {2373-8227},
mesh = {*Mycobacterium abscessus/genetics/drug effects ; Microbial Sensitivity Tests ; Mycobacterium Infections, Nontuberculous/microbiology/drug therapy ; *Anti-Bacterial Agents/pharmacology ; Drug Discovery/methods ; Bacterial Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems ; Phenotype ; Humans ; },
abstract = {The rise of difficult-to-treat Mycobacterium abscessus infections presents a growing clinical challenge due to the immense arsenal of intrinsic, inducible and acquired antibiotic resistance mechanisms that render many existing antibiotics ineffective against this pathogen. Moreover, the limited success in discovery of novel compounds that inhibit novel pathways underscores the need for innovative drug discovery strategies. Here, we report a strategic advancement in PROSPECT (PRimary screening Of Strains to Prioritize Expanded Chemistry and Targets), which is an antimicrobial discovery strategy that measures chemical-genetic interactions between small molecules and a pool of bacterial mutants, each depleted of a different essential protein target, to identify whole-cell active compounds with high sensitivity. Applying this modified strategy to M. abscessus, in contrast to previously described versions of PROSPECT which utilized protein degradation or promoter replacement strategies for generating engineered hypomorphic strains, here we leveraged CRISPR interference (CRISPRi) to more efficiently generate mutants each depleted of a different essential gene involved in cell wall synthesis or located at the bacterial surface. We applied this platform to perform a pooled PROSPECT pilot screen of a library of 782 compounds using CRISPRi guides as mutant barcodes. We identified a range of active hits, including compounds targeting InhA, a well-known mycobacterial target but under-explored in the M. abscessus space. The unexpected susceptibility to isoniazid, traditionally considered to be ineffective in M. abscessus, suggested a complex interplay of several intrinsic resistance mechanisms. While further complementary efforts will be needed to change the landscape of therapeutic options for M. abscessus, we propose that PROSPECT with CRISPRi engineering provides an increasingly accessible, high-throughput target-based phenotypic screening platform and thus represents an important step toward accelerating early stage drug discovery.},
}
@article {pmid41141388,
year = {2026},
author = {Wang, T and Yu, M and Liu, P and Song, Z and Li, C and Yang, J and Liu, N},
title = {In vivo gene therapy: A strategy for mutations, degenerations, and tumors.},
journal = {Genes &amp; diseases},
volume = {13},
number = {1},
pages = {101808},
pmid = {41141388},
issn = {2352-3042},
abstract = {Gene mutations, organ function degeneration, and carcinogenesis are the primary threats to human health. Gene therapy, which involves the addition, deletion, regulation, and editing of genes, as well as the development of genetic vaccines, can potentially cure genetic mutation disorders, degenerative diseases, and cancers. Ex vivo gene therapy has recently been used to treat monogenetic mutation diseases of the hematopoietic system and cancers. However, in vivo gene therapy remains inapplicable. The primary elements of in vivo gene therapy include deoxyribonucleic acid (DNA) nucleases (e.g., zinc finger nucleases, transcription activator-like effector nucleases), CRISPR-Cas system, base editors, prime editors, and delivery vectors (e.g., viral and non-viral vehicles). According to the development of DNA nucleases and delivery vectors, in vivo gene therapy can be made available for future clinical use. The current review summarizes the development of DNA nucleases and delivery vectors for in vivo gene therapy, emphasizing recent progress.},
}
@article {pmid41141466,
year = {2025},
author = {Mohiuddin, M},
title = {Monitoring and Assessment of Circulating Tumor DNA in Cancers Using Ultrarapid Sensitivity as an Innovative Practice.},
journal = {Health science reports},
volume = {8},
number = {10},
pages = {e71409},
pmid = {41141466},
issn = {2398-8835},
abstract = {BACKGROUND: Liquid biopsy with circulating tumor DNA (ctDNA) has rapidly emerged as a new paradigm for assessing tumor burden, genetic heterogeneity, and therapeutic response in a real-time, noninvasive manner. However, ctDNA is often visually low (sometimes < 0.1% of the total circulating cell-free DNA), creating a significant challenge for reliable detection (especially for early-stage disease and minimal residual disease).<br><br>DISCUSSION: New technologies for structural variant (SV)-based ctDNA assays, nanomaterial-based electrochemical sensors, magnetic nano-electrode platforms, and fragment-enriched library preparation have improved sensitivity to attomolar concentrations and less in some populations. In some cancers, ctDNA may provide early evidence of recurrence (i.e., >&#x2009;1 year) before being clinically evident using traditional metrics. These technologies allow for unprecedented opportunities and sensitivity for early detection, monitoring of treatment response, and early detection of molecular recurrence. Nevertheless, a barrier remains for widespread clinical application owing to pre-analytical technique variability, analytical platform variability, cost, and the necessity of large-scale, prospective trials.<br><br>CONCLUSION: This study will analyze new innovative technology-based ultrasensitive ctDNA assay detection and future research and clinical applications for breast, colorectal, lung, lymphoid, and gastroesophageal cancers, and studies assessing ctDNA for monitoring treatment. Prospects for ctDNA detection utilizing multiplexed CRISPR-Cas ctDNA assays, microfluidic point-of-care (POC) devices, and AI-based error suppression methods may be the next horizon for ctDNA liquid biopsy technology.},
}
@article {pmid41144208,
year = {2026},
author = {Shaw, S and Sateriale, A and Pawlowic, MC and Vinayak, S and Brooks, CF and Byerly, JH and Striepen, B},
title = {Genetic Manipulation of Cryptosporidium parvum.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2978},
number = {},
pages = {173-185},
pmid = {41144208},
issn = {1940-6029},
mesh = {*Cryptosporidium parvum/genetics ; Animals ; Mice ; CRISPR-Cas Systems ; *Cryptosporidiosis/parasitology ; Gene Editing/methods ; Transfection/methods ; Humans ; },
abstract = {Cryptosporidium parvum can be reliably genetically manipulated using CRISPR/Cas9 driven homologous repair coupled to in vivo propagation in immunodeficient mice. This chapter will guide through foundational procedures for excystation, transfection, infection, collection, and purification of transgenic Cryptosporidium parvum. The genetic tools for Cryptosporidium research were expanded significantly over the last 5 years. While we cannot cover all advances in detail, we will highlight novel selection markers, conditional mutagenesis strategies, and genetic crosses.},
}
@article {pmid41144643,
year = {2025},
author = {Huang, C and Guo, LJ and Li, JY and Chen, YW and Wu, B and Liu, YW and Zhang, CZ and Lin, WW and Yang, Y and Chen, JY and Liu, ZJ},
title = {A CRISPR-Cas12a Fluorescent Aptasensor for Point-of-Care Drug Concentration Detection: A Synergistic Regulation by DNA Aptamer and RNA Blocker.},
journal = {Analytical chemistry},
volume = {97},
number = {44},
pages = {24415-24424},
doi = {10.1021/acs.analchem.5c03744},
pmid = {41144643},
issn = {1520-6882},
mesh = {*Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Point-of-Care Systems ; *Vancomycin/analysis/blood ; Limit of Detection ; *RNA/chemistry/antagonists &amp; inhibitors ; Humans ; *Fluorescent Dyes/chemistry ; *Anti-Bacterial Agents/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The combination of CRISPR-Cas12a with aptamers can potentially improve the sensitivity, specificity, and speed of non-nucleic acid target detection. Nevertheless, current CRISPR-Cas12a aptasensors, solely dependent on aptamer affinity and overlooking the enzymatic regulation of CRISPR-Cas12a, may produce false-positive signals. We proposed a CRISPR-Cas12a aptasensor synergistically regulated by an aptamer and RNA, which incorporated aptamer-mediated drug recognition and CRISPR-powered signal amplification in a one-pot format. Herein, a regulatory RNA probe synergized with the conformational switching of a drug-induced aptamer, enabling precise regulation of Cas12a trans-cleavage activity via toehold-mediated strand displacement (TMSD). This dual regulatory mechanism transformed the aptamer-activated CRISPR-Cas12a sensing process into a TMSD-driven conditional reaction, avoiding false-positive signals and thus achieving better detection performance with a lower detection limit. With the vancomycin (VCM) aptamer as a model, the aptasensor can detect VCM within 30 min from 2% serum, 1% synovial fluid, and 1% cerebrospinal fluid, with a detection limit of 13.62, 7.56, and 6.75 nM, respectively. The proposed aptasensor was incorporated into a custom 3D-printed portable fluorometer and underwent clinical validation in 22 VCM serum samples, reporting no significant difference when compared with the enzyme-multiplied immunoassay technique, confirming the reliability for point-of-care quantification. It further received cross-validation with a quinine aptamer, suggesting universality [a linear range of 10-250 nM (R[2] = 0.985) and a detection limit of 0.42 nM]. By integration of aptamer selectivity with CRISPR programmability, this work presents a novel robust biosensing paradigm for point-of-care drug concentration detection.},
}
@article {pmid41145060,
year = {2026},
author = {Monteiro Belo Dos Santos, S and Van Tricht, C and Lammertyn, J and Spasic, D},
title = {Zoonotic disease detection at the point-of-care: the best of RPA and CRISPR-Cas.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118139},
doi = {10.1016/j.bios.2025.118139},
pmid = {41145060},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Biosensing Techniques/methods/instrumentation ; SARS-CoV-2/isolation &amp; purification/genetics ; *COVID-19/diagnosis/virology ; Animals ; *Point-of-Care Systems ; *Zoonoses/diagnosis/virology ; Recombinases ; Molecular Diagnostic Techniques/methods ; Point-of-Care Testing ; Pandemics ; },
abstract = {Biosensors are increasingly crucial in detecting biomarkers for emerging zoonotic diseases at the point-of-care (POC). This imminence was recently highlighted by the deficient response during the SARS-CoV-2 pandemic. While polymerase chain reaction (PCR) is the common nucleic acid (NA) testing method for zoonotic diseases in laboratory settings, it is impractical for the POC settings due to the equipment-related cost, lack of portability and user-friendliness. Recent advances in NA amplification introduced isothermal methods, such as recombinase polymerase amplification (RPA), which is known for its low temperature (37-42 °C), short incubation time (5-20 min) and suitability for integration in miniaturized, portable, low-cost, highly sensitive diagnostic platforms. However, RPA susceptibility to false positive results steered to its combination with CRISPR-Cas12/13, leading to the rise of SHERLOCK and DETECTR. This review first explores RPA-CRISPR-Cas bioassay development as either two- or one-step. This is followed by a discussion on the integration of canonical RPA, or its combination with CRISPR-Cas, into different diagnostic platforms towards NA amplification at the POC (e.g., mobile laboratories, centrifugal, or pump-free platforms). Finally, the advantages, limitations, and outlook for POC-based diagnostics of zoonotic diseases with RPA(-CRISPR-Cas) are discussed, highlighting the need for innovative technologies to address global health challenges. While promising, many of these approaches still require further research to achieve streamlined, single-step reactions and seamless integration into diagnostic platforms. Moreover, despite two decades of RPA(-CRISPR-Cas) development, technology readiness is limited, still missing validated platforms, integrated sample preparation, and AI-powered results analysis enabling real time epidemiological monitoring.},
}
@article {pmid41145673,
year = {2026},
author = {Benz, T and Larghero, P and Meyer, C and Hanewald, T and Brüggmann, D and Hentrich, AE and Louwen, F and Marschalek, R},
title = {CRISPR/Cas9-mediated t(4;11) translocation in human hematopoietic stem/precursor cells demonstrates plasticity to differentiate into either the myeloid or lymphoid lineage.},
journal = {Leukemia},
volume = {40},
number = {1},
pages = {72-86},
pmid = {41145673},
issn = {1476-5551},
support = {2022.070.1//Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Translocation, Genetic ; *Hematopoietic Stem Cells/metabolism/cytology ; *Chromosomes, Human, Pair 11/genetics ; *Cell Differentiation/genetics ; *Chromosomes, Human, Pair 4/genetics ; Myeloid-Lymphoid Leukemia Protein/genetics ; Histone-Lysine N-Methyltransferase/genetics ; *Myeloid Cells/cytology/metabolism ; *Lymphocytes/cytology/metabolism ; Cell Lineage/genetics ; Oncogene Proteins, Fusion/genetics ; DNA-Binding Proteins/genetics ; Transcriptional Elongation Factors ; },
abstract = {The chromosomal translocation t(4;11)(q21;q23) is frequently diagnosed in KMT2A-r Acute Leukemia patients. Although we understand much about the function of both wildtype KMT2A and AFF1 multiprotein complexes, little is known about the molecular actions the two fusion proteins KMT2A::AFF1 and AFF1::KMT2A during the very early steps of disease onset and progression. Most published data have been generated in t(4;11) cell lines or transplanted mouse models, where exactly this process remains a black box. Here, we present the results of our efforts to establish a t(4;11) chromosomal translocation in human hematopoietic stem/precursor cells by CRISPR/Cas9. These genetically modified cells can be expanded over 5-6 months in vitro and their potential to differentiate was examined with IL-7 supplementation. The benefit of this model system is that (1) both reciprocal fusion proteins are concomitantly present, and (2) a molecular surveillance is possible at any timepoint through analysis of RNA, DNA or protein. Thus, the CRISPR/Cas9 technique allowed us to create a bona fide model system to study the very early steps of leukemia onset at the molecular level. In conclusion, this approach is the fastest way to investigate and characterize KMT2A-r fusions in primary human cells.},
}
@article {pmid41147512,
year = {2025},
author = {Mirzaei, F and Mosaffa Jahromi, A and Molavi, H and Kabelitz, D and Kalantar, K and Meri, S},
title = {Targeting RNA-Binding proteins Roquin-1 and Regnase-1 could enhance CAR-iPSC-derived macrophage immunotherapy for solid tumors: a perspective and challenges.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-7},
pmid = {41147512},
issn = {1555-8584},
mesh = {Animals ; Humans ; CRISPR-Cas Systems ; Gene Editing ; *Immunotherapy/methods ; *Immunotherapy, Adoptive/methods ; *Induced Pluripotent Stem Cells/cytology/metabolism/immunology ; *Macrophages/immunology/metabolism ; *Neoplasms/therapy/immunology/genetics ; Receptors, Chimeric Antigen/genetics/metabolism/immunology ; *Ribonucleases/genetics/metabolism ; *RNA-Binding Proteins/genetics ; Tumor Microenvironment/immunology ; *Ubiquitin-Protein Ligases/genetics ; Transcription Factors ; },
abstract = {Solid tumours present major treatment obstacles because of their immunosuppressive microenvironment and poor response to traditional chimeric antigen receptor (CAR)-based immunotherapies. Recent advances in cellular engineering have introduced CAR-macrophages derived from induced pluripotent stem cells (CAR-iMacs) as a promising approach to get around these obstacles. CAR-iMacs are designed to attack tumours, but their phenotypic plasticity can cause them to transform into M2-like macrophages in the tumour environment (TME), where they may instead suppress immune responses and promote tumour progression and metastasis. Roquin-1 and Regnase-1 are RNA-binding proteins that act as negative regulators of inflammatory genes that contribute to the phenotypic plasticity of macrophages. This perspective highlights a novel approach to augmenting anti-tumour responses of CAR-iMacs by simultaneously knocking out Roquin-1 and Regnase-1 via CRISPR-Cas9 gene editing. This approach drives a shift from an immunosuppressive M2-like state to an M1 state, promoting sustained pro-inflammatory signalling, boosting phagocytic and cytotoxic capabilities within the tumour microenvironment. Addressing a serious constraint in conventional adoptive cell therapies, this dual-targeting platform could provide a potent and scalable immunotherapeutic treatment for solid malignancies.},
}
@article {pmid41147788,
year = {2026},
author = {Saika, H and Hara, N and Yasumoto, S and Muranaka, T and Yoshimi, K and Mashimo, T and Toki, S},
title = {Versatile genome editing using Type I-E CRISPR-Cas3 in rice.},
journal = {Plant &amp; cell physiology},
volume = {67},
number = {1},
pages = {82-92},
pmid = {41147788},
issn = {1471-9053},
support = {JPJ008000//Ministry of Agriculture, Fisheries and Food/ ; //Cross-ministerial Strategic Innovation Promotion Program/ ; JPJ012287//Bio-oriented Technology Research Advancement Institution/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; Plants, Genetically Modified ; },
abstract = {The Type I-E CRISPR-Cas3 derived from Escherichia coli (Eco CRISPR-Cas3) can introduce large deletions in target sites and is available for mammalian genome editing. The use of Eco CRISPR-Cas3 in plants is challenging because seven CRISPR-Cas3 components (six Cas proteins and CRISPR RNA) must be expressed simultaneously in plant cells. To date, application has been limited to maize protoplasts, and no mutant plants have been produced. In this study, we developed a genome editing system in rice using Eco CRISPR-Cas3 via Agrobacterium-mediated transformation. Deletions in the target gene were detected in 39%-71% of transformed calli by polymerase chain reaction (PCR) analysis, and the frequency of alleles lacking a region 7.0 kb upstream of the protospacer adjacent motif sequence was estimated as 21%-61% by quantifying copy number by droplet digital PCR, suggesting that mutant plants could be obtained with reasonably high frequency. Deletions were determined in plants regenerated from transformed calli, and stably inherited to the progenies. Sequencing analysis showed that deletions of 0.1-7.2 kb were obtained, as reported previously in mammals. Interestingly, deletions separated by intervening fragments or with short insertion and inversion were also determined, suggesting the creation of novel alleles. Moreover, we demonstrated C to T base editing based on Type I-E CRISPR-Cas3 in rice, whereas base editing based on Type I-C and Type I-F2 CRISPR-Cas3 has been reported previously only in human cells. Overall, Eco CRISPR-Cas3 could be a promising genome editing tool for gene knockout, gene deletion, base editing, and genome rearrangement in plants.},
}
@article {pmid41148377,
year = {2025},
author = {Baars, J and Kurm, V and Scholten, B and Griekspoor, Y and Lavrijssen, B and Steens, JA and Smulders, MJM and van Peer, A},
title = {On site discrimination between two closely related commercial strains of oyster mushroom using a loop-mediated isothermal amplification (LAMP) test.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {17},
pmid = {41148377},
issn = {1573-4978},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *Pleurotus/genetics/classification ; DNA, Fungal/genetics ; Molecular Diagnostic Techniques ; },
abstract = {BACKGROUND: Protection of the intellectual property (IP) rights on new crop varieties is important as it allows the breeding company or entity that produced the variety to earn back (part of) the investment. Infringement on the IP rights of mushroom varieties is not uncommon. In order to combat infringement of the IP rights on two strains of Oyster mushroom (SPOPPO and ALLERPO) it is important to be able to readily recognize and discriminate the two strains in commercial practice. This article describes the development of tools for the on-site identification of two closely related sporeless strains of Oyster mushroom.<br><br>METHODS AND RESULTS: To develop a reliable method of discriminating between SPOPPO and ALLERPO, we used either the LAMP technique or a modification of that technique. It allows for fast (within 30&#xa0;min) identification of the commercially used strains SPOPPO and ALLERPO with high specificity. Fast on-site answers on strain identity can be important when experiencing unexpected strain behavior or when strains are of suspect origin. Both strains are discriminated from sporulating strains by a LAMP reaction on the intact version of the msh4 gene; sporeless strains contain a msh4 gene with a large insert that renders the associated protein inactive.<br><br>CONCLUSIONS: SPOPPO and ALLERPO are distinguished from each other and other commercially used Pleurotus ostreatus strains by LAMP reactions that target genomic regions with strain specific recombinations. To our knowledge, this is the first time LAMP reactions have been developed to discriminate between Pleurotus ostreatus strains.},
}
@article {pmid41148514,
year = {2025},
author = {Tripathi, A and Vishwakarma, K and Tripathi, S and Jadaun, JS and Nayak, AK},
title = {Utilization of MADS-Box genes for agricultural advancement: current insights and future prospects.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {20},
pmid = {41148514},
issn = {1573-4978},
mesh = {*MADS Domain Proteins/genetics/metabolism ; Gene Expression Regulation, Plant/genetics ; Plant Proteins/genetics/metabolism ; *Crops, Agricultural/genetics ; CRISPR-Cas Systems ; Oryza/genetics ; *Agriculture/methods ; Solanum lycopersicum/genetics ; Phylogeny ; Plants, Genetically Modified/genetics ; Gene Editing/methods ; Genes, Plant ; },
abstract = {MADS-box genes constitute a highly conserved family of transcription factors integral to the regulation of a diverse array of plant developmental processes, encompassing floral organ specification, fruit maturation, root architecture and adaptation to abiotic stresses. These transcription factors encode proteins containing the distinctive MADS (MCM1, AGAMOUS, DEFICIENS, and SRF) domain, which mediates DNA binding and orchestrates interaction with co-regulators, thereby enabling the precise transcriptional control of developmental gene networks. Functional characterization through transgenic approaches including overexpression, knockdown, and CRISPR/Cas9-based mutagenesis-has revealed the capacity of MADS-box gene manipulation to modulate key agronomic traits, such as yield potential, as well as resilience to salinity, drought, and temperature fluctuations. In rice, targeted editing of OsMADS18 using CRISPR/Cas9 generated a substantial quantitative variation in tiller and panicle number, demonstrating the direct contribution of MADS-box gene function to biomass and yield performance. Similarly, CRISPR/Cas9-mediated disruption of the RIPENING INHIBITOR (RIN) gene in tomato (Solanum lycopersicum) underscored its central role in regulating fruit ripening, linking MADS-box gene activity to postharvest quality and development. Phylogenomic studies reveal strong conservation of MADS-box gene lineages in monocot grasses, as evidenced by clustered short internal branches, whereas eudicots, particularly Solanaceae present well-differentiated subclades, reflecting lineage-specific diversification events. Notably, network analysis highlight the high connectivity and central regulatory position of many MADS-box proteins, underlining their roles as master integrators of developmental and environmental signalling involved in both floral and vegetative transitions. A mechanistic understanding of these regulatory circuits offers translational opportunities to engineer crops with improved performance and resilience, reinforcing the pivotal role of MADS-box genes in crop improvement.},
}
@article {pmid41148632,
year = {2026},
author = {Jin, Z and Yang, Y and Chen, C and Zhang, Z and Ren, Q and Cui, Z and Huang, CZ and Zuo, H},
title = {Gene silencing regulated by aggregates of Corn aptamer at 3' UTR of mRNA.},
journal = {Nanoscale horizons},
volume = {11},
number = {1},
pages = {232-242},
doi = {10.1039/d5nh00510h},
pmid = {41148632},
issn = {2055-6764},
mesh = {*RNA, Messenger/genetics/chemistry/metabolism ; *3' Untranslated Regions/genetics ; *Gene Silencing ; *Aptamers, Nucleotide/chemistry/genetics ; Humans ; CRISPR-Cas Systems ; *Zea mays/genetics/chemistry ; },
abstract = {Gene therapy, as a cutting-edge approach for disease intervention, relies heavily on advancements in gene silencing techniques. For instance, CRISPR-Cas9 has emerged as a leading gene-editing tool due to its ability to introduce precise cuts at specific genomic loci, enabling targeted gene insertion, deletion, or modification. In this study, we developed a simple and effective gene silencing strategy by introducing a nucleic acid self-assembly module into the 3' untranslated region (UTR) of mRNA. This module demonstrated significant gene silencing efficacy in eukaryotic cells through the formation of RNA aggregates. To systematically investigate its regulatory mechanism on translation efficiency through the formation of higher-order RNA structures, we quantitatively analyzed both mRNA and protein expression levels. Furthermore, our modular 3' UTR sequences can be integrated with classical 5' UTR elements (e.g., TOP sequences) to construct a multidimensional post-transcriptional regulatory network. This technology expands the diversity of existing UTR element libraries and offers a reservoir of programmable regulatory elements for applications in synthetic biology. It enables the construction of orthogonal combinations of multidimensional elements, tailored to specific gene expression regulation needs.},
}
@article {pmid41148687,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {Beyond the Resistome: Molecular Insights, Emerging Therapies, and Environmental Drivers of Antibiotic Resistance.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
pmid = {41148687},
issn = {2079-6382},
abstract = {Antibiotic resistance remains one of the most formidable challenges to modern medicine, threatening to outpace therapeutic innovation and undermine decades of clinical progress. While resistance was once viewed narrowly as a clinical phenomenon, it is now understood as the outcome of complex ecological and molecular interactions that span soil, water, agriculture, animals, and humans. Environmental reservoirs act as silent incubators of resistance genes, with horizontal gene transfer and stress-induced mutagenesis fueling their evolution and dissemination. At the molecular level, advances in genomics, structural biology, and systems microbiology have revealed intricate networks involving plasmid-mediated resistance, efflux pump regulation, integron dynamics, and CRISPR-Cas interactions, providing new insights into the adaptability of pathogens. Simultaneously, the environmental dimensions of resistance, from wastewater treatment plants and aquaculture to airborne dissemination, highlight the urgency of adopting a One Health framework. Yet, alongside this growing threat, novel therapeutic avenues are emerging. Innovative β-lactamase inhibitors, bacteriophage-based therapies, engineered lysins, antimicrobial peptides, and CRISPR-driven antimicrobials are redefining what constitutes an "antibiotic" in the twenty-first century. Furthermore, artificial intelligence and machine learning now accelerate drug discovery and resistance prediction, raising the possibility of precision-guided antimicrobial stewardship. This review synthesizes molecular insights, environmental drivers, and therapeutic innovations to present a comprehensive landscape of antibiotic resistance. By bridging ecological microbiology, molecular biology, and translational medicine, it outlines a roadmap for surveillance, prevention, and drug development while emphasizing the need for integrative policies to safeguard global health.},
}
@article {pmid41148810,
year = {2025},
author = {Liu, H and Zhang, P},
title = {Advances in β-Thalassemia Gene Therapy: CRISPR/Cas Systems and Delivery Innovations.},
journal = {Cells},
volume = {14},
number = {20},
pages = {},
pmid = {41148810},
issn = {2073-4409},
mesh = {*beta-Thalassemia/therapy/genetics ; Humans ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Animals ; *Gene Transfer Techniques ; beta-Globins/genetics ; },
abstract = {β-thalassemia is an inherited blood disorder caused by mutations in the β-globin (HBB) gene, leading to reduced or absent β-globin production, resulting in chronic anemia. While current therapies, including blood transfusions and hematopoietic stem cell transplantation, offer symptomatic relief, they are limited by complications and their limited accessibility. CRISPR-based gene editing technologies provide new therapeutic avenues by enabling the precise correction of HBB mutations or the reactivation of fetal hemoglobin (HbF) through the targeting of regulatory elements such as BCL11A. These approaches have shown promising preclinical and clinical outcomes. However, efficient and safe delivery remains a major challenge. Viral vectors offer high efficiency but raise concerns about immunogenicity and insertional mutagenesis, whereas non-viral systems such as lipid nanoparticles and engineered exosomes offer lower toxicity and modularity but face targeting limitations. This review highlights recent progress in CRISPR-based therapies for β-thalassemia and emerging delivery strategies to enhance clinical translation.},
}
@article {pmid41149303,
year = {2025},
author = {Zhu, Z and Ma, H and Yao, H and Yuan, Y and Miao, X and Su, S},
title = {CRISPR-Enhanced Colorimetric Aptasensor for Adenosine Triphosphate Detection Based on MoS2-Based Nanozymes.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149303},
issn = {2079-6374},
support = {SYW2025037//the Science and Technology Program of Suzhou/ ; 2024GRFX045//the Visiting Scholar Project for Higher Vocational Colleges in Jiangsu Province/ ; TC2024JCYL23//the Science and Technology Program of Taicang/ ; 2023JXKYTD01//the Innovation Team Funds of Suzhou Chien-shiung Institute of Technology/ ; },
mesh = {Colorimetry/methods ; *Adenosine Triphosphate/analysis ; *Biosensing Techniques/methods ; *Aptamers, Nucleotide ; Molybdenum/chemistry ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Disulfides/chemistry ; CRISPR-Cas Systems ; },
abstract = {As the direct energy source in organisms, accurate and simple detection of adenosine triphosphate (ATP) is of great significance. Herein, a colorimetric aptasensor for ATP determination was designed by integrating the CRISPR/Cas12a system with an aptamer, and with Prussian blue nanocube and gold nanoparticle co-functionalized MoS2 (MoS2-PBNCs-AuNPs) nanozymes. As expected, the introduced CRISPR/Cas12a system and aptamer could efficiently amplify the detection signal and improve the specific recognition ability, respectively. Meanwhile, the catalytic activity of the MoS2-PBNCs-AuNPs nanozymes can be regulated with the concentration of ATP. The high-affinity binding of ATP to the aptamer competitively inhibited aptamer-crRNA hybridization, causing fewer Cas12 proteins to be activated. As a result, the uncleaved single-stranded DNA (ssDNA) adsorbed onto the surface of nanozymes to effectively enhance their catalytic oxidation capability toward 3,3',5,5'-tetramethylbenzidine (TMB). According to this phenomenon, this CRISPR-enhanced colorimetric aptasensor can detect down to 0.14 μM ATP with high selectivity, reproducibility, and stability. In addition, acceptable recoveries and low relative standard deviations of the aptasensor for ATP determination suggest that it is promising for application in early detection of clinical-related diseases.},
}
@article {pmid41149312,
year = {2025},
author = {Zhou, Z and Cho, IH and Kadam, US},
title = {CRISPR-Cas-Based Diagnostics in Biomedicine: Principles, Applications, and Future Trajectories.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149312},
issn = {2079-6374},
mesh = {*CRISPR-Cas Systems ; Humans ; *Biosensing Techniques ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) systems, originally identified as prokaryotic adaptive immune mechanisms, have rapidly evolved into powerful tools for molecular diagnostics. Leveraging their precise nucleic acid targeting capabilities, CRISPR diagnostics offer rapid, sensitive, and specific detection solutions for a wide array of targets. This review delves into the fundamental principles of various Cas proteins (e.g., Cas9, Cas12a, Cas13a) and their distinct mechanisms of action (cis- and trans-cleavage). It highlights the diverse applications spanning infectious disease surveillance, cancer biomarker detection, and genetic disorder screening, emphasizing key advantages such as speed, high sensitivity, specificity, portability, and cost-effectiveness, particularly for point-of-care (POC) testing in resource-limited settings. The report also addresses current challenges, including sensitivity limitations without pre-amplification, specificity issues, and complex sample preparation, while exploring promising future trajectories like the integration of artificial intelligence (AI) and the development of universal diagnostic platforms to enhance clinical translation.},
}
@article {pmid41149352,
year = {2025},
author = {Qin, Y and Xie, J and Zhen, S},
title = {CRISPR-Cas12a and DNA Tetrahedron Assemblies Amplified Fluorescence Anisotropy for the Sensitive Detection of Hepatitis B Virus DNA.},
journal = {Biosensors},
volume = {15},
number = {10},
pages = {},
pmid = {41149352},
issn = {2079-6374},
support = {22322409//National Natural Science Foundation of China/ ; 21974109//National Natural Science Foundation of China/ ; SWU-KF25011//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Hepatitis B virus/genetics ; *DNA, Viral/analysis ; *Fluorescence Polarization/methods ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques ; Limit of Detection ; Hepatitis B ; },
abstract = {Fluorescence anisotropy (FA) has been widely used for analyzing biomolecules due to its high throughput, homogeneous detection, and strong resistance to photobleaching. However, the traditional FA method suffers from low sensitivity when the target molecules are small and rotate rapidly, often producing insignificant changes in the FA value. In this study, by combining double signal amplification through the trans-cleavage of CRISPR-Cas12a and DNA tetrahedron assemblies with a large molecular size, a new, fast, simple and highly sensitive FA method was constructed to achieve the quantitative detection of hepatitis B virus DNA (HBV-DNA). The experimental results showed that the linear range of this method was 0.5-9 nmol/L, and the detection limit (LOD = 3σ/k) was 48 pmol/L. In addition, the method demonstrated excellent selectivity and anti-interference, and it was successfully applied to detect HBV-DNA in human serum, indicating that this method has the potential for clinical diagnosis.},
}
@article {pmid41150843,
year = {2026},
author = {Butt, H and Sathish, S and London, E and Le, A and Li, Q and Gudmundsdottir, B and Lee, DY and Burke, EV and Yates, BP and Liu, DR and Hsieh, M and Leonard, A and Eaton, WA and Uchida, N and Pierciey, FJ and Newby, GA and Tisdale, JF and Demirci, S},
title = {Comparative analysis of CRISPR-Cas9, lentiviral transduction, and base editing for sickle cell disease in a murine model.},
journal = {Blood advances},
volume = {10},
number = {2},
pages = {289-300},
pmid = {41150843},
issn = {2473-9537},
mesh = {Animals ; *Anemia, Sickle Cell/therapy/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Mice ; *Lentivirus/genetics ; Disease Models, Animal ; Humans ; *Transduction, Genetic ; Hematopoietic Stem Cells/metabolism ; Hematopoietic Stem Cell Transplantation ; },
abstract = {Sickle cell disease (SCD) is a red blood cell disorder caused by a mutation in the β-globin gene, leading to sickle hemoglobin polymerization under low oxygen conditions. Both CRISPR-Cas9 editing and lentiviral transduction have shown promising clinical outcomes, but it remains unclear which approach is superior. Alternatively, new editing tools such as base editing may also be promising and reduce risks of genotoxicity. To compare these approaches, we studied them in an immunocompromised mouse model. We optimized ex vivo conditions in CD34+ hematopoietic stem and progenitor cells (HSPC) and infused edited SCD HSPC into busulfan-conditioned nonirradiated NOD,B6.SCID Il2rγ-/-KitW41/W41 (NBSGW) mice. Ex vivo analysis confirmed successful editing and transduction. At 16 weeks, bone marrow analysis showed similar human CD45+ cell engraftment across all groups (75%-90%). In the competitive transplantation group, there was a lower amount of B-cell lymphoma/leukemia 11A enhancer editing than base editing and lentiviral transduction. A secondary transplantation model yielded similar results. An antisickling assay showed significantly higher red blood cell sickling reduction in the base editing, transduction, and competitive transplantation groups compared to CRISPR-Cas9. In conclusion, although all methods showed therapeutic potential, base editing and lentiviral transduction provided superior outcomes over CRISPR-Cas9-mediated editing in a competitive murine transplantation model.},
}
@article {pmid41151122,
year = {2025},
author = {Cea Salazar, VI and Boender, AJ and Seelke, AMH and Gaard, L and Mederos, SL and Rogers, S and Gutierrez, XZ and Bales, KL and Young, LJ and Trainor, BC},
title = {CRISPR-mediated knockdown of oxytocin receptor in extended amygdala reduces stress-induced social avoidance in female California mice.},
journal = {Hormones and behavior},
volume = {176},
number = {},
pages = {105845},
pmid = {41151122},
issn = {1095-6867},
support = {R01 MH112788/MH/NIMH NIH HHS/United States ; R01 MH121829/MH/NIMH NIH HHS/United States ; },
mesh = {Animals ; *Receptors, Oxytocin/genetics/metabolism ; Female ; *Stress, Psychological/genetics/metabolism ; Peromyscus ; Mice ; *Amygdala/metabolism ; *Social Behavior ; Gene Knockdown Techniques ; Nucleus Accumbens/metabolism ; CRISPR-Cas Systems ; Septal Nuclei/metabolism ; *Avoidance Learning/physiology ; Behavior, Animal/physiology ; },
abstract = {Oxytocin receptors (OTRs) within the extended amygdala and nucleus accumbens (NAc) have been implicated in modulating social behaviors, particularly following stress. The effects of OTR could be mediated by modulating the activity of pre-synaptic axon terminals or via receptors in post-synaptic neurons or glia. Using a viral-mediated CRISPR/Cas9 gene editing system in female California mice (Peromyscus californicus), we selectively knocked down OTR in the anteromedial bed nucleus of the stria terminalis (BNST) or NAc to examine their roles modulating social approach and vigilance behaviors. Knockdown of OTR in the BNST attenuated stress-induced decreases of social approach and had less robust effects on vigilance when interacting with a target mouse behind a wire barrier. In this large arena, where mice could control their proximity to a target mouse, BNST OTR knockdown also increased investigation of a non-social stimulus (empty cage). Behavioral effects of BNST OTR knockdown were weaker in the small arena where focal mice physically interacted with target mice. Interestingly, OTR knockdown in the NAc, reduced stress-induced social vigilance without affecting social approach. These effects could mediate altered encoding of socially aversive experiences, as knockdown manipulations were performed before stress exposure. Together, these results highlight effects of local OTR on social behavior that are region-specific.},
}
@article {pmid41151616,
year = {2025},
author = {Terhalle, E and Rademacher, J},
title = {[Nontuberculous Mycobacteria: Diagnostic Challenges and Individualized Therapeutic Approaches].},
journal = {Deutsche medizinische Wochenschrift (1946)},
volume = {150},
number = {22},
pages = {1360-1366},
doi = {10.1055/a-2502-6525},
pmid = {41151616},
issn = {1439-4413},
mesh = {Humans ; *Mycobacterium Infections, Nontuberculous/diagnosis/drug therapy/epidemiology/therapy ; *Nontuberculous Mycobacteria/isolation &amp; purification ; Anti-Bacterial Agents/therapeutic use ; Precision Medicine ; },
abstract = {Non-tuberculous mycobacteria (NTM) are increasingly recognized as clinically relevant pathogens, particularly in countries with a low tuberculosis incidence. Recent data from Denmark demonstrate a continuous annual rise in NTM-related pulmonary disease (NTM-PD) of 4.6% over 3 decades, with more than half of the isolates associated with true disease. Structural lung diseases such as bronchiectasis, prior tuberculosis, and chronic pulmonary conditions are major risk factors, alongside immunodeficiencies and immunosuppressive therapies. The diagnosis of NTM-PD requires a combination of clinical symptoms, radiological findings, and the microbiological confirmation. Novel diagnostic tools, such as anti-GPL IgA serology and a CRISPR-Cas-based cfDNA assay, show promise for differentiating colonization from disease and monitoring treatment response, but the sputum culture remains essential for species identification and drug susceptibility testing. Treatment is complex and species-specific, with macrolides forming the backbone of most regimens. Refractory cases, particularly those involving Mycobacterium abscessus, pose therapeutic challenges and often require multidisciplinary management. Inhaled liposomal amikacin (ALIS) has shown benefit in refractory MAC disease. Clinical decision-making must balance efficacy, tolerability, and long-term adherence, highlighting the need for individualized treatment strategies and regular monitoring. This review outlines current evidence and practical recommendations for clinicians managing NTM-PD.},
}
@article {pmid41152217,
year = {2025},
author = {Song, Z and Guo, J and Fan, Z and Huang, S and Li, G and Zhao, Z and Chen, B and Huang, S and Zheng, W and Wei, Y and Chen, Y and Huang, X and Liu, J and Wu, L and Wang, X},
title = {Noncanonical target-strand cytosine base editing via engineered Un1Cas12f1 platform.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9499},
pmid = {41152217},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Animals ; *CRISPR-Cas Systems/genetics ; *Cytosine/metabolism ; Mice ; Male ; Humans ; *CRISPR-Associated Proteins/metabolism/genetics ; Protein Engineering ; },
abstract = {CRISPR/Cas-derived base editors harness various deaminase or glycosylase activities to target bases within non-target strand (NTS) of the R-loop, catalyzing base conversions independent of double-strand break formation. To develop miniature BEs compatible with therapeutic viral vectors, we explore the compact Cas12f system. Through computational modeling and mutagenesis, we establish a highly active enUn1Cas12f1 protein, and subsequently construct the derivative cytosine BE (CBE). Remarkably, the engineered CBE exhibits an unexpected activity to also edit the target strand (TS), indicating its substantially expanded editable space. We refine this activity via a focused alanine scan, establishing a nickase-CBE that preferentially install TS edits (TSminiCBE). Further engineering with a non-specific DNA binding domain yields an optimized TS-editing BE that enables in vivo base edits in mice (male). Overall, through extensive engineering of the Cas12f platform and repurposing its intrinsic dynamics, our work establishes a strand-selectable miniature CBE toolkit with strong potential for diverse applications.},
}
@article {pmid41152284,
year = {2025},
author = {Mishal, R and Meléndez-Zajgla, J and Rueda-Zarazúa, B and Labra-Barrios, ML and Castañón-Sánchez, CA and Uribe Carvajal, S and Padierna-Mota, L and Hernández-Hernández, JM and Leon-Avila, G and Pérez Rangel, A and Hernández-Martínez, E and Angeles-Morales, EB and Albalawi, IK and Luna-Arias, JP},
title = {RNA-seq analysis of wild-type and mutated TBPL1 gene in breast cancer cells lines through CRISPR/Cas9 approach reveals novel molecular signatures.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {37578},
pmid = {41152284},
issn = {2045-2322},
support = {Ph.D. fellowship 465228//Consejo Nacional de Humanidades, Ciencia y Tecnologia, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; PhD. fellowship 752614//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; Ciencia de Frontera 2019_Proyecto No. 116337//Consejo Nacional de Humanidades, Ciencia y Tecnolog&#xed;a, Mexico/ ; },
mesh = {Humans ; *Breast Neoplasms/genetics/pathology ; *CRISPR-Cas Systems ; Female ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; *TATA-Box Binding Protein/genetics/metabolism ; *Mutation ; RNA-Seq ; Transcriptome ; Cell Proliferation/genetics ; Gene Expression Profiling ; Cell Movement/genetics ; },
abstract = {Breast cancer is the leading cause of death among women globally. Several genes have been found to be transcriptionally dysregulated in cancer, according to recent studies. TATA-box binding protein (TBP) and its two paralogs, TBPL1 and TBPL2, play roles in human transcription. The TBPL1 gene is implicated in colorectal carcinomas by suppressing the expression of miR-18a. However, its function in breast cancer remains undisclosed. TBPL1 is distantly related to TBP and possesses a 40% similarity with TBP's core domain. In the present study, we explored the potential role of the TBPL1 gene in transcriptome regulation by knocking out the TBPL1 gene through the CRISPR/Cas9 method. Following the knockout of the TBPL1 gene, we examined the gene transcription patterns and compared them to wild-type cell lines. We observed disparate signatures of upregulated and downregulated genes in wild-type and mutated conditions. Healthy breast MCF-12F, and T47D, SKBR3, and MDA-MB-231 breast cancer cell lines were assessed, as these cancer cells exhibit overexpression of the TBPL1 gene. Next-generation sequencing data revealed distinct marker genes regulated by the TBPL1 gene and their potential involvement in cell migration, proliferation, anti-apoptosis, and metastasis. Additionally, we also discovered novel lncRNAs implicated in the transcriptome analysis of the TBPL1 knocked-out gene. Our investigation indicated that this gene might affect varied stages of breast cancer cell lines' cellular properties, such as cell duplication, morphology, and growth. It might also contribute to tumor formation in more aggressive cell lines like MDA-MB-231 in vivo.},
}
@article {pmid41152294,
year = {2025},
author = {Nemoto, A and Imaizumi, K and Miya, F and Hiroi, Y and Yamada, M and Ideno, H and Saitoh, S and Kosaki, K and Okuno, H and Okano, H},
title = {Rescue of imprinted genes by epigenome editing in human cellular models of Prader-Willi syndrome.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9442},
pmid = {41152294},
issn = {2041-1723},
mesh = {*Prader-Willi Syndrome/genetics/therapy ; Humans ; *Genomic Imprinting/genetics ; Induced Pluripotent Stem Cells/metabolism ; *Gene Editing/methods ; DNA Methylation/genetics ; CRISPR-Cas Systems ; *Epigenome/genetics ; Hypothalamus/metabolism ; Organoids/metabolism ; Chromosomes, Human, Pair 15/genetics ; Epigenesis, Genetic ; Epigenome Editing ; },
abstract = {Prader-Willi syndrome (PWS) is a genomic imprinting disorder caused by the loss of function of the paternal chromosome 15q11-13, resulting in a spectrum of symptoms associated with hypothalamic dysfunction. PWS patients lack the expression of paternally expressed genes (PEGs) in the 15q11-13 locus but possess an epigenetically silenced set of these genes in the maternal allele. Thus, activation of these silenced genes can serve as a therapeutic target for PWS. Here, we leverage CRISPR-based epigenome editing system to modulate the DNA methylation status of the PWS imprinting control region (PWS-ICR) in induced pluripotent stem cells (iPSCs) derived from PWS patients. Successful demethylation in the PWS-ICR restores the PEG expression from the maternal allele and reorganizes the methylation patterns in other PWS-associated imprinted regions beyond the PWS-ICR. Remarkably, these corrected epigenomic patterns and PEG expression are maintained following the differentiation of these cells into hypothalamic organoids. Finally, the single-cell transcriptomic analysis of epigenome-edited organoids demonstrates a partial restoration of the transcriptomic dysregulation observed in PWS. This study highlights the utility of epigenome editing technology as a therapeutic approach in addressing PWS and potentially other imprinting disorders.},
}
@article {pmid41152563,
year = {2026},
author = {Erbasan, E and Aliciaslan, M and Erendor, F and Sanlioglu, AD and Sanlioglu, S},
title = {Therapeutic precision gene editing of cholesterol pathways as a gene therapy strategy for cardiovascular disease.},
journal = {Gene therapy},
volume = {33},
number = {2},
pages = {218-237},
pmid = {41152563},
issn = {1476-5462},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *Cardiovascular Diseases/therapy/genetics ; CRISPR-Cas Systems ; Animals ; Proprotein Convertase 9/genetics ; *Hypercholesterolemia/therapy/genetics ; *Cholesterol/metabolism/genetics ; Precision Medicine ; Cholesterol, LDL ; },
abstract = {Hypercholesterolemia, defined by high low-density lipoprotein cholesterol levels, critically increases the risk of atherosclerotic cardiovascular disease, which represents the foremost cause of worldwide morbidity and mortality. While established lipid-lowering therapies, primarily statins, are effective for many patients, a significant proportion either fail to achieve optimal LDL-C targets, experience dose-limiting side effects, or face challenges with the long-term adherence required for sustained cardiovascular benefit. The recent emergence and rapid advancement of precise gene editing technologies most notably CRISPR-Cas9 and its advanced variants like base editing and prime editing offer a revolutionary therapeutic paradigm. These tools have the potential to achieve durable modification of the expression or function of genes fundamentally involved in cholesterol metabolism. This comprehensive overview integrates the current knowledge of critical cholesterol regulatory pathways and the main protein targets that are suitable for gene editing. The fundamental mechanisms, relative advantages, and inherent limitations of gene editing platforms and delivery systems for clinical translation are examined. The expanding preclinical data and groundbreaking clinical evidence highlighting the transformative potential of gene editing to achieve significant and lasting reductions in LDL-C, especially through promising therapies like VERVE base editors targeting PCSK9 and ANGPTL3 are critically evaluated. The challenges including off-target effects, delivery efficiency and specificity, long-term safety and durability, complex ethical considerations, and evolving regulatory landscapes that must be rigorously navigated for these therapies to become mainstream clinical practice are thoroughly addressed. Successfully overcoming these challenges could mark the beginning of a new era of personalized, one-time treatments for hypercholesterolemia.},
}
@article {pmid41153385,
year = {2025},
author = {Boti, MA and Diamantopoulos, MA and Scorilas, A},
title = {RNA-Targeting Techniques: A Comparative Analysis of Modern Approaches for RNA Manipulation in Cancer Research and Therapeutics.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
pmid = {41153385},
issn = {2073-4425},
mesh = {Humans ; *Neoplasms/genetics/therapy ; RNA Interference ; CRISPR-Cas Systems ; Oligonucleotides, Antisense/therapeutic use/genetics ; Animals ; *RNA/genetics ; },
abstract = {RNA-targeting techniques have emerged as powerful tools in cancer research and therapeutics, offering precise and programmable control over gene expression at the post-transcriptional level. Once viewed as passive intermediates in the central dogma, RNA molecules are now recognized as dynamic regulators of cellular function, capable of influencing transcription, translation, and epigenetic regulation. Advances in high-throughput sequencing technologies, transcriptomics, and structural RNA biology have uncovered a diverse landscape of coding and non-coding RNAs involved in oncogenesis, drug resistance, and tumor progression. In response, several RNA-targeting strategies have been developed to modulate these transcripts, including antisense oligonucleotides (ASOs), RNA interference (RNAi), CRISPR-Cas13 systems, small molecules, and aptamers. This review provides a comparative analysis of these technologies, highlighting their molecular mechanisms, therapeutic potential, and current limitations. Emphasis is placed on the translational progress of RNA-targeting agents, including recent FDA approvals and ongoing clinical trials for cancer indications. Through a critical comparison of these strategies, this review underscores the growing significance of RNA-targeting technologies as a foundation for next-generation cancer therapeutics and precision oncology.},
}
@article {pmid41153439,
year = {2025},
author = {Stunf Pukl, S},
title = {Genetic Therapy of Fuchs Endothelial Corneal Dystrophy: Where Are We? A Review.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
pmid = {41153439},
issn = {2073-4425},
mesh = {*Fuchs' Endothelial Dystrophy/therapy/genetics ; Humans ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Collagen Type VIII/genetics ; Mutation ; Transcription Factor 4/genetics ; Animals ; Gene Editing ; },
abstract = {OBJECTIVES: The incidence of Fuchs endothelial corneal dystrophy (FECD) is growing, and with it, the unmet need for a corneal transplant. Among alternative treatment modalities, only genetic therapy represents a causal therapy.<br><br>METHODS: Following the SNARA protocol, the PubMed and ClinicalTrials databases were searched using the keywords Fuchs endothelial corneal dystrophy, FECD, genetic therapy, and CRISPR-Cas9.<br><br>RESULTS: FECD is polyfactorial disease and mutations or polymorphisms in at least 15 different genes were connected to the disease. For the early-onset form of the disease, exclusive connection to mutations in COL8A2 was confirmed, while for the late-onset form, the most characteristic mutation is the expansion of the CTG18.1 triplet in the TCF4 gene, making these two possible targets. While the CRISPR-Cas9 approach represents the mainstay of genetic therapy development recently, the application of this method to FECD contains several obstacles, studied in preclinical settings. DT-168 and the Ad-Cas9-Col8a2gRNA molecules were developed for FECD treatment and preclinically tested, and phase I and II clinical studies for DT-168 are also already being performed.<br><br>CONCLUSIONS: The review of the literature proved that genetic therapy for FECD is at the level of preclinical research and that there are several specific challenges connected to the target genetic mutation as well as the delivery of possible treatment and duration of the effect. Further studies in the field might bring solutions in the future for alternative treatments for this common corneal disease.},
}
@article {pmid41153450,
year = {2025},
author = {Nardon, E and Azzalini, E and Paladin, D and Boscarino, D and Bonin, S},
title = {CRISPR/Cas Tools for the Detection of Borrelia sensu lato in Human Samples.},
journal = {Genes},
volume = {16},
number = {10},
pages = {},
pmid = {41153450},
issn = {2073-4425},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Lyme Disease/diagnosis/microbiology/genetics ; *Borrelia burgdorferi Group/genetics/isolation &amp; purification ; DNA, Bacterial/genetics ; Real-Time Polymerase Chain Reaction/methods ; Sensitivity and Specificity ; Bacterial Outer Membrane Proteins/genetics ; Antigens, Surface ; Bacterial Vaccines ; Lipoproteins ; },
abstract = {BACKGROUND/OBJECTIVES: Lyme disease diagnosis remains challenging due to the limitations of current methods. While PCR-based assays are widely used, their sensitivity can be affected by sample type and the inhibition of host DNA. This study aimed to evaluate the feasibility and sensitivity of a CRISPR/Cas12-based detection system for Borrelia burgdorferi sensu lato, comparing its performance with real-time PCR.<br><br>METHODS: DNA from three Borrelia genospecies (B. burgdorferi, B. garinii, and B. afzelii) was amplified targeting the OspA gene. Detection was performed using a Cas12/crRNA system with a fluorescent ssDNA reporter. Sensitivity assays were conducted on serial dilutions of Borrelia DNA, with and without human genomic DNA, and results were compared with qPCR.<br><br>RESULTS: Direct detection of Borrelia DNA without amplification was not feasible. However, when combined with PCR, the Cas12/crRNA system reliably detected as few as 5 genome copies per reaction. End-point PCR extended to 60 cycles improved detection robustness for B. garinii and B. afzelii, although sensitivity decreased in the presence of human genomic DNA.<br><br>CONCLUSIONS: The Cas12/crRNA-based system offers a sensitive and accessible alternative to qPCR, especially in settings lacking real-time PCR instrumentation. Future developments may include integration with isothermal amplification and microfluidic platforms to enhance direct detection capabilities.},
}
@article {pmid41154642,
year = {2025},
author = {Zhang, H and Yang, Y and Yang, T and Cao, P and Yu, C and Liang, L and Liu, R and Chen, Z},
title = {Engineering a High-Fidelity MAD7 Variant with Enhanced Specificity for Precision Genome Editing via CcdB-Based Bacterial Screening.},
journal = {Biomolecules},
volume = {15},
number = {10},
pages = {},
pmid = {41154642},
issn = {2218-273X},
support = {2023YFC3402300//National Key R&amp;D Program of China/ ; 22208044//National Natural Science Foundation of China/ ; 22278058//National Natural Science Foundation of China/ ; XLYC2203075//"Xingliao Talent Plan"project/ ; 2024-MSBA-09//Natural Science Foundation of Liaoning Province/ ; 2025JH2/101330156//Natural Science Foundation of Liaoning Province/ ; 2023JJ12SN030//Science and Technology Innovation Foundation of Dalian/ ; DUT24YG131//Fundamental Research Funds for the Central Universities/ ; DUT25LAB105//Fundamental Research Funds for the Central Universities/ ; DUT25RC(3)019//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Bacterial Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/genetics/metabolism ; Mutation ; },
abstract = {CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) nucleases enable precise genome editing, but off-target cleavage remains a critical challenge. Here, we report the development of MAD7_HF, a high-fidelity variant of the MAD7 nuclease engineered through a bacterial screening system leveraging the DNA gyrase-targeting toxic gene ccdB. This system couples survival to efficient on-target cleavage and minimal off-target activity, mimicking the transient action required for high-precision editing. Through iterative selection and sequencing validation, we identified MAD7_HF, harboring three substitutions (R187C, S350T, K1019N) that enhanced discrimination between on- and off-target sites. In Escherichia coli assays, MAD7_HF exhibited a >20-fold reduction in off-target cleavage across multiple mismatch contexts while maintaining on-target efficiency comparable to wild-type MAD7. Structural modeling revealed that these mutations stabilize the guide RNA-DNA hybrid at on-target sites and weaken interactions with mismatched sequences. This work establishes a high-throughput bacterial screening strategy that allows the identification of Cas12a variants with improved specificity at a given target site, providing a useful framework for future efforts to develop precision genome-editing tools.},
}
@article {pmid41155240,
year = {2025},
author = {Carbone, F},
title = {Special Issue: Latest Research on Plant Genomics and Genome Editing.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
pmid = {41155240},
issn = {1422-0067},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *Genomics/methods ; *Plants/genetics ; CRISPR-Cas Systems ; High-Throughput Nucleotide Sequencing ; },
abstract = {Over the past ten years, plant science has undergone a remarkable transformation driven by the convergence of next-generation sequencing, increasingly sophisticated bioinformatics tools, and the rise of targeted genome editing platforms [...].},
}
@article {pmid41155368,
year = {2025},
author = {Hossain, MM and Sultana, F and Mostafa, M and Khan, I and Tran, LP and Mostofa, MG},
title = {Reinforced Defenses: R-Genes, PTI, and ETI in Modern Wheat Breeding for Blast Resistance.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
pmid = {41155368},
issn = {1422-0067},
mesh = {*Triticum/genetics/microbiology/immunology ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics/immunology ; *Plant Breeding ; *Plant Immunity/genetics ; *Genes, Plant ; Magnaporthe/pathogenicity ; Host-Pathogen Interactions/genetics/immunology ; },
abstract = {Wheat blast, caused by Magnaporthe oryzae pathotype Triticum (MoT), poses a major threat to wheat (Triticum aestivum) cultivation, particularly in South America and Bangladesh. The rapid evolution and spread of the pathogen necessitate the development of durable and broad-spectrum resistance in wheat cultivars. This review summarizes current insights into the multi-layered defense mechanisms of wheat, encompassing resistance (R) genes, pattern-triggered immunity (PTI), and effector-triggered immunity (ETI) against MoT. The R-genes provide race-specific resistance through ETI, while both ETI and PTI are required to form integral layers of the plant immune system that synergistically reinforce host defense network. Recent advances in genomics, transcriptomics, and molecular breeding have facilitated the discovery and deployment of key R-genes and signaling components involved in PTI and ETI pathways. Integrating these immune strategies through gene pyramiding, marker-assisted selection (MAS), and genome editing offers a promising route towards enhanced and durable resistance in hosts. Harnessing and optimizing these multilayered immune systems will be pivotal to securing wheat productivity amid the growing threat of wheat blast.},
}
@article {pmid41155374,
year = {2025},
author = {Honjo, A and Yako, H and Miyamoto, Y and Yagi, M and Yamamoto, M and Nishi, A and Sakagami, H and Yamauchi, J},
title = {Knocking Down FRMD4A, a Factor Associated with the Brain Development Disorder and a Risk Factor for Alzheimer's Disease, Using RNA-Targeting CRISPR/Cas13 Reveals Its Role in Cell Morphogenesis.},
journal = {International journal of molecular sciences},
volume = {26},
number = {20},
pages = {},
pmid = {41155374},
issn = {1422-0067},
mesh = {*Alzheimer Disease/genetics/metabolism/pathology ; Animals ; *CRISPR-Cas Systems ; Neurons/metabolism/cytology ; Mice ; *Morphogenesis/genetics ; *Cytoskeletal Proteins/genetics/metabolism ; *Membrane Proteins/genetics/metabolism ; Gene Knockdown Techniques ; Humans ; *Brain/metabolism ; Cell Line ; },
abstract = {Genetic truncation or mutation of the gene encoding band 4.1, ezrin, radixin, and moesin (FERM) domain protein containing 4A (FRMD4A) is associated with brain developmental diseases, including microcephaly with global developmental delay. It has also been identified as a risk factor for Alzheimer's disease. By analogy with other FERM domain-containing proteins, FRMD4A is believed to regulate cell morphogenesis and/or cell polarization in central nervous system (CNS) cells; however, it remains unclear whether and how dysfunction of FRMD4A and/or its closely homologous protein FRMD4B causes abnormal morphogenesis in neuronal cells. Here, we describe for the first time the roles of FRMD4A and FRMD4B in process elongation in neuronal cells. Knockdown of Frmd4a or Frmd4b using specific RNA-targeting clustered regularly interspaced short palindromic repeat (CRISPR) and Cas13-fitted gRNAs led to decreased process elongation in primary cortical neurons. Similar decreases in neuronal marker expression were observed in the N1E-115 cell line, a model of neuronal differentiation. Furthermore, hesperetin, an aglycone of the citrus flavonoid hesperidin known to promote neuroprotective signaling, recovered the decreased process elongation induced by the knockdown of Frmd4a or Frm4b. Hesperetin also stimulated phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPKs/ERKs), which could help promote neuronal processes. These results suggest that FRMD4A and FRMD4B regulate process elongation through a possible signaling pathway linked to the sustained phosphorylation of MAPKs/ERKs. Crucially, this study reveals that, at the molecular and cellular levels, hesperetin can restore normal phenotypes when FRMD4A protein or FRMD4B protein is impaired.},
}
@article {pmid41155971,
year = {2025},
author = {Serrano, DR and Juste, F and Anaya, BJ and Ramirez, BI and Sánchez-Guirales, SA and Quispillo, JM and Hernandez, EM and Simon, JA and Trallero, JM and Serrano, C and Rawat, S and Lalatsa, A},
title = {Exosome-Based Drug Delivery: A Next-Generation Platform for Cancer, Infection, Neurological and Immunological Diseases, Gene Therapy and Regenerative Medicine.},
journal = {Pharmaceutics},
volume = {17},
number = {10},
pages = {},
pmid = {41155971},
issn = {1999-4923},
support = {PID2024-156769OB-I00//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; Innovation in Pharmacology, Nanotechnology, and personalized medicine by 3D printing//Universidad Complutense de Madrid/ ; },
abstract = {Exosomes, naturally derived extracellular vesicles, have emerged as powerful bio-nanocarriers in precision medicine. Their endogenous origin, biocompatibility, and ability to encapsulate and deliver diverse therapeutic payloads position them as transformative tools in drug delivery, gene therapy, and regenerative medicine. This review presents a comprehensive analysis of exosome-based therapeutics across multiple biomedical domains, including cancer, neurological and infectious diseases, immune modulation, and tissue repair. Exosomes derived from stem cells, immune cells, or engineered lines can be loaded with small molecules, RNA, or CRISPR-Cas systems, offering highly specific and low-immunogenic alternatives to viral vectors or synthetic nanoparticles. We explore endogenous and exogenous loading strategies, surface functionalization techniques for targeted delivery, and innovations that allow exosomes to traverse physiological barriers such as the blood-brain barrier. Furthermore, exosomes demonstrate immunomodulatory and regenerative properties in autoimmune and degenerative conditions, with promising roles in skin rejuvenation and cosmeceuticals. Despite their potential, challenges remain in large-scale production, cargo loading efficiency, and regulatory translation. Recent clinical trials and industry efforts underscore the accelerating momentum in this field. Exosomes represent a promising platform in precision medicine, though further standardization and validation are required before widespread clinical use. This review offers critical insights into current technologies, therapeutic mechanisms, and future directions to unlock the full translational potential of exosomes in clinical practice.},
}
@article {pmid41156655,
year = {2025},
author = {Peláez Sánchez, RG and González Restrepo, J and Pineda, S and Cuartas-López, AM and Martínez Garro, JM and Torres-Castro, M and Urrego, R and López-Rojas, LE and Salazar Florez, JE and Monroy, FP},
title = {Bioinformatic Identification of CRISPR-Cas Systems in Leptospira Genus: An Update on Their Distribution Across 77 Species.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {10},
pages = {},
pmid = {41156655},
issn = {2076-0817},
mesh = {*CRISPR-Cas Systems/genetics ; *Leptospira/genetics/classification ; *Computational Biology/methods ; Genome, Bacterial ; Bacteriophages/genetics ; Leptospirosis/microbiology ; },
abstract = {UNLABELLED: Leptospirosis is a globally distributed zoonotic disease caused by pathogenic bacteria of the Leptospira genus. Genome editing in Leptospira has been difficult to perform. Currently, the functionality of the CRISPR-Cas system has been demonstrated in species such as Leptospira interrogans. However, the different CRISPR-Cas systems present in most of the 77 species are unknown. Therefore, the objective of this study was to identify these arrays across the genomes of all described Leptospira species using bioinformatics tools.<br><br>METHODS: a bioinformatics workflow was followed: genomes were downloaded from the NCBI database; Cas protein detection was carried out using the CRISPR-CasFinder and RAST web servers; functional analyses of Cas proteins were performed with InterProScan, ProtParam, Swiss Model, Alphafold3, Swiss PDB Viewer, and Pymol; conservation pattern detection was conducted using MEGA12, and Seqlogos; spacer identification was carried out with the Actinobacteriophages database and BLAST version 1.4.0; and bacteriophage detection was performed using PHASTER, and PHASTEST.<br><br>RESULTS: Cas proteins were detected in 36 out of the 77 species of the Leptospira species, including Cas1 to Cas9 and Cas12. These proteins were classified into Class 1 and Class 2 systems, corresponding to types I, II, and V. Direct repeats and spacers were detected in 19 species, with the direct repeats exhibiting two conserved nucleotide motifs. Analysis of spacer sequences revealed 323 distinct bacteriophages. Additionally, three intact bacteriophages were detected in the genomes of four Leptospira species. Notably, two saprophytic species have complete CRISPR-Cas systems.<br><br>CONCLUSIONS: The presence of Cas proteins, direct repeats, and spacer sequences with homology to bacteriophage genomes provides evidence for a functional CRISPR-Cas system in at least 19 species.},
}
@article {pmid41157107,
year = {2025},
author = {Shpiliukova, K and Kachanov, A and Brezgin, S and Chulanov, V and Ivanov, A and Kostyushev, D and Kostyusheva, A},
title = {m[6]A RNA Modification: Technologies Behind Future Anti-Cancer Therapy.},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {20},
pages = {},
pmid = {41157107},
issn = {1420-3049},
support = {075-15-2025-519//Ministry of Science and Higher Education of the Russian Federation (Federal scientific and technical program for the development of genetic technologies for 2019-2030/ ; },
mesh = {Humans ; *Adenosine/analogs &amp; derivatives/metabolism/genetics ; *Neoplasms/genetics/drug therapy/therapy/metabolism ; Methylation ; Animals ; Epigenesis, Genetic ; *Antineoplastic Agents/pharmacology/therapeutic use ; *RNA/genetics/metabolism ; *RNA Processing, Post-Transcriptional ; RNA Methylation ; },
abstract = {N6-methyladenosine (m[6]A) modifications are among the most prevalent epigenetic marks in eukaryotic RNAs, regulating both coding and non-coding RNAs and playing a pivotal role in RNA metabolism. Given their widespread influence, m[6]A modifications are deeply implicated in the pathogenesis of various cancers, including highly aggressive malignancies such as lung cancer, melanoma, and liver cancer. Dysregulation of m[6]A dynamics-marked by an imbalance in methylation and demethylation-can drive tumor progression, enhance metastatic potential, increase aggressiveness, and promote drug resistance, while also exerting context-dependent tumor-suppressive effects. Given this dual role, precise modulation of m[6]A levels and the activity of its regulatory enzymes (writers, erasers, and readers) represent a promising therapeutic avenue. In this review, we highlight recent advances in targeting m[6]A machinery, including small-molecule inhibitors, antisense oligonucleotides, and CRISPR/Cas-based editing tools, capable of both writing and erasing m[6]A marks and altering m[6]A methylation sites per se. By evaluating these strategies, we aim to identify the most effective approaches for restoring physiological m[6]A homeostasis or for strategically manipulating the m[6]A machinery for therapeutic benefit.},
}
@article {pmid41159426,
year = {2026},
author = {Kato, M and Kleidon, J and Phillips, A and Tabrett, A and Harding, R and Dale, J and Paul, JY},
title = {Transgene-Free, Gene-Edited Cavendish Bananas (Musa acuminata, AAA).},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1620-1634},
pmid = {41159426},
issn = {1467-7652},
support = {//Cooperative Research Centre Projects/ ; //Australian Banana Research Pty Ltd/ ; },
mesh = {*Gene Editing/methods ; *Musa/genetics ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Transgenes/genetics ; DNA, Bacterial/genetics ; Oxidoreductases/genetics ; Intramolecular Lyases/genetics ; Carotenoids/metabolism ; },
abstract = {Global consumer acceptance of gene-edited food crops is increasing with new breeding technologies that can modify the genome without foreign DNA integration. Here, we report an Agrobacterium-based system for transgene-free, gene editing of the banana cultivar, Cavendish. The protocol uses a three-tiered approach whereby cells containing T-DNA are enriched by positive antibiotic selection over a 48-h period, CRISPR/Cas9-mediated gene editing occurs over a short transient window and negative selection of cells containing T-DNA is achieved by the addition of 5-FC, which is converted to cytotoxic 5-FU by the co-expressed CODA enzyme. Two key enzymes in the carotenoid biosynthesis pathway were targeted as visual markers of editing: phytoene desaturase (pds) and lycopene β-cyclase (LCYb). Disruptive edits of the LCYb gene were easily identifiable at the calli stage of the regeneration process with cells appearing pink due to lycopene accumulation. Eight of 32 plants for pds and 34 of 125 plants for LCYb contained edits and were likely free of integrated vector DNA as determined by targeted genome sequencing and T-DNA-specific PCR, respectively. Plants containing disruptive tri-allelic edits in either gene displayed an albino phenotype. A subset of potentially transgene-free, edited plants was verified by whole genome sequencing. The transient editing protocol has an estimated 17.6% to 21.9% efficiency in generating plants containing disruptive tri-allelic modifications that are free of novel DNA. The protocol overcomes the regulatory limitations associated with the release of gene-edited, vegetatively propagated crops and provides an effective means of creating new disease-resistant and agronomically superior Cavendish cultivars.},
}
@article {pmid41159723,
year = {2025},
author = {Duan, Z and Yang, R and Lai, T and Jiang, W and Zhang, J and Chen, B and Liao, L},
title = {Development of a CRISPR/Cas9-induced gene editing system for Pseudoalteromonas fuliginea and its applications in functional genomics.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {11},
pages = {e0177125},
pmid = {41159723},
issn = {1098-5336},
support = {2022YFC2807501//National Key Research and Development Program of China/ ; 41976224//National Natural Science Foundation of China/ ; 42476264//National Natural Science Foundation of China/ ; },
mesh = {*Pseudoalteromonas/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Genomics/methods ; Genome, Bacterial ; },
abstract = {Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is laborious and time-consuming in this bacterial system. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we established a CRISPR/Cas9 system in Pseudoalteromonas and carried out an extensive investigation of the Type II CRISPR/Cas9 platform for gene editing in Pseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea, multiple genes were selected as targets, and the gene editing effects were confirmed through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea, encompassing the deletion of genes such as fliJ, indA, and genes encoding Pf sRNAs, as well as the in vivo insertion of 3×FLAG and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70%. In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea, which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications.IMPORTANCEPseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in P. fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.},
}
@article {pmid41159851,
year = {2026},
author = {Coppola, MR and Bellitto, D and Asgari, E and Bazzurro, V and Casucci, G and Piacente, F and Bozzo, M and Ceresa, D and Parisi, C and Winata, C and Candiani, S and Tonetti, M},
title = {Zebrafish as a model for Catel-Manzke syndrome-identification and characterization of the zebrafish TGDS ortholog.},
journal = {The FEBS journal},
volume = {293},
number = {8},
pages = {2248-2269},
pmid = {41159851},
issn = {1742-4658},
support = {GMR22T1065//Fondazione Telethon/ ; PRIN 2022 - grant 22ETJJES//Ministero dell'Universit&#xe0; e della Ricerca/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; Disease Models, Animal ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Gene Expression Regulation, Developmental ; Humans ; },
abstract = {Catel-Manzke syndrome (CMS) is a rare genetic disorder associated with mutations in the TDP-glucose 4,6-dehydratase (TGDS) gene, the function of which in vertebrates remains unclear. This study investigated the zebrafish ortholog tgds to assess its suitability for modeling the disease. During development, the tgds transcript exhibits a conserved biphasic expression pattern with an initial maternal contribution followed by a second wave of expression after gastrulation. Recombinant zebrafish Tgds expressed in Escherichia coli demonstrated UDP-D-glucose 4,6-dehydratase (EC4.2.1.76) activity, similar to TGDS orthologs in lower eukaryotes, where it acts as the first step in the L-rhamnose biosynthetic pathway. This finding suggests the presence of a yet unidentified pathway in vertebrates. Furthermore, CMS-associated mutations in conserved residues significantly impair enzyme activity and stability. CRISPR/Cas9-mediated F0 knockout of tgds resulted in a range of developmental defects in zebrafish. In particular, craniofacial cartilage alterations, associated with a decrease in sulfate glycosaminoglycan content, mirrored some skeletal features observed in humans with CMS. These findings establish the zebrafish as a relevant model to further explore CMS pathogenesis and the in vivo function of tgds.},
}
@article {pmid41160062,
year = {2025},
author = {Yilmaz Çolak, &#xc7;},
title = {Harnessing CRISPR technology for the diagnosis of Bordetella pertussis: advances and implications.},
journal = {Future microbiology},
volume = {20},
number = {16},
pages = {1145-1152},
pmid = {41160062},
issn = {1746-0921},
mesh = {*Bordetella pertussis/genetics/isolation &amp; purification ; *Whooping Cough/diagnosis/microbiology ; Humans ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Molecular Diagnostic Techniques/methods ; Gene Editing/methods ; },
abstract = {Following the discovery of the prokaryotic adaptive immune system known as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) proteins, this technology has revolutionized biotechnology as a multifaceted genome-editing tool with a wide range of applications. CRISPR technology has not only provided novel treatment options, especially for genetic diseases, but also transformed molecular diagnostic platforms. The specific, sensitive, and adaptable nature of the CRISPR-Cas systems has led to the development of innovative solutions for the detection of diseases, including viral and bacterial infections. This review provides an overview of the CRISPR-Cas systems and mainly focuses on the application of CRISPR-based assays for the detection of Bordetella pertussis, which is the main causative agent of a highly infectious disease, whooping cough. The review emphasizes the need for novel diagnostic tools for B. pertussis, along with highlighting some future perspectives, since its diagnosis can be challenging due to nonspecific early symptoms and interference from closely related Bordetella species. In this regard, CRISPR-based diagnostic platforms can offer a promising avenue for rapid and accurate detection of B. pertussis, helping the management of whooping cough.},
}
@article {pmid41160201,
year = {2026},
author = {Wang, YY and Lin, YH and Ke, CC and Lai, TH and Yu, IS and Au, CF and Singh, R and Lin, YH},
title = {Human teratozoospermia-related AGTPBP1 R791H mutation is associated with sperm head and tail defects in a CRISPR-engineered murine model.},
journal = {Journal of assisted reproduction and genetics},
volume = {43},
number = {1},
pages = {133-142},
pmid = {41160201},
issn = {1573-7330},
support = {NSTC 111-2320-B-030 -007-MY3//National Science and Technology Council/ ; CTH-109A-2215//Cardinal Tien Hospital/ ; },
mesh = {Animals ; Male ; *Teratozoospermia/genetics/pathology ; Mice ; *Sperm Head/pathology ; Humans ; *Sperm Tail/pathology ; Disease Models, Animal ; Spermatozoa/pathology ; CRISPR-Cas Systems/genetics ; Mutation ; *Infertility, Male/genetics/pathology ; *Poly-ADP-Ribose Binding Proteins/genetics ; },
abstract = {BACKGROUND: Infertility is a pervasive global health concern affecting millions of couples worldwide. Approximately 7% of the male population is infertile. Teratozoospermia, defined by > 96% abnormal sperm morphology, is a major cause of infertility often linked to genetic defects. In our previous study, we identified three AGTPBP1 mutations (p.Glu423Asp, p.Pro631Leu, and p.Arg811His) in teratozoospermia cases. AGTPBP1 is a key enzyme involved in regulating tubulin polyglutamylation and generating Δ-2 tubulin, a major structural component of the sperm tail and an essential structure for sperm head differentiation. However, functional proof of the impact of AGTPBP1 Arg811His on sperm head and tail impairment remained unestablished.<br><br>METHODS: Knock-in mice carrying the equivalent mutation, Arg791His (R791H) corresponding to the human mutation (R811H), in the Agtpbp1 gene were generated and analyzed for sperm morphological abnormalities.<br><br>RESULTS: Sperm morphological evaluation revealed a significant increase in the proportion of morphologically abnormal sperm in the Agtpbp1[R791H/R791H] mice. Detailed morphological analysis revealed a significantly higher incidence of sperm head abnormalities and abnormal attachment of the head to the midpiece in the Agtpbp1[R791H/R791H] mice relative to wild-type controls. Further, sperm with head defects from Agtpbp1[R791H/R791H] mice exhibited abnormal accumulation of polyglutamylated tubulin within the sperm head. The mutant mice showed exactly the same morphological defects as seen in human patients and those displayed by mice lacking the complete carboxypeptidase A domain of AGTPBP1 but at a relatively lesser frequency.<br><br>CONCLUSIONS: We conclude that the R791H mutation in the Agtpbp1 gene impairs sperm head and tail differentiation, resulting in sperm morphological defects.},
}
@article {pmid41160625,
year = {2025},
author = {Bish, LM and Fuss, JL and Panaccione, DG},
title = {Gene editing of the thioester reductase step in the biosynthesis of lysergic acid amides.},
journal = {PloS one},
volume = {20},
number = {10},
pages = {e0334651},
pmid = {41160625},
issn = {1932-6203},
support = {R15 GM114774/GM/NIGMS NIH HHS/United States ; },
mesh = {*Gene Editing/methods ; CRISPR-Cas Systems ; *Lysergic Acid/analogs &amp; derivatives/metabolism ; *Amides/metabolism ; *Oxidoreductases/genetics/metabolism ; Peptide Synthases/genetics/metabolism ; Fungal Proteins/genetics/metabolism ; },
abstract = {Ergot alkaloids derived from lysergic acid are important in agriculture, as food and feed contaminants, and in medicine, as the foundation of several pharmaceuticals. The fungus Metarhizium brunneum makes several lysergic acid amides, with lysergic acid α-hydroxyethylamide (LAH) being produced in by far the highest concentration. The multifunctional enzyme lysergyl peptide synthetase 3 (Lps3) has multiple domains that play important roles in lysergic acid amide synthesis. We hypothesized a role for the reductase domain of Lps3 in liberating LAH from an enzyme-bound precursor and tested this hypothesis with CRISPR/Cas9-based gene editing experiments. We transformed M. brunneum with a Cas9/single guide RNA complex and a donor DNA that replaced the tyrosine at the active site of the reductase domain of Lps3 with a phenylalanine. Sanger sequencing of edited and wild-type genes demonstrated successful editing of the reductase domain without non-target mutations in Lps3. High performance liquid chromatography of the edited strain showed a significant reduction of LAH and accumulation of the precursor lysergic acid. The phenotype was similar when the edited allele of lpsC was in a wild-type background or in backgrounds with late pathway genes easO or easP knocked out, except no LAH was detectable when the edit was in the easO knockout background. The data demonstrate that the reductase domain plays a key role or roles in formation of LAH. The abundant lysergic acid accumulating in the mutants, as opposed to later pathway intermediates in LAH biosynthesis (such as lysergyl-alanine), indicated severe debilitation of Lps3. The data indicate a requirement for the reductase domain of Lps3 in synthesis of lysergic acid amides and demonstrate the feasibility of the CRISPR/Cas9-based approach for editing genes in Metarhizium species.},
}
@article {pmid41160667,
year = {2025},
author = {Du, X and Goh, PK and Ma, C and Coughlan, E and Greatorex, S and Porter, LH and Russ, B and Cummins, KD and Sek, K and Slaney, CY and Scott, AM and Oliaro, J and Neeson, PJ and Risbridger, GP and Taylor, RA and Trapani, JA and Turner, SJ and Darcy, PK and Wiede, F and Tiganis, T},
title = {Targeting PTPN2 enhances human CAR T cell efficacy and the development of long-term memory in mouse xenograft models.},
journal = {Science translational medicine},
volume = {17},
number = {822},
pages = {eadk0627},
doi = {10.1126/scitranslmed.adk0627},
pmid = {41160667},
issn = {1946-6242},
mesh = {Animals ; Humans ; *Protein Tyrosine Phosphatase, Non-Receptor Type 2/metabolism/antagonists &amp; inhibitors ; *Xenograft Model Antitumor Assays ; Mice ; *Immunologic Memory ; *Receptors, Chimeric Antigen/metabolism/immunology ; Immunotherapy, Adoptive ; CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; *T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T cells have been ineffective against solid tumors, where the hostile tumor microenvironment limits CAR T cell function and persistence. Protein tyrosine phosphatase N2 (PTPN2) attenuates T cell receptor and cytokine signaling to maintain T cell tolerance. Here, we used CRISPR-Cas9 gene editing or an inhibitor to target PTPN2 in human CAR T cells specific for the Lewis Y (LeY) neoantigen, which is expressed in most epithelial tumors. Targeting PTPN2 increased CAR and cytokine signaling, including interferon signaling, and enhanced the antigen-induced expansion, activation, and cytotoxicity of anti-LeY CAR T cells in vitro and in vivo. The deletion of PTPN2 in CAR T cells repressed the growth of human tumor and patient-derived xenografts in mice, when compared with unedited CAR T cells, and prolonged mouse survival. The administration of inhibitor also enhanced the ability of α-LeY CAR T cells to repress tumor growth. Cellular indexing of transcriptomes and epitopes by sequencing analysis of splenic PTPN2-deficient CD8[+] CAR T cells in tumor-bearing mice revealed that PTPN2 deficiency favored the generation of CD45RA[+] CAR T cells expressing markers of long-lived stem cell memory (SCM) CAR T cells. Flow cytometric analysis reaffirmed that the deletion or inhibition of PTPN2 promoted the intratumoral accumulation of SCM CD8[+] CAR T cells and the overall persistence of CD8[+] CAR T cells. These data support the use of gene editing or small-molecule inhibitors targeting PTPN2 in human CAR T cells to treat solid tumors.},
}
@article {pmid41160700,
year = {2025},
author = {Elliott, SD and Ready, PJ and Wrinn, CM and Ma, Q and Edward, M and Niescier, RF and Escobar, I and Sun, J and Ganga, AK and McAtee, CK and Atiş, &#x130;S and Koleske, AJ and Bordey, A and Breslow, DK},
title = {A CRISPR activation screen reveals a cilia disassembly pathway mutated in focal cortical dysplasia.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eaeb7238},
pmid = {41160700},
issn = {2375-2548},
support = {R01 MH133562/MH/NIMH NIH HHS/United States ; R35 GM137956/GM/NIGMS NIH HHS/United States ; T32 MH014276/MH/NIMH NIH HHS/United States ; T32 NS007224/NS/NINDS NIH HHS/United States ; },
mesh = {*Cilia/metabolism/genetics/pathology ; Humans ; *Mutation ; *Malformations of Cortical Development/genetics/metabolism/pathology ; Cytoskeletal Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; rhoA GTP-Binding Protein/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Armadillo Domain Proteins/genetics/metabolism ; Animals ; Signal Transduction ; Focal Cortical Dysplasia ; },
abstract = {Defective assembly of primary cilia causes ciliopathies, but cilia disassembly and its role in disease remain poorly understood. From a genome-wide CRISPR activation (CRISPRa) screen for negative regulators of ciliary function, we find here that the F2R G protein-coupled receptor, sterile alpha and TIR motif-containing 1 (SARM1) hydrolase, ryanodine receptors, peri-centrosomal calcium signaling, and RhoA form a functional pathway that is necessary and sufficient for cilia disassembly. Highlighting the significance of this pathway, several components are somatically mutated in focal cortical dysplasia (FCD), a neurological disorder characterized by intractable epilepsy. Supporting the functional impact of these variants, patient-derived SARM1 and RhoA mutations potentiate cilia loss, and a RhoA variant impairs cortical development. Conversely, SARM1 inhibition restores cilia in cells with FCD-associated alterations. Together, our work identifies a pathway for cilia disassembly, implicates aberrant pathway activation as a feature of FCD-associated mutations, and illustrates the potential of CRISPRa screening to provide insight into diseases caused by somatic mutations.},
}
@article {pmid41161091,
year = {2025},
author = {Hong, SH and Kim, I and Lee, G and Kim, EH and Bae, E and Suh, JY},
title = {Structural and mechanistic investigation of the anti-CRISPR protein AcrIE5 using NMR spectroscopy and AlphaFold modeling.},
journal = {Biochemical and biophysical research communications},
volume = {789},
number = {},
pages = {152854},
doi = {10.1016/j.bbrc.2025.152854},
pmid = {41161091},
issn = {1090-2104},
mesh = {*Pseudomonas aeruginosa/virology/genetics/metabolism ; *CRISPR-Cas Systems ; Models, Molecular ; *Viral Proteins/chemistry/metabolism/genetics ; Amino Acid Sequence ; *CRISPR-Associated Proteins/chemistry/metabolism ; Nuclear Magnetic Resonance, Biomolecular ; Magnetic Resonance Spectroscopy ; },
abstract = {The CRISPR-Cas system employs RNA-guided endonucleases to protect bacteria and archaea from invading bacteriophages and plasmids. In response, bacteriophages have evolved anti-CRISPR proteins that inhibit diverse types of the CRISPR-Cas system. AcrIE5 was discovered from the mobile genetic elements of Pseudomonas aeruginosa, and potently inhibits the type I-E CRISPR-Cas system of P. aeruginosa. Here, we determined the solution structure of AcrIE5 using NMR spectroscopy, which adopts a novel αβ fold comprising three α-helices and two β-strands. AcrIE5 harbors a mobile loop between the β-strands that is conserved among homologs encoded by MGEs infecting Pseudomonas species, but truncated in homologs from MGEs of other bacteria. AlphaFold correctly reproduced the experimental structure of AcrIE5 and predicted its binding at a cleft formed by Cas8e, Cas7e, and Cas5e within the P. aeruginosa type I-E Cascade. The mobile loop and α-helices of AcrIE5 mediated key interactions with Cas8e at the PAM recognition site, as well as with adjacent Cas7e and Cas5e. AcrIE5 did not bind individual subunits of Cascade with high affinity, suggesting that it recognizes a composite interface of the functional Cascade assembly. Taken together, our findings suggest that AcrIE5 may compete with DNA binding to the PAM recognition site of type I-E Cascade, similar to AcrIE3 and AcrIE4, and also highlight a potential functional role of the conserved mobile loop in host-specific anti-CRISPR activity.},
}
@article {pmid41161233,
year = {2026},
author = {Shan, J and Wang, W and Sheng, Y and Hong, B and Luo, L and Liu, X and Ma, Y and Wang, J},
title = {Ultra-fast one-pot isothermal detection of respiratory virus: ADNA-initiated CRISPR/Cas12a-mediated RCA cycle.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118141},
doi = {10.1016/j.bios.2025.118141},
pmid = {41161233},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *SARS-CoV-2/isolation &amp; purification/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; *Molecular Diagnostic Techniques/methods ; *COVID-19/diagnosis/virology ; Endodeoxyribonucleases/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Respiratory virus detection is a prominent area of molecular diagnostics, yet current clinical diagnostic methods lack ultra-rapid and highly sensitive detection capabilities. Here, we reported an ultra-rapid, one-pot isothermal assay called "ADNA-initiated CRISPR-Cas12a-mediated RCA cycle" (ACRE). ACRE was developed through computational studying, engineered design of nucleic acid, and enzyme kinetics analysis, combining rolling circle amplification (RCA) with Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a system for respiratory virus detection. The assay involved an engineered assistant DNA (ADNA) initiation reaction, followed by leveraging the cis-cleavage activity of Cas12a coupled with an engineered Padlock, which converted the linear RCA into the RCA cycle. When the RCA cycle is continuously in operation, the trans-cleavage activity of Cas12a facilitates both signal output and amplification. The limit of detection (LOD) for three respiratory viruses (SARS-CoV-2, Inf A, and Inf B) was as low as several hundred attomoles (751 aM, 3.7 fM, and 863 aM), with single-nucleotide specificity. Remarkably, the current assay can detect targets with concentrations above 10 pM within 2.5 min, without the reverse transcription step or specialized instrumentation. Given its exceptional speed, sensitivity, and specificity, ACRE could serve as a robust assay for detecting respiratory virus, enabling molecular diagnostics in clinical settings.},
}
@article {pmid41161575,
year = {2025},
author = {Verma, R and Das, G and H V, M and Muwel, N and Choudhary, R and Kumar, S and Nath, S and Gattani, A and Gupta, V and Sharma, RK and Ajith, Y},
title = {CRISPR-Cas systems: Pioneering next-generation diagnostic tools for parasitic diseases.},
journal = {Molecular and biochemical parasitology},
volume = {264},
number = {},
pages = {111708},
doi = {10.1016/j.molbiopara.2025.111708},
pmid = {41161575},
issn = {1872-9428},
mesh = {*CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Humans ; Animals ; *Parasitic Diseases/diagnosis/parasitology ; Nucleic Acid Amplification Techniques/methods ; Parasites/genetics ; },
abstract = {Parasitic diseases pose significant threats to both human and veterinary health, causing morbidity, mortality, and economic losses. Effective diagnostics are critical, yet conventional methods such as microscopy, serology, and polymerase chain reaction (PCR) are limited by low sensitivity, cross-reactivity, or dependence on costly equipment and skilled personnel. Isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), have improved point-of-care (POC) applications but remain limited by nonspecific amplification and reduced sensitivity for low-copy targets. Clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) systems have emerged as transformative tools in molecular diagnostics, offering high sensitivity, specificity, rapidity, and cost-effectiveness. This review presents an overview of CRISPR-Cas systems, their historical development, classification (Class 1 and Class 2, Types I-VI), molecular mechanisms, and diagnostic potential in parasitic diseases, with illustrative examples from studies published between 2017 and May 2025. Despite significant progress, CRISPR-based diagnostics face challenges such as off-target activity, dependence on nucleic acid amplification, and complex sample preparation. Future directions focus on amplification-free detection, multiplexed assay development, and integration with nanotechnology, microfluidics, smartphone-based devices, and artificial intelligence. CRISPR-Cas technologies thus represent a promising frontier in next-generation diagnostics for parasitic disease surveillance, control, and personalized healthcare in both human and veterinary health.},
}
@article {pmid41161594,
year = {2025},
author = {Fu, H and Xu, W and Huang, M and Cong, Y},
title = {Molecular Detection of Salmonella.},
journal = {Journal of food protection},
volume = {88},
number = {12},
pages = {100659},
doi = {10.1016/j.jfp.2025.100659},
pmid = {41161594},
issn = {1944-9097},
mesh = {*Salmonella/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods ; *Foodborne Diseases/microbiology/prevention &amp; control ; Epidemiological Monitoring ; Polymerase Chain Reaction/methods ; DNA, Bacterial/isolation &amp; purification ; Genotyping Techniques/methods ; Whole Genome Sequencing/methods ; Food Safety ; *Food Microbiology/methods ; Humans ; },
abstract = {The genus Salmonella consists of a group of globally significant foodborne pathogens that pose substantial public health risks. Traditional detection methods are inadequate for rapid diagnosis and effective epidemic surveillance due to limitations such as time-consuming procedures and insufficient sensitivity. In recent years, the development of molecular techniques has driven innovations in Salmonella detection. Nucleic acid-based detection methods including polymerase chain reaction (PCR), real-time fluorescent quantitative PCR (qPCR), whole genome sequencing (WGS) and more emerge as crucial approaches for Salmonella detection due to their high sensitivity, specificity, and rapidity. Our review systematically summarized technological advancements in molecular detection of Salmonella, including specific genetic targets and drug resistance genes used for molecular detection, typing technologies, and emerging techniques such as CRISPR-Cas systems and microfluidic chips. This review comprehensively covers a wide array of molecular detection and characterization technologies, including conventional PCR, qPCR, multiplex PCR, digital PCR (dPCR), isothermal amplification techniques (such as loop-mediated isothermal amplification, recombinase polymerase amplification), genotyping methods (including pulsed-field gel electrophoresis, multilocus sequence typing, etc.), WGS, melting curve analysis (MCA), and other emerging technologies. The review also discusses the balance between sensitivity and specificity in complex samples, challenges regarding the cost and accessibility of advanced technologies, as well as prospects for future development directions including portable point-of-care testing devices, automated detection equipment. Ongoing optimization of molecular detection technologies will provide critical support for the prevention and control of Salmonella infections.},
}
@article {pmid41162070,
year = {2025},
author = {Gautam, V and Jambagi, SR and Muthugounder, M},
title = {Genome editing of detoxification gene repertoires in insects using clustered regularly interspaced short palindromic repeats (CRISPR): A systematic review and meta-analysis.},
journal = {Pesticide biochemistry and physiology},
volume = {215},
number = {},
pages = {106687},
doi = {10.1016/j.pestbp.2025.106687},
pmid = {41162070},
issn = {1095-9939},
mesh = {Animals ; *Gene Editing ; *Insecta/genetics ; *Insecticide Resistance/genetics ; Insecticides/pharmacology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Inactivation, Metabolic/genetics ; },
abstract = {Decoding the molecular mechanisms underlying insect resistance to insecticides and host plant adaptation is essential for effective and sustainable Insecticide Resistance Management (IRM). Reverse genetic approaches targeting "target site resistance" can help control pests without harming pollinators and beneficial biocontrol agents. Understanding the role of mutations involved in xenobiotic resistance enables the judicious use of pesticides. CRISPR-based genome editing allows precise manipulation of detoxification genes, helping to decipher their roles in resistance development. This review provides a comprehensive overview of CRISPR-mediated genome editing in insect detoxification genes and their involvement in resistance mechanisms. In addition to synthesizing overall data trajectories, we present study-level effect sizes that highlight context-specific responses to gene editing, offering insights that can inform future experimental designs and functional validation studies.},
}
@article {pmid41162404,
year = {2025},
author = {Yan, X and Liu, B and Zhou, S and Fan, Y and Wei, S and Qiu, D and Xiang, H and Zhou, J and Mergny, JL and Monchaud, D and Ju, H and Zhou, J},
title = {A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics.},
journal = {Analytical chemistry},
volume = {97},
number = {44},
pages = {24634-24642},
doi = {10.1021/acs.analchem.5c04782},
pmid = {41162404},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Human papillomavirus 16/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; G-Quadruplexes ; *CRISPR-Associated Proteins/genetics/metabolism ; Photochemical Processes ; Papillomavirus Infections/diagnosis/virology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {The potential of clustered regularly interspaced short palindromic repeats (CRISPR) and corresponding CRISPR-associated (Cas) protein systems (CRISPR/Cas) systems for biomedical applications is tremendous; however, precise control of their activity is essential to better harness this potential and, beyond this, to develop reliable diagnostic reagents. Herein, we report on such a strategy by controlling the CRISPR/Cas12a activity using a photo-controllable CRISPR RNA (crRNA). To this end, the 3' end of crRNA was conjugated to a G-quadruplex (G4) block through a photocleavable linker: upon photo irradiation, the G4 trigger is removed, thus allowing for the DNA target to access and hybridize with the crRNA, and thus be processed by the CRISPR/Cas12a system. The efficiency of this approach was demonstrated by the detection of human papillomavirus 16 DNA in 50 clinical samples: our one-pot strategy was found to be as efficient as the routinely implemented method (qPCR), with 95.7% sensitivity and 100% specificity, in addition to be faster (25 versus 60 min) and both simpler and less expensive (being implementable as lateral flow test strips). Collectively, this new and fully controllable CRISPR/Cas system holds great potential for next-generation clinical diagnostics.},
}
@article {pmid41162659,
year = {2025},
author = {Campbell, IW and Basta, DW and Zingl, FG and Sullivan, EJ and Doranga, S and Waldor, MK},
title = {Anoxia activates CRISPR-Cas immunity in the mouse intestine.},
journal = {Nature microbiology},
volume = {10},
number = {12},
pages = {3069-3074},
pmid = {41162659},
issn = {2058-5276},
support = {P30 DK034854/DK/NIDDK NIH HHS/United States ; R01 AI042347/AI/NIAID NIH HHS/United States ; T32 AI007061/AI/NIAID NIH HHS/United States ; Investigator program//Howard Hughes Medical Institute (HHMI)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems ; *Citrobacter rodentium/genetics/immunology ; *Intestines/microbiology/immunology ; Gastrointestinal Microbiome ; Bacterial Proteins/genetics/metabolism ; Oxygen/metabolism ; Gene Expression Regulation, Bacterial ; *Hypoxia ; Mice, Inbred C57BL ; Enterobacteriaceae Infections/microbiology ; },
abstract = {The natural context in which CRISPR-Cas systems are active in Enterobacteriaceae has remained enigmatic. Here we find that the Citrobacter rodentium type I-E CRISPR-Cas system is activated by the oxygen-responsive transcriptional regulator Fnr in the anoxic environment of the mouse intestine. Since Fnr-dependent regulation is predicted in ~41% of Enterobacteriaceae cas3 orthologues, we propose that anoxic regulation of CRISPR-Cas immunity is an adaptation that protects Enterobacteriaceae against threats from foreign DNA within the intestinal microbiome.},
}
@article {pmid41162706,
year = {2026},
author = {Chong, Z and Hui, S and Qiu, X and Palakurty, S and Sariol, A and Kaszuba, T and Nguyen, MN and Li, P and Raju, S and Hall, PD and Nelson, CA and Baltazar-Perez, I and Price, DA and Rothlauf, PW and Crowe, JE and Whelan, SPJ and Leung, DW and Amarasinghe, GK and Bailey, AL and Fremont, DH and Diamond, MS},
title = {Multiple LDLR family members act as entry receptors for yellow fever virus.},
journal = {Nature},
volume = {649},
number = {8095},
pages = {173-182},
pmid = {41162706},
issn = {1476-4687},
support = {//Vir Biotechnology, Bavarian Nordic and Moderna./ ; },
mesh = {Animals ; *Yellow fever virus/physiology/pathogenicity/metabolism ; Humans ; *Virus Internalization ; Mice ; *Receptors, LDL/metabolism/genetics/chemistry/deficiency ; *Yellow Fever/virology/metabolism ; Female ; Low Density Lipoprotein Receptor-Related Protein-1/metabolism/genetics/deficiency/chemistry ; Male ; *Receptors, Virus/metabolism ; Viral Envelope Proteins/metabolism/chemistry ; Protein Domains ; Hepatocytes/metabolism/virology ; CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Infection by yellow fever virus (YFV), the prototype Orthoflavivirus, induces a febrile syndrome in humans that can progress to liver failure, haemorrhage and death[1]. Despite decades of study, the entry receptors for YFV remain unclear. Here, using a surface protein-targeted CRISPR-Cas9 screen, we identified LRP4, a low-density lipoprotein receptor (LDLR) family member, as a candidate entry receptor for YFV. Genetic ablation of LRP4 impaired YFV infection of cells and, reciprocally, complementation or ectopic expression of LRP4 increased infection. Related viruses in the YFV antigenic complex also showed LRP4-dependent infection. LRP4 promoted YFV entry into cells through LDLR type A (LA) domain binding to domain III of the YFV envelope protein. Soluble LRP4-Fc decoy receptors neutralized YFV infection in cell culture and reduced viral burden in vivo. As we observed residual YFV infection in LRP4-deficient cells, we evaluated whether other LDLR family members promote YFV entry. This approach identified LRP1 and VLDLR as additional receptors for YFV infection in cell culture. LRP1-Fc, LRP4-Fc and VLDLR-Fc decoys protected mice from YFV challenge, and LRP1-Fc decoys inhibited YFV infection and liver pathogenesis in mice engrafted with human hepatocytes. A genetic deficiency of LRP1 in primary human hepatocyte cultures also resulted in reduced YFV infection. Our findings establish a role for multiple LDLR family members in YFV entry, infection and pathogenesis, which has implications for receptor use and countermeasure development for multiple emerging orthoflaviviruses.},
}
@article {pmid41162785,
year = {2025},
author = {Coda, DM and Watt, L and Glauser, L and Batiuk, MY and Burns, AM and Stahl, CL and Wong, LY and Gräff, J},
title = {Cell-type- and locus-specific epigenetic editing of memory expression.},
journal = {Nature genetics},
volume = {57},
number = {11},
pages = {2661-2668},
pmid = {41162785},
issn = {1546-1718},
support = {101043457//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; 310030_219342//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; 310030_197752//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Epigenesis, Genetic/genetics ; *Memory/physiology ; Animals ; *Gene Editing/methods ; Mice ; CRISPR-Cas Systems/genetics ; Promoter Regions, Genetic ; Nerve Tissue Proteins/genetics ; Neuronal Plasticity/genetics ; Male ; Cytoskeletal Proteins/genetics ; Neurons/metabolism/physiology ; Epigenome Editing ; },
abstract = {Epigenetic mechanisms have long been proposed to act as molecular mnemonics[1-3], but whether the epigenetic makeup of a single genomic site can guide learnt behaviors remains unknown. Here we combined CRISPR-based epigenetic editing tools[4,5] with c-Fos-driven engram technologies[6,7] to address this question in memory-bearing neuronal ensembles. Focusing on the promoter of Arc, which encodes a master regulator of synaptic plasticity[8], we found that its locus-specific and temporally controlled epigenetic editing is necessary and sufficient to regulate memory expression. Such effects occurred irrespective of the memory phase-during the initially labile period after learning and for fully consolidated memories-and were reversible within subject, testifying to their inherent plasticity. These findings provide a proof-of-principle that site-specific epigenetic dynamics are causally implicated in memory expression.},
}
@article {pmid41163074,
year = {2025},
author = {Dashti, M and Mohammaddust Sarab, M and Shad, F and Dehnavi, S},
title = {CRISPR-mediated engineering of mesenchymal stromal/stem cells: a summary of recent progress in immunological applications for regenerative medicine and cancer therapy.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {592},
pmid = {41163074},
issn = {1757-6512},
mesh = {Humans ; *Mesenchymal Stem Cells/immunology/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Neoplasms/therapy/immunology ; *Regenerative Medicine/methods ; Gene Editing ; Animals ; *Mesenchymal Stem Cell Transplantation ; },
abstract = {Mesenchymal stromal/stem cells (MSCs) have introduced as a cornerstone of regenerative medicine, owing to their immunomodulatory properties and therapeutic potential in autoimmune and inflammatory disorders. Although, their clinical application is often restricted due to immune rejection and heterogeneity in immunoregulatory responses. The advent of Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology has revolutionized MSC engineering, enabling precise genetic modifications to enhance their immunological efficacy. This review explores how CRISPR-mediated editing of MSCs can mitigate immunogenicity, amplify anti-inflammatory functions, and repurpose MSCs for targeted immunotherapy. Key strategies include knockout of β2-microglobulin to evade T-cell recognition, augmentation of anti-inflammatory mediators like interleukin (IL)-10 and TNF-alpha stimulated gene/protein 6 (TSG-6), and disruption of pro-inflammatory pathways such as toll-like receptor 4 (TLR4)/NF-κB. In addition, CRISPR-engineered MSCs demonstrate promise in reshaping tumor microenvironments and combating bacterial infections through enhanced innate immunity. Despite challenges including off-target effects and delivery optimization, CRISPR-tailored MSCs represent a transformative approach to overcoming immunological barriers, paving the way for universal, off-the-shelf therapies in rheumatoid arthritis, cancer, and beyond.},
}
@article {pmid41165418,
year = {2025},
author = {Kumar, U and Dwivedi, D and Das, U},
title = {Advancements in CRISPR-Mediated Multiplex Genome Editing: Transforming Plant Breeding for Crop Improvement and Polygenic Trait Engineering.},
journal = {Biotechnology journal},
volume = {20},
number = {11},
pages = {e70148},
doi = {10.1002/biot.70148},
pmid = {41165418},
issn = {1860-7314},
support = {DBTHRDPMU/JRF/BET-24/I/2024-25/376//Department of Biotechnology/ ; 24J/01/00130//Council of Scientific and Industrial Research/ ; 3/1/3/BRET-2024/HRD (L1)//Indian Council of Medical Research/ ; AICE-JRF/SRF-KK04002406//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; Multifactorial Inheritance/genetics ; Genome, Plant ; },
abstract = {With accelerating climate change and the urgent need to stack polygenic traits, multiplex CRISPR/Cas offers a scalable route to resilient crops-yet low editing efficiency and regeneration bottlenecks remain critical constraints. This review centers on multiplex strategies for polygenic trait engineering in plants, surveying compact nucleases (Cas9, Cas12, Cas13 and emerging ultra-compact variants), polycistronic gRNA platforms (tRNA-gRNA arrays, self-cleaving ribozymes, Csy4 processing), and delivery routes (Agrobacterium, biolistics, protoplast transfection, viral vectors). We highlight concrete outcomes-for example, targeted edits in PYL ABA-receptors increased rice grain yield by up to 31% in field tests-and applications from yield and disease resistance to abiotic-stress tolerance, nutrient biofortification and de novo domestication. Technical risks (off-targets, mosaicism, chromosomal rearrangements, transformability) are appraised alongside emerging fixes: compact/engineered nucleases, RNA-processing arrays, morphogenic regulators, and AI-driven sgRNA design integrated with multi-omics. By prioritizing multiplex approaches for polygenic trait stacking, the review argues that these tools are essential to accelerate precision breeding for climate-adapted agriculture.},
}
@article {pmid41165493,
year = {2025},
author = {Zhang, J and Li, X and Wang, M and Ren, S and Li, M and Liu, Y and Lu, F and Li, Q and Li, Y},
title = {Rational Construction of a Robust Bacillus amyloliquefaciens Cell Factory for Acid-Stable α Amylase Production.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {45},
pages = {28883-28895},
doi = {10.1021/acs.jafc.5c04227},
pmid = {41165493},
issn = {1520-5118},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism/enzymology ; *Bacterial Proteins/genetics/metabolism/chemistry ; *alpha-Amylases/genetics/chemistry/metabolism/biosynthesis ; Fermentation ; Gene Editing ; Prophages/genetics/metabolism ; Enzyme Stability ; CRISPR-Cas Systems ; Multigene Family ; },
abstract = {With the development of synthetic biology and biotechnology, chassis engineering has become the main means of industrial protein production, but it has been limited by the lack of efficient gene editing methods and effective engineering strategies. Bacillus amyloliquefaciens shows potential for expressing heterologous proteins, but its cells undergo early autolysis, hindering further application. In this study, an autolysis-related prophage gene cluster was rationally deleted by establishing an efficient CRISPR-nCas9 editing process, and the prophage mutant strain was constructed, which prevented cell lysis. Based on the prophage mutant strain, we screened secondary metabolite biosynthetic gene clusters that hindered the expression of heterologous proteins, and we made reasonable deletions to further improve their efficient expression. Finally, an optimized yield of acid-stable α amylase (2,46,089.21 U/mL) was obtained in a 5-L fed-batch fermentation. Therefore, we successfully constructed an ideal candidate strain for the expression of heterologous proteins, which provides an important research basis for the development of more chassis strains.},
}
@article {pmid41165983,
year = {2026},
author = {Radoeva, T and Rigola, D and Op den Camp, RHM and van Dijk, PJ and Underwood, CJ},
title = {Targeted Mutagenesis in Natural Apomicts.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2987},
number = {},
pages = {181-191},
pmid = {41165983},
issn = {1940-6029},
mesh = {*Apomixis/genetics ; CRISPR-Cas Systems ; *Mutagenesis ; *Taraxacum/genetics ; Parthenogenesis/genetics ; Chromosome Mapping ; Genes, Plant ; },
abstract = {Apomixis-clonal reproduction through seeds-is an alternative reproductive strategy that takes place in less than 0.1% of plant species and has evolved independently in diverse plant lineages. To date, the genetic basis of apomixis has been unraveled genetically in only a few genera. The identification of causal apomixis genes is technically challenging, as apomictic species are typically polyploid and the genetic loci associated with apomixis are often in low-recombination regions limiting conventional fine-mapping. In triploid apomictic dandelion (Taraxacum officinale), after conventional genetic mapping, deletion mapping, and complete apomixis loci haplotype assembly, we made use of targeted mutagenesis using CRISPR/Cas9 technology to identify the Taraxacum officinale PARTHENOGENESIS (ToPAR) gene that is responsible for embryogenesis in the absence of fertilization. Here, we report the methods used to clone the ToPAR gene by targeted mutagenesis and we expect that the general principles could be applied in other systems to identify novel apomixis genes.},
}
@article {pmid41165985,
year = {2026},
author = {Bicknell, R and Koltunow, AMG},
title = {Pilosella: A Dicotyledonous Model for Studying Aposporous, Autonomous Apomixis.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2987},
number = {},
pages = {209-220},
pmid = {41165985},
issn = {1940-6029},
mesh = {*Apomixis/genetics ; Seeds/genetics/growth &amp; development ; *Asteraceae/genetics/physiology/growth &amp; development ; Ovule/genetics ; CRISPR-Cas Systems ; },
abstract = {Pilosella, a member of the Compositae, is a model system used to study the molecular genetics of aposporous apomixis. These plants are small, rapidly growing perennials that are easy to cultivate both in the greenhouse and in tissue culture. Apomixis in Pilosella occurs by apospory where mitotically derived embryo sacs arise adjacent to cells undergoing female gamete meiosis in the ovule. Seed initiation is autonomous, where both embryo and endosperm form without fertilization in the aposporous embryo sac. Apomixis is not fully penetrant in Pilosella. Instead, plants are facultatively apomictic, and apomixis can be easily scored through the simple decapitation of the immature capitulum bud. Natural sexual and facultatively apomictic forms are readily cross-compatible, facilitating comparative studies of inheritance and allele function. A wide range of experimental methods have been described for these plants, including histological techniques for studying the cytological aspects of apomixis, an efficient Agrobacterium-mediated transformation system, CRISPR/Cas9 mutagenesis, and mapping approaches that use deletion mutation and segregation in polyhaploid populations. Freely available online resources include a genome assembly, a molecular map based on cDNA markers and a transcriptome database. Collectively, these resources make Pilosella a highly tractable experimental system for studying the genetic control of native apomixis.},
}
@article {pmid41167015,
year = {2025},
author = {Song, X and Peng, Q and Zhang, X and Zhao, Y and Zhang, G and Guo, R and Li, Y and Shen, Y and Yang, S and Hu, M and Zhang, X and Li, J and Fan, B and Li, B},
title = {Enhanced immunogenicity of swine coronavirus PEDV by engineering transcriptional regulatory sequences.},
journal = {Vaccine},
volume = {68},
number = {},
pages = {127914},
doi = {10.1016/j.vaccine.2025.127914},
pmid = {41167015},
issn = {1873-2518},
mesh = {Animals ; *Porcine epidemic diarrhea virus/immunology/genetics ; Swine ; Antibodies, Viral/blood/immunology ; *Coronavirus Infections/prevention &amp; control/veterinary/immunology ; *Viral Vaccines/immunology/genetics/administration &amp; dosage ; *Swine Diseases/prevention &amp; control/immunology/virology ; Antibodies, Neutralizing/blood/immunology ; Spike Glycoprotein, Coronavirus/immunology/genetics ; Female ; *Immunogenicity, Vaccine ; Vaccines, Inactivated/immunology/administration &amp; dosage ; Immunoglobulin G/blood ; CRISPR-Cas Systems ; Pregnancy ; Immunoglobulin A/blood ; Gene Editing ; },
abstract = {Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus that can cause severe diarrhea and high mortality in neonatal piglets. Therefore, the development of safe and effective vaccines remains a top priority for controlling PEDV infection. Previous studies have found that the transcription regulatory sequence (TRS) of the viral M gene has the strongest regulatory ability for the expression of exogenous gene. In order to investigate whether the transcription level of the S gene is increased after swapping body TRS of the S gene (TRS-S) with the body-TRS of M gene (TRS-M), CRISPR/Cas9 gene editing technology were used to replace the TRS-S of PEDV with TRS-M, and found that replacing TRS-S by TRS-M, as well as the downstream bases of TRS-M, can effectively promote the expression of S protein. Subsequently, the recombinant strain with high expression of S protein was prepared into an inactivated vaccine, which induced higher levels of S-specific immunoglobulin G (IgG), IgA, and neutralizing antibodies in immunized pregnant sows. The newborn spiking pigs also obtained high levels of antibodies through breastfeeding, and after being challenged, it was found that clinical symptoms and microscopic lesions were significantly reduced. In conclusion, these data suggest that replacing TRS-S with the transcriptionally efficient TRS-M effectively promotes PEDV S protein expression, and this modified strain exhibits improved immunogenicity and better protection against viral challenge, which provides new method for the research and development of PEDV vaccines.},
}
@article {pmid41167048,
year = {2025},
author = {Wei, S and Ma, R and Zhang, T and Wen, L},
title = {Targeted large-fragment genomic deletion in human pluripotent stem cells (hPSCs) via CRISPR/Cas9.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103859},
doi = {10.1016/j.scr.2025.103859},
pmid = {41167048},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Cell Differentiation ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Large genomic deletions are increasingly recognized as contributors to human disease. Here, we describe a CRISPR/Cas9-based protocol to induce targeted large-fragment deletions in human pluripotent stem cells (hPSCs). Two sgRNAs flanking the target region were designed, synthesized, and co-delivered with Cas9 protein into single-cell hPSCs via electroporation. Deletions were identified using junction PCR. Successfully edited cell pools were expanded as single-cell colonies and assessed for genomic deletion, stem cell identity, karyotype integrity, and tri-lineage differentiation capacity. This approach provides a robust method for modeling genomic deletions in hPSCs for disease research and functional genomics.},
}
@article {pmid41167358,
year = {2025},
author = {Rahangdale, S and Vishwakarma, A and Chauhan, R and Singh, S and Singh, PK},
title = {Engineered sgRNA captures single-stranded donor template and delivers at the DSB site to enhance HDR.},
journal = {International journal of biological macromolecules},
volume = {332},
number = {Pt 2},
pages = {148614},
doi = {10.1016/j.ijbiomac.2025.148614},
pmid = {41167358},
issn = {1879-0003},
mesh = {Saccharomyces cerevisiae/genetics ; *Gene Editing/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems/genetics ; *DNA Breaks, Double-Stranded ; *Recombinational DNA Repair ; *DNA, Single-Stranded/genetics ; Streptococcus pyogenes/genetics ; },
abstract = {The CRISPR-Cas9 system from Streptococcus pyogenes has revolutionized genome modification through precise editing across a wide range of organisms. Yeast supports efficient genome editing via plasmid-based Cas9-gRNA expression, while higher eukaryotes often require genome-integrated cassettes or RNP delivery. In this study, we engineered CRISPR components to enhance nuclear targeting and editing efficiency. We demonstrated the proof of concept in Saccharomyces cerevisiae using its CAN1 locus. We developed a dual-host compatible vector, encoding Cas9 nuclease fused with three nuclear localization signals (Cas9-3xNLS). The recombinant protein, expressed in E. coli and purified on a Ni-NTA column, showed DNA cleavage in an in vitro assay. Genome editing efficacy of Cas9-3xNLS was demonstrated in S. cerevisiae AH109 strain. Further, we engineered sgRNAs by extending their ends to facilitate the annealing to ssODN. We synthesized ssODNs having a complementary sequence either at 3' or 5' to anchor with sgRNAs. sgRNAs (unmodified and end extended) and ssODNs were introduced into yeast in various combinations. sgRNA with a 3' ssDNA-anchoring motif and ssODN representing the antisense strand of the target gene with sgRNA complementary motif at the 5' end (free homology arm at 3') improved HDR efficiency significantly. This combination yielded about a 1.64-fold increase in canavanine-resistant colonies as compared to the control via precise insertion of a stop codon. In contrast, extension of sgRNA at the 5' end did not show any advantage. This approach is flexible and easy to use and has the potential to enhance homology-directed repair in diverse organisms.},
}
@article {pmid41167881,
year = {2025},
author = {Meng, X and Yue, Y and Huang, M and Duan, Z and Liu, K and Wu, L},
title = {DNAzyme-CRISPR driven dual-mode biosensor with nanozyme signal amplification for on-site Pb[2+] detection.},
journal = {Analytica chimica acta},
volume = {1379},
number = {},
pages = {344711},
doi = {10.1016/j.aca.2025.344711},
pmid = {41167881},
issn = {1873-4324},
mesh = {*Lead/analysis ; *DNA, Catalytic/chemistry/metabolism ; *Biosensing Techniques/methods ; Electrochemical Techniques ; *CRISPR-Cas Systems ; Food Contamination/analysis ; Limit of Detection ; Colorimetry ; Cerium/chemistry ; },
abstract = {BACKGROUND: Lead ion (Pb[2+]) is a toxic heavy metal that poses severe threats to food safety. Traditional methods like inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) rely on bulky instrumentation, which are limited by high costs, complex sample preparation requirements, and inability to meet the demands for rapid on-site testing. Nanozyme-based biosensors have emerged as promising alternatives, yet single-mode sensors often suffer from matrix interference in complex food samples. Therefore, developing a rapid and reliable on-site method is critical for Pb[2+] detection.<br><br>RESULTS: To address this challenge, a CRISPR/Cas12a-driven dual-mode biosensor integrating Pt/CeO2 nanozyme-mediated peroxidase activity with GR-5 DNAzyme recognition was developed. The biosensor's design capitalizes on GR-5 DNAzyme for Pb[2+]-specific recognition, triggering CRISPR/Cas12a-mediated cleavage of electrochemical/colorimetric signal probe SH-ssDNA-Pt/CeO2 to generate dual signals. This innovative platform synergizes electrochemical precision and colorimetric simplicity for Pb[2+] detection in complex food matrices. The biosensor achieved an ultra-sensitive electrochemical response (linear range: 0.002-200&#xa0;nM; limit of detection: 0.14 pM) alongside a robust colorimetric readout (linear range: 0.5-2000&#xa0;nM; limit of detection: 0.47&#xa0;nM), representing significant sensitivity improvements over conventional single-mode sensors. Crucially, the intrinsic cross-verification mechanism between orthogonal signal modalities minimized false positives while ensuring >90.5&#xa0;% recovery in spiked corn, edible oil, beef and red wine samples with RSD <5&#xa0;%. These results highlight the potential of the dual-mode sensor as a practical, field deployable sensing platform for the detection of Pb[2+].<br><br>SIGNIFICANCE: This work innovatively integrates the specificity of CRISPR with the catalytic properties of nanozymes into a self-validating electrochemical/colorimetric dual-mode system. The detection method not only establishes a robust platform for highly sensitive, reliable and visualized detection of Pb[2+] in complex food matrices, but also pioneers a new paradigm for multi-modal biosensor design.},
}
@article {pmid41168338,
year = {2025},
author = {Elsayed, EM and Stukenberg, D and Meier, D and Schmeck, B and Becker, A},
title = {RECKLEEN is a lambda Red/CRISPR-Cas9 based single plasmid platform for enhanced genome editing in Klebsiella pneumoniae.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1509},
pmid = {41168338},
issn = {2399-3642},
support = {LOEWE/2/13/519/03/06.001(0002)/74//Hessisches Ministerium f&#xfc;r Wissenschaft und Kunst (Hessen State Ministry of Higher Education, Research and the Arts)/ ; },
mesh = {*Klebsiella pneumoniae/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *Plasmids/genetics ; *Bacteriophage lambda/genetics ; *Genome, Bacterial ; },
abstract = {Klebsiella pneumoniae (Kp) has evolved as a major public health threat due to its multidrug-resistance (MDR) and hypervirulence. Current Kp genome-editing tools are constrained by cumbersome workflows, low flexibility, and limited scalability. Here, we present the RECKLEEN system -Recombineering/CRISPR-based KLebsiella Engineering for Efficient Nucleotide editing - as a single plasmid platform designed for precise genetic manipulation of Kp. RECKLEEN combines lambda Red recombineering with powerful CRISPR-Cas9-based targeted counterselection, achieving up to 99.998% killing efficiency. By implementing the near PAM-less SpG Cas9 variant in RECKLEEN, the compatible target sequence spectrum was significantly broadened. This approach enables deletions, point mutations, and DNA integrations, with efficiencies reaching 100% of the counter-selected clones. Simultaneous multi-target deletions were accomplished with up to 72% efficiency. To streamline the process, we developed a toolbox of eleven plasmids based on a modular cloning standard, enabling time- and resource-efficient assembly of editing constructs. This allows a 5-days workflow, from plasmid construction to the generation of strains with the desired genetic modification(s). The efficacy of RECKLEEN extends to various MDR Kp strains, such as ATCC 700721, ATCC BAA-1705, and ATCC 700603, demonstrating its broad applicability. RECKLEEN significantly enhances genome-editing capabilities for Kp, advancing research into its pathology and MDR mechanisms.},
}
@article {pmid41168408,
year = {2026},
author = {Ke, Z and Zhang, Q and Chen, X and Jin, R and Han, G and Cao, Q and Zhu, K and Wei, S and Chen, J and Wang, Q and Zhang, M and Huang, W and Li, K and Xiong, K and Lu, K and Chen, Y and Cao, C},
title = {CRISPR/Cas9 library screening uncovered CCT2 as a critical driver of acquired resistance to EGFR-targeted therapy by stabilizing TMX1 in non-small cell lung cancer.},
journal = {Cell death and differentiation},
volume = {33},
number = {4},
pages = {860-876},
pmid = {41168408},
issn = {1476-5403},
mesh = {*Carcinoma, Non-Small-Cell Lung/drug therapy/genetics/metabolism/pathology ; Humans ; *Lung Neoplasms/drug therapy/genetics/pathology/metabolism ; *Drug Resistance, Neoplasm/drug effects/genetics ; *CRISPR-Cas Systems/genetics ; ErbB Receptors/metabolism/antagonists &amp; inhibitors ; Animals ; Mice ; Protein Kinase Inhibitors/pharmacology/therapeutic use ; Cell Line, Tumor ; Mice, Nude ; Protein Stability ; *Thioredoxins/metabolism ; *Membrane Proteins/metabolism ; },
abstract = {In the treatment of non-small cell lung cancer (NSCLC) with epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), the emergence of acquired resistance remains a significant challenge. Elucidating the underlying mechanisms of resistance is crucial for developing novel strategies to overcome or delay therapeutic escape. To this end, this study aimed to identify key drivers of EGFR-TKIs resistance and explore actionable targets for intervention. We investigated resistance mechanisms by integrating CRISPR/Cas9-based genome-wide screening with tandem mass tag (TMT) proteomic analysis, and virtually screened bioactive small molecule libraries to identify compounds capable of restoring EGFR-TKIs sensitivity. The multi-omics approach revealed that CCT2 is a critical mediator of resistance to third-generation EGFR-TKIs in lung cancer, with higher expression of CCT2 observed in resistant cells compared to sensitive cells. Mechanistically, CCT2 recruits tripartite motif-containing protein 28 (TRIM28) to catalyze SUMO2 modification of thioredoxin-related transmembrane protein 1 (TMX1), inhibiting its ubiquitination and enhancing protein stability. This post-translational modification (PTM) promotes TMX1-dependent reactive oxygen species (ROS) clearance, thereby conferring resistance. Importantly, pharmacological inhibition with the compound HY-10127, identified through virtual screening, effectively restored EGFR-TKIs sensitivity in resistant cell lines and delayed the development of resistance in xenograft models. The findings establish the CCT2/TRIM28/TMX1/ROS axis as a novel resistance mechanism in EGFR-mutated lung cancer, and targeting this pathway with HY-10127 represents a promising strategy to overcome resistance to third-generation EGFR-TKIs, providing preclinical rationale for clinical translation. These discoveries advance our understanding of molecular resistance mechanisms and offer potential therapeutic targets for improving lung cancer prognosis.},
}
@article {pmid41170433,
year = {2025},
author = {Zhang, S and Chu, M and Sun, X},
title = {The arms race in bacteria-phage interaction: deciphering bacteria defense and phage anti-defense mechanisms through metagenomics.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1687307},
pmid = {41170433},
issn = {1664-302X},
abstract = {Bacteriophages are viruses that specifically infect bacteria and co-evolve with their hosts through mutual interactions. They represent one of the most significant drivers of microbial diversity, influencing its evolution, generation, and maintenance. To counter bacteriophage infection, bacteria have developed sophisticated immune systems, including both passive adaptations, such as inhibiting phage adsorption and preventing DNA entry, and active defense systems such as restriction-modification systems and CRISPR-Cas systems. The ongoing arms race between bacteriophages and bacteria has left distinct evolutionary signatures in their genomic sequences. Advances in large-scale genomic and metagenomic sequencing technologies, coupled with bioinformatics approaches, have greatly enhanced our understanding of bacteria-phage interaction mechanisms, driving progress in bacteriophage biology. This review systematically analyses the diverse immune strategies bacteria employ against phage infection, elucidates the coordination and interrelationships among different anti-phage mechanisms, and highlights potential directions for future research.},
}
@article {pmid41170434,
year = {2025},
author = {Araújo, MRB and Dos Santos, LS and Prates, FD and Perini, HF and Silva, JS and Ramos, JN and Bokermann, S and Sacchi, CT and de Mattos Guaraldi, AL and Campos, KR and Cardoso, TDCS and Castro, DLC and Silva, MA and Sousa, M&#xc2;B and Vieira, VV and Santos, MBN and Camargo, CH and Andrade, BS and da Silva, MV and Sant'Anna, LO and Viana, MVC and Azevedo, V},
title = {Virulence and mutations analysis based on the whole genome of a Brazilian Corynebacterium diphtheriae strain isolated from a cutaneous infection.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1579154},
pmid = {41170434},
issn = {1664-302X},
abstract = {Corynebacterium diphtheriae is the main etiological agent of diphtheria, a potentially fatal disease whose most severe signs and symptoms result from the action of an exotoxin, the diphtheria toxin (DT). Although non-toxigenic C. diphtheriae strains have been associated with several diseases, including cutaneous infections and endocarditis, they are not monitored in many countries, and their mechanisms of virulence and antimicrobial resistance remain underexplored. Therefore, this study aimed to provide a comprehensive characterization -through genomic, in vitro, and in vivo analyses - of a non-toxigenic C. diphtheriae strain (46855) isolated from a leg lesion, highlighting its pathogenic potential and resistance profile. The isolate was assigned to a novel sequence type (ST-925) and was found to be resistant to tetracycline and rifampin. Multiple antimicrobial resistance genes were predicted in the genome, such as tet(33), rbpA, and rpoB2, in addition to mutations in the rpoB gene. A diverse set of virulence-associated genes related to adhesion, iron uptake systems, gene regulation, and post-translational modification was also identified. The isolate was able to form biofilm in vitro and exhibited strong virulence in Galleria mellonella larvae and A549 human pneumocyte cells. Finally, the structural analysis of the rpoB gene, carried out for the first time in this study, linked the observed mutations to rifampin resistance in C. diphtheriae. In summary, the data revealed that C. diphtheriae 46855, although non-toxigenic, harbors multiple genes associated with antimicrobial resistance and virulence, emphasizing the need for greater surveillance and functional studies on non-toxigenic strains.},
}
@article {pmid41170598,
year = {2025},
author = {Yoo, D and Park, H and Lim, H and Kim, G and Kim, D and Seo, Y and An, J and Kang, S and Park, C and Lee, MH and Lee, T},
title = {Versatile Biosensing Tool: CRISPR-Cas12a System-Integrated Electrochemical Biosensor for Severe Fever with Thrombocytopenia Syndrome Virus Detection in Clinical and Environmental Conditions.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {49},
pages = {e06860},
doi = {10.1002/smll.202506860},
pmid = {41170598},
issn = {1613-6829},
support = {2021R1C1C1005583//National Research Foundation of Korea (NRF)/ ; RS-2024-00416117//National Research Foundation of Korea (NRF)/ ; No.2020R1A5A1018052//National Research Foundation of Korea (NRF)/ ; RS-2024-00507931//Materials &amp; Components Technology Development Program/ ; },
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Phlebovirus/isolation &amp; purification/genetics ; *Severe Fever with Thrombocytopenia Syndrome/virology/diagnosis ; *Electrochemical Techniques/methods ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly lethal zoonotic virus with a maximum mortality rate of 30%. Despite its risks and potential for human-to-human transmission, the standard diagnostic method has been absent for SFTSV detection. Therefore, this study introduces a versatile SFTSV biosensing technology using the electrochemical-clustered regularly interspaced short palindromic repeat (EC-CRISPR) system. The sensing membrane is functionalized with the 2WJ DNA@Au-MoS2 signal probe as a strategy to amplify the EC response resulting from target detection events of the CRISPR system. The sensor exhibits selective, sensitive, and reproducible detection capabilities in phosphate-buffered saline, human serum, and Haemaphysalis longicornis genomic DNA diluted conditions with detection limits of 210.7, 189.0, and 285.1 fM, respectively. This verifies the versatility of the fabricated system, which significantly contributes to the early SFTSV detection in various fields. In the meantime, the sufficient sensing performance is demonstrated in identifying of SFTSV from infectious agent DNA. Furthermore, the proposed EC-CRISPR biosensing platform can be considered as a flexible foundational technique for the diagnosis of zoonotic infectious diseases, as it demonstrates practical applicability for the detection of Orientia tsutsugamushi by utilizing a customized CRISPR system programming strategy.},
}
@article {pmid41170612,
year = {2025},
author = {Pu, J and Zhou, S and Ma, W and Liu, C and Guo, L and Yan, B and Huo, Z},
title = {Light-controlled CRISPR-Cas12a one-pot platform for ultrasensitive cell-free DNA detection in systemic lupus erythematosus diagnosis.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {44},
pages = {8916-8922},
doi = {10.1039/d5ay01286d},
pmid = {41170612},
issn = {1759-9679},
mesh = {*Lupus Erythematosus, Systemic/diagnosis/blood/genetics ; Humans ; *Cell-Free Nucleic Acids/blood/genetics/analysis ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {Systemic lupus erythematosus (SLE), as a complex autoimmune disease with heterogeneous clinical manifestations, presents significant challenges for early diagnosis. Circulating cell-free DNA (cfDNA) has emerged as a promising disease monitoring biomarker due to its correlation with SLE pathological progression in terms of concentration and fragmentation patterns. However, existing detection methods lack sufficient sensitivity and practicality for clinical application. To address this, we developed a spatiotemporally resolved light-controlled biosensor by integrating a photoactivatable CRISPR-Cas12a system with TdT-mediated poly-A tail extension, achieving three major innovations: (1) implementation of NPOM-dt modified crRNA for precise regulation; (2) optimization of 365 nm UV activation protocol to eliminate interference in one-pot reactions; and (3) establishment of a three-phase "Extension-Activation-Detection" workflow. The platform demonstrates outstanding performance with a detection limit of 0.42 pM, excellent linearity (R[2] = 0.9956) in the 0-0.1 nM range, and the novel DNA Integrity Index (DII) as a diagnostic indicator - showing significantly higher values in SLE patients (9.82 × 10[3] nmol g[-1]) versus healthy controls (4.2 × 10[3] nmol g[-1], P < 0.0001) with an AUC of 0.8947. This study provides an innovative detection platform combining high sensitivity with clinical utility for early SLE diagnosis.},
}
@article {pmid41170688,
year = {2025},
author = {Wang, S and Guan, X and Sun, S},
title = {A CRISPR/Cas12a-based aptasensor enhanced by functionalized AuNPs for sensitive full-range C-reactive protein detection.},
journal = {Journal of materials chemistry. B},
volume = {13},
number = {45},
pages = {14749-14760},
doi = {10.1039/d5tb01727k},
pmid = {41170688},
issn = {2050-7518},
mesh = {*C-Reactive Protein/analysis ; *Gold/chemistry ; *Aptamers, Nucleotide/chemistry/metabolism ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; Particle Size ; *Endodeoxyribonucleases/metabolism ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {C-reactive protein (CRP) is a well-established biomarker whose plasma levels increase significantly during inflammatory and infectious episodes. It plays a vital role in the diagnosis of bacterial infections and autoimmune diseases, and cardiovascular risk assessment. However, conventional detection techniques often struggle to balance high sensitivity with a broad dynamic range. Here, we developed and validated a novel fluorescence-based aptasensor named AuCA (AuNP-enhanced CRISPR Aptasensor), which integrates nucleic acid aptamers, magnetic beads (MBs), gold nanoparticles (AuNPs), and the CRISPR/Cas12a system for robust CRP quantification. In this system, MBs functionalized with aptamer 1 selectively captured the target protein, while AuNPs co-modified with aptamer 2 and a Cas12a activation sequence (Trigger) enabled efficient signal transduction. Upon target binding, the Trigger activated the Cas12a/crRNA complex, initiating trans-cleavage of fluorescent reporters and producing a markedly amplified signal. The optimized AuCA platform achieved a low detection limit of 60 ng mL[-1] and a quantifiable range of 0.1-150 μg mL[-1]. It exhibited excellent specificity and resistance to biological interference, ensuring reliable measurements even in complex sample matrices. When applied to clinical human plasma, AuCA demonstrated results that are in strong concordance with results from a commercial immunoturbidimetric assay. AuCA allowed the simultaneous detection of both standard and hypersensitive CRP (hsCRP), supporting comprehensive full-range CRP (frCRP) analysis with strong potential for clinical applications.},
}
@article {pmid41170849,
year = {2025},
author = {Yu, T and Xie, J and Huang, X and Huang, J and Bao, G and Yuan, W and Gao, C and Liu, C and Hu, J and Yang, W and Li, G},
title = {BaeR and H-NS control CRISPR-Cas-mediated immunity and virulence in Acinetobacter baumannii.},
journal = {mSystems},
volume = {10},
number = {11},
pages = {e0106725},
pmid = {41170849},
issn = {2379-5077},
support = {82373637//National Natural Science Foundation of China/ ; 82073611//National Natural Science Foundation of China/ ; 82002186//National Natural Science Foundation of China/ ; BK20231241//Jiangsu Provincial Department of Science and Technology/ ; YZ2023104//YangZhou Municipal Science and Technology Bureau (YangZhou Science and Technology Bureau)/ ; },
mesh = {*Acinetobacter baumannii/pathogenicity/genetics/immunology ; *CRISPR-Cas Systems/genetics ; *Bacterial Proteins/genetics/metabolism ; Virulence/genetics ; Gene Expression Regulation, Bacterial ; Biofilms/growth &amp; development ; *DNA-Binding Proteins/genetics/metabolism ; Acinetobacter Infections/microbiology ; Humans ; },
abstract = {Acinetobacter baumannii balances its remarkable ability to acquire antibiotic resistance genes via horizontal gene transfer (HGT) with the immune defense functions of its CRISPR-Cas system, forming a dynamic equilibrium governed by intricate transcriptional regulation. However, the regulatory mechanisms underlying the I-Fb CRISPR-Cas system in A. baumannii remain poorly understood. This study elucidated a multitiered regulatory axis mediated by BaeR and H-NS that coordinates immune defense and virulence expression in the I-Fb CRISPR-Cas system. Using DNA pull-down and electrophoretic mobility shift assay (EMSA), we demonstrated that H-NS directly binds AT-rich regions within the cas3 promoter, suppressing both interference activity and adaptive immunity of the I-Fb CRISPR-Cas system. Intriguingly, the two-component regulator BaeR controlled this suppression by positively regulating H-NS expression. The results revealed that Δcas3 mutants exhibited increased biofilm thickness, elevated the extracellular matrix component poly N-acetyl glucosamine (PNAG) production, upregulated pilus expression, and significantly enhanced epithelial cell adhesion. Strikingly, Δh-ns-cas3 and ΔbaeR-cas3 double-knockout strains showed no statistically significant differences in virulence phenotypes compared to the Δcas3 single mutants. These findings indicate CRISPR-Cas-mediated inhibition of biofilm formation is abolished upon cas3 deletion, thereby releasing the regulatory constraints imposed by BaeR and H-NS. This dysregulation leads to excessive biofilm and extracellular matrix component accumulation, ultimately amplifying bacterial colonization capacity and pathogenicity in host environments. This discovery reveals the dual regulatory roles of BaeR and H-NS in the A. baumannii I-Fb CRISPR-Cas system, mediating both immune defense and virulence modulation. These insights establish a theoretical foundation for novel antimicrobial strategies targeting CRISPR-Cas regulatory networks.IMPORTANCEA. baumannii, a leading cause of drug-resistant nosocomial infections, evolves antibiotic resistance through horizontal gene transfer (HGT) while employing CRISPR-Cas systems to limit foreign DNA invasion. This study reveals that the I-Fb CRISPR-Cas system, typically a defense mechanism, functions as a repressor of virulence traits in A. baumannii. We demonstrate that the transcriptional regulators H-NS and BaeR form a hierarchical axis suppressing Cas3 expression, thereby constraining biofilm formation and host adhesion. Strikingly, CRISPR-Cas deficiency enhances virulence, thickens biofilms, elevates PNAG production, and enhances epithelial colonization through escape from BaeR-/H-NS-mediated control. This work redefines CRISPR-Cas as a dual-function module balancing immune defense and pathogenicity, exposing the BaeR-H-NS-Cas3 axis as a druggable target for novel anti-infectives aimed at disrupting bacterial adaptive evolution.},
}
@article {pmid41170863,
year = {2025},
author = {Liu, J and Feng, G and Guo, C and Liu, D and Li, Y},
title = {Opportunities and Challenges of CRISPR-Cas9 in the Sustainable Development of Animal Husbandry.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {45},
pages = {28575-28587},
doi = {10.1021/acs.jafc.5c09076},
pmid = {41170863},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; Animals ; Animal Husbandry/methods ; *Gene Editing ; Animals, Genetically Modified/genetics/metabolism ; Sustainable Development ; Livestock/genetics/growth &amp; development/metabolism ; },
abstract = {Food security is of vital importance to the destiny of the entire globe. However, the realization of the United Nations' Sustainable Development Goals, which advocate for the eradication of hunger and the improvement of nutrition, appears somewhat bleak. The advent of gene editing has made precise modification of biological genomes a reality. Among these technologies, CRISPR-Cas9, due to its convenience and high efficiency, has been widely applied in fields such as agriculture. The application of CRISPR-Cas9 in genetically engineered animals can rapidly improve animal traits, reduce resource consumption, and has great potential in addressing food security issues. Therefore, this review focuses on the editing principles of CRISPR-Cas9, its developmental trajectory, and the opportunities and challenges it presents in animal husbandry, aiming to provide a theoretical reference for the further application of CRISPR-Cas9 to promote the sustainable development of animal husbandry.},
}
@article {pmid41170937,
year = {2026},
author = {Soliman, H and Akram, N and Saleh, M},
title = {CRISPR-Enhanced RAA-SHERLOCK Assay for Point-of-Care Detection of Cyprinid Herpesvirus-3: Development, Validation and Clinical Application.},
journal = {Journal of fish diseases},
volume = {49},
number = {4},
pages = {e70079},
pmid = {41170937},
issn = {1365-2761},
support = {//Veterinary Medicine University/ ; //Academy of Scientific Research and Technology/ ; },
mesh = {*Fish Diseases/diagnosis/virology ; Animals ; *Herpesviridae/isolation &amp; purification/genetics ; *Herpesviridae Infections/veterinary/diagnosis/virology ; *Carps ; CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Point-of-Care Systems ; Recombinases/metabolism ; },
abstract = {Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), poses a significant threat to global aquaculture due to its high mortality rates and economic impact. Current diagnostic methods, such as PCR, are limited by equipment dependency and procedural complexity, hindering point-of-care (POC) applications. To address this, we developed an integrated assay combining recombinase-aided amplification (RAA) with CRISPR-Cas13a-mediated SHERLOCK technology and lateral flow detection (LFD) for rapid and visual detection of CyHV-3 in clinical samples. The KHV-SHERLOCK assay targets a conserved region of the CyHV-3 thymidine kinase (TK) gene, demonstrating exceptional specificity with no cross-reactivity to related pathogens or host DNA. Sensitivity evaluations revealed a detection limit of 100 ag/μL for CyHV-3 plasmid DNA, tenfold more sensitive than the conventional PCR (1 fg/μL) assay, even in the presence of 100 ng of carp genomic DNA as background interference. Clinical validation using 50 archived samples showed 100% concordance with reference PCR results, confirming diagnostic reliability. The assay's isothermal RAA step (37°C, 40 min) and CRISPR-Cas13a detection (37°C, 1 h) enable equipment-free operation, while LFD provides unambiguous visual results within minutes. This platform merges high sensitivity with POC practicality, offering a transformative tool for field-based KHVD surveillance.},
}
@article {pmid41171167,
year = {2025},
author = {Tharmatt, A and Guha, S and Kumeria, T and Yadav, S and Mittal, A and Chitkara, D},
title = {Lipopolymeric Nanoplex-Mediated CRISPR/Cas9 Delivery for VEGF-A Knockdown in Psoriatic Angiogenesis.},
journal = {ACS applied bio materials},
volume = {8},
number = {11},
pages = {10137-10157},
doi = {10.1021/acsabm.5c01478},
pmid = {41171167},
issn = {2576-6422},
mesh = {*CRISPR-Cas Systems ; *Psoriasis/drug therapy/metabolism/genetics/pathology ; Humans ; *Vascular Endothelial Growth Factor A/genetics/metabolism/antagonists &amp; inhibitors ; Animals ; *Biocompatible Materials/chemistry/pharmacology/chemical synthesis ; Particle Size ; *Neovascularization, Pathologic/drug therapy/genetics ; Mice ; Materials Testing ; *Polymers/chemistry ; Gene Knockdown Techniques ; *Nanoparticles/chemistry ; Angiogenesis ; },
abstract = {Psoriasis is a chronic, incurable inflammatory skin disease characterized by immune cell infiltration, aberrant keratinocyte differentiation, and enhanced angiogenesis. Overexpression of the vascular endothelial growth factor-A (VEGF-A) gene promotes angiogenesis and is essential for endothelial cell migration, adhesion, and proliferation. Therefore, downregulating VEGF-A represents a promising therapeutic strategy for angiogenesis-related disorders. We investigated the application of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein complexes (sgRNA/eGFP-Cas9 RNPs) targeting VEGF-A in psoriasis. To enable efficient delivery in vitro and in vivo, we developed lipopolymeric nanoplexes (NPXs) encapsulating sgRNA/eGFP-Cas9 RNPs. These NPXs exhibited a particle size of 142.2 nm (polydispersity index: 0.144), a zeta potential of +4.27 mV, and achieved >70% transfection efficiency in HaCaT (human immortalized keratinocyte) cells. Ex vivo skin permeation studies demonstrated 66% of permeation after 24 h. The optimized NPX formulation was incorporated into a Carbopol-based gel, which displayed non-Newtonian, shear-thinning behavior with variable thixotropy and achieved 48% of skin permeation after 24 h. In vivo efficacy assessment in an imiquimod-induced psoriasis model in Swiss albino mice showed significantly improved Psoriasis Area and Severity Index (PASI) scores, reduced epidermal damage, and suppressed keratinocyte proliferation compared to naked RNPs and blank gel controls. Gene editing analysis revealed an indel frequency of 40.7% by T7 endonuclease I assay and 14% by Sanger sequencing. Enhanced cellular uptake, efficient skin permeation and retention, and improved therapeutic efficacy collectively highlight the potential of NPX-mediated CRISPR/Cas9 delivery as a noninvasive strategy for psoriasis treatment.},
}
@article {pmid41171314,
year = {2025},
author = {Zaheer, A and AboQuella, NM and Wadan, AS and Saad, HA and Kumar, D and Panjwani, S and Rath, S and Ahmed, SI},
title = {CRISPR-based gene therapy for huntington's disease: current advances and future prospects.},
journal = {Neurogenetics},
volume = {26},
number = {1},
pages = {76},
pmid = {41171314},
issn = {1364-6753},
mesh = {*Huntington Disease/therapy/genetics ; Humans ; *Genetic Therapy/methods/trends ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Huntingtin Protein/genetics ; Disease Models, Animal ; },
abstract = {CRISPR-Cas9 technology offers transformative potential in treating Huntington's Disease (HD) by directly addressing its genetic root causes. This manuscript explores the pathophysiological mechanisms of HD, characterized by toxic mutant huntingtin (mHTT) protein resulting from expanded CAG repeats in the HTT gene, and the challenges posed by current therapeutic limitations. We comprehensively review the mechanisms of CRISPR-based therapeutic strategies, including excision of expanded repeats, allele-specific targeting, and epigenome editing, highlighting their efficacy in preclinical studies using animal models and human iPSCs. Delivery methods, such as viral and non-viral vectors, are analysed for their role in optimizing therapeutic outcomes while minimizing off-target effects and immune responses. Ethical and safety considerations, especially regarding precision and long-term impacts, are critically examined alongside emerging strategies to enhance specificity. With ongoing clinical trials and advancements in delivery systems, CRISPR technology represents a paradigm shift in addressing HD and broader neurodegenerative conditions. This review underscores the promise of gene editing in overcoming existing barriers and paving the way for transformative therapeutic approaches.},
}
@article {pmid41171707,
year = {2025},
author = {Vadakkethil, AA and Panda, S and Mitra, A and Dash, M and Baig, MJ and Angadi, UB and Kumar, D and Jaiswal, S and Asif Iquebal, M and Molla, KA},
title = {CRISPR-GATE: a one-stop repository and guide to computational resources for genome editing experimentation.},
journal = {Briefings in bioinformatics},
volume = {26},
number = {5},
pages = {},
pmid = {41171707},
issn = {1477-4054},
support = {//Indian Council of Agricultural Research/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Computational Biology/methods ; *Software ; *Databases, Genetic ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Internet ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated protein (CRISPR-Cas) has emerged and evolved as a revolutionary genome editing technology, transforming research across diverse biological disciplines. Over the past decade, this technology has unveiled numerous opportunities for precise genome manipulation. However, the processes of discovering Cas proteins, repurposing them as editing tools, selecting appropriate candidate tool from the CRISPR-toolbox, designing experiments, and analyzing data are often complex and require careful consideration. To support researchers at every stage of CRISPR experimentation, a wide array of web resources has been developed. In this article, we provide a comprehensive overview of standalone and web-based tools that assist in the identification of CRISPR-Cas systems and the design of guide RNAs (gRNAs). We also highlight tools for evaluating gRNA efficiency, predicting CRISPR-Cas9 mutation profiles, as well as tools for base editing and prime editing, and the analysis and visualization of experimental results. Additionally, we introduce CRISPR-Gateway for Accessing Tools and Resources (CRISPR-GATE), an all-inclusive web repository that consolidates publicly available tools for genome editing research. This repository offers a categorized and user-friendly interface, allowing researchers to quickly access relevant tools based on their specific needs. CRISPR-GATE aims to streamline the search for CRISPR resources, facilitating both education and accelerating innovation. The web repository can be accessed from https://crispr-gate.daasbioinfromaticsteam.in/.},
}
@article {pmid41171899,
year = {2025},
author = {Gil-Campillo, C and Mignolet, J and Domínguez-San Pedro, A and Rapún-Araiz, B and Janssen, AB and de Bakker, V and Veening, JW and Garmendia, J},
title = {CRISPRi-seq in Haemophilus influenzae reveals genome-wide and medium-specific growth determinants.},
journal = {PLoS pathogens},
volume = {21},
number = {10},
pages = {e1013650},
pmid = {41171899},
issn = {1553-7374},
mesh = {*Haemophilus influenzae/genetics/growth &amp; development ; *Genome, Bacterial ; *CRISPR-Cas Systems/genetics ; Humans ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Expression Regulation, Bacterial ; Genomics/methods ; },
abstract = {Work in the human pathobiont Haemophilus influenzae has pioneered functional genomics in bacteria such as genome-wide transposon mutagenesis combined with deep sequencing. These approaches unveiled a large set of likely essential genes, but functional studies are hampered due to a limited molecular toolbox. To bridge this gap, we engineered a titratable anhydrotetracycline-inducible CRISPRi (Clustered Regularly Interspaced Short Palindromic Repeats interference) platform for efficient regulation of gene expression in H. influenzae. Genome-wide fitness analyses in two different in vitro culture media by CRISPRi-seq revealed growth medium-dependent fitness cost for a panel of H. influenzae genes. We demonstrated that CRISPRi-programmed fitness defects can be rescuable, and we refined previous Tn-seq based essentialome studies. Finally, we introduce HaemoBrowse, an extensive user-friendly online resource for visual inspection of H. influenzae genome annotations, including sgRNA spacers. The inducible CRISPRi platform described here represents a valuable tool enabling functional genomics and the study of essential genes, thereby contributing to the identification of therapeutic targets for developing drugs and vaccines against H. influenzae.},
}
@article {pmid41171921,
year = {2025},
author = {Zhang, H and You, J and Zhou, H and Zhang, Z and Wu, H and Zhang, D and Pan, X and Zhang, W and Zhang, X and Rao, Z},
title = {The two-component nuclease-active KELShedu system confers broad antiphage activity via abortive infection.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eadv4747},
pmid = {41171921},
issn = {2375-2548},
mesh = {*Escherichia coli/virology/genetics/metabolism ; *Bacteriophages/physiology ; *Escherichia coli Proteins/metabolism/genetics ; *DNA-Binding Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Nucleotides/metabolism ; },
abstract = {Bacteriophages and bacteria engage in a continuous evolutionary arms race, driving the development of intricate bacterial defense systems such as CRISPR-Cas, BREX (Bacteriophage Exclusion), Gabija, and Shedu. Here, we characterize a two-component KELShedu system in Escherichia coli that confers resistance to phages via abortive infection. The KELShedu system comprises KELA, a double-stranded DNA-binding protein, and KELB, a metal ion-dependent nuclease harboring the DUF4263 domain. In addition, we find that physiological levels of nucleotide triphosphates (NTPs) inhibit the DNA cleavage activity of the KELShedu system, suggesting that KELShedu's activation depends on reduced intracellular NTP levels during phage invasion. Our research demonstrates that the KELShedu system responds to nucleotide depletion triggered by phage replication, leading to nonspecific degradation of cellular DNA and ultimately inducing abortive infection. These insights into the KELShedu system expand the repertoire of bacterial antiphage mechanisms and lay the groundwork for applications in microbial engineering and therapeutic development.},
}
@article {pmid41171933,
year = {2025},
author = {Li, S and Vonesch, SC and Roy, KR and Tu, CS and Steudle, F and Nguyen, M and Jann, C and Steinmetz, LM},
title = {The editable landscape of the yeast genome reveals hotspots of structural variant formation.},
journal = {Science advances},
volume = {11},
number = {44},
pages = {eady9875},
pmid = {41171933},
issn = {2375-2548},
support = {R01 HG012446/HG/NHGRI NIH HHS/United States ; },
mesh = {*Genome, Fungal ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; *Saccharomyces cerevisiae/genetics ; *Genomic Structural Variation ; Whole Genome Sequencing ; INDEL Mutation ; },
abstract = {It is unclear how CRISPR editing outcomes vary across the genome and whether undesirable events such as structural variants (SVs) are predictable or preventable. To define a genome-wide map of editability, we performed whole-genome sequencing on 1875 budding yeast clones edited across 16 chromosomes by CRISPR-Cas9 and donor-templated repair. We found that unintended edits, including short indels and SVs, were enriched in specific genomic and sequence contexts. We developed a predictive model, SCORE (System for CRISPR Outcome and Risk Evaluation), which revealed 4.8% of the genome as SV prone, consisting of 562 SV hotspots. Donor repair-enhancing strategies suppressed SV formation in regions with moderate, but not high, predicted risk. Applying SCORE to the Sc2.0 synthetic yeast genome revealed a markedly altered SV landscape due to the removal of endogenous repetitive elements and the insertion of loxP sites. Our study provides the genome-scale map of SV hotspots after CRISPR editing and predictive and experimental tools to mitigate their formation.},
}
@article {pmid41172903,
year = {2026},
author = {Zhang, J and Zhu, M and Yan, H and Qiao, J and Liu, Y},
title = {Split CRISPR/Cas systems: Pioneering solutions for molecular diagnostics challenges.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118177},
doi = {10.1016/j.bios.2025.118177},
pmid = {41172903},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods/instrumentation ; *Pathology, Molecular/methods ; *Molecular Diagnostic Techniques/methods ; *RNA/genetics/analysis ; },
abstract = {Split CRISPR/Cas systems have recently emerged as revolutionary tools in molecular diagnostics, addressing the limitations of conventional CRISPR-based detection methods, such as low sensitivity and specificity for low-abundance targets and limited regulatory flexibility. This review highlights "split-activation" strategies that enhance analytical performance without requiring pre-amplification. It elaborates on two key approaches: split activator-mediated Cas systems for ultrasensitive RNA detection and split crRNA architectures for multiplex nucleic acid analysis. These innovations achieve significant improvements in sensitivity for femtomolar-level biomarkers while maintaining superior single-base discrimination. We examine their applications in clinical and on-site monitoring, analyze current challenges including background noise, multiplex capacity, and cost considerations. Future directions include developing customized Cas variants, nanomaterial-based amplification-free workflows, and integrated microfluidic platforms. This technology holds great promise for precision diagnostics, particularly in resource-limited settings.},
}
@article {pmid41172978,
year = {2025},
author = {Sancho-Shimizu, V},
title = {VUS no more: Decoding inborn errors of immunity for clinical action.},
journal = {Cell},
volume = {188},
number = {22},
pages = {6105-6106},
doi = {10.1016/j.cell.2025.09.009},
pmid = {41172978},
issn = {1097-4172},
mesh = {Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Phenotype ; },
abstract = {Saturation genome editing meets functional phenotyping to turn sequencing ambiguity into actionable diagnoses.},
}
@article {pmid41173524,
year = {2026},
author = {Dai, J and Molloy, EK},
title = {StarCDP: Dynamic Programming Algorithms for Fast and Accurate Cell Lineage Tree Reconstruction from CRISPR-Based Lineage Tracing Data.},
journal = {Journal of computational biology : a journal of computational molecular cell biology},
volume = {33},
number = {1},
pages = {48-66},
doi = {10.1177/15578666251386082},
pmid = {41173524},
issn = {1557-8666},
mesh = {*Cell Lineage/genetics ; *Algorithms ; Animals ; Mice ; Humans ; Phylogeny ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Software ; *Computational Biology/methods ; Lung Neoplasms/genetics/pathology ; Single-Cell Analysis/methods ; Dynamic Programming ; },
abstract = {CRISPR-based lineage tracing, coupled with single-cell RNA sequencing, has emerged as a promising approach for studying development and disease progression at the cellular level. Thus, cell lineage tree (CLT) reconstruction has attracted significant attention in recent years, including the introduction of Star Homoplasy Parsimony (SHP) to model the unique properties of CRISPR-induced mutations, along with the Startle family of methods. However, CLT reconstruction continues to be challenged by technological limitations in producing consistent phylogenetic signals across CLTs. To address these issues, we present Star-CDP, a collection of dynamic programming algorithms that enable researchers to seek, count, sample, and build consensus trees from solutions to SHP within a constrained search space, defined by subsets of cells from which a solution must draw its clades. When using our procedure to construct clade constraints, Star-CDP runs in polynomial time, enabling scalability to larger numbers of cells than Startle-ILP (integer linear programming), the leading method for SHP. In simulations, Star-CDP's strict consensus achieved the same or higher accuracy (f1-score) compared to the leading parsimony methods, with the greatest gains in accuracy occurring when the phylogenetic signal was limited due to the high ratio of cells to mutations. On lineage tracing data from a mouse model of lung adenocarcinoma, Star-CDP's strict consensus achieved the lowest SHP score and comparable numbers of metastatic reseedings compared to PAUP*'s strict consensus and Startle-NNI (nearest neighbor interchange), all benchmarked on a standard data processing pipeline (although our study also revealed that the pipeline can impact relative performance for migrations/reseedings). Star-CDP is available on GitHub: https://github.com/molloy-lab/Star-CDP.},
}
@article {pmid41173724,
year = {2026},
author = {Chen, Y and Duan, Q and Wang, L and He, Q and Fang, L and Xiao, L and Song, H and Cao, Y},
title = {Genome-scale CRISPRi and base-editing libraries for genetic decoding and strain engineering in Shewanella.},
journal = {Trends in biotechnology},
volume = {44},
number = {2},
pages = {496-520},
doi = {10.1016/j.tibtech.2025.10.005},
pmid = {41173724},
issn = {1879-3096},
mesh = {*Shewanella/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Bacterial/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Library ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-based libraries with diverse gene-editing functions, such as gene knockdown and mutation, can significantly accelerate our understanding of complex metabolic networks in microorganisms, particularly for species beyond classical model organisms. Here, three distinct CRISPR-based libraries were designed in the electroactive microorganism Shewanella oneidensis MR-1: a CRISPR interference (CRISPRi) library covering 99.6% of genes in the genome, a protein mutation library focused on genes involved in carbon metabolism, and an inactivation library, with sizes of 30 804, 5963, and 4072 single guide (sg)RNAs, respectively. The principles for the design and construction of libraries were validated, and a conjugation-based library transformation method with high coverage and uniformity was developed. For the first time, we explored the potential essential genes of S. oneidensis MR-1, and expanded the substrate spectrum available for electricity generation, including glucose and chitin. These efforts enable deeper genomic interrogation of Shewanella, and provide a framework for applying genome-scale CRISPR-based tools to other undercharacterized microbial species.},
}
@article {pmid41173850,
year = {2025},
author = {Brown, BC and Tokolyi, A and Morris, JA and Lappalainen, T and Knowles, DA},
title = {Large-scale causal discovery using interventional data sheds light on gene network structure in k562 cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9628},
pmid = {41173850},
issn = {2041-1723},
support = {K99HG012792//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; K99 HG012373/HG/NHGRI NIH HHS/United States ; R01 MH106842/MH/NIMH NIH HHS/United States ; R01 AG057422/AG/NIA NIH HHS/United States ; U01AG068880//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; R01MH106842//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; K99HG012373//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; R01AG057422//U.S. Department of Health &amp; Human Services | NIH | National Institute on Aging (U.S. National Institute on Aging)/ ; U01 AG068880/AG/NIA NIH HHS/United States ; K99 HG012792/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Gene Regulatory Networks ; K562 Cells ; CRISPR-Cas Systems ; },
abstract = {Inference of directed biological networks is an important but notoriously challenging problem. The recent proliferation of large-scale CRISPR perturbation data provides a new opportunity to tackle this problem by leveraging the transcriptional response to the presence of a gene-targeting guide. Here, we introduce inverse sparse regression (inspre), an approach to learning causal networks that leverages large-scale intervention-response data. Applied to 788 genes from the genome-wide perturb-seq dataset, inspre discovers a network with small-world and scale-free properties. We integrate our network estimate with external data, finding relationships between gene eigencentrality and both measures of gene essentiality and gene expression heritability. Our analysis helps to elucidate the structure of networks that may underlie complex traits.},
}
@article {pmid41173880,
year = {2025},
author = {Binenbaum, J and Adamkova, V and Fryer, H and Xu, L and Gorringe, N and Włodzimierz, P and Burns, R and Papikian, A and Jacobsen, SE and Henderson, IR and Harris, CJ},
title = {CRISPR targeting of H3K4me3 activates gene expression and unlocks centromere-proximal crossover recombination in Arabidopsis.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9587},
pmid = {41173880},
issn = {2041-1723},
support = {ERC Starting Grant (TransPlantMemory)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; EP/Z001749/1//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; ERC Advanced Grant (EvoPanCen)//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; EP/X025306/1//RCUK | Engineering and Physical Sciences Research Council (EPSRC)/ ; URF\R1\201016//Royal Society/ ; doctoral studentship//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; BB/V003984/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {*Arabidopsis/genetics/metabolism ; *Arabidopsis Proteins/genetics/metabolism ; *Histones/metabolism/genetics ; *Gene Expression Regulation, Plant ; *Centromere/genetics/metabolism ; *Crossing Over, Genetic ; *CRISPR-Cas Systems/genetics ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; Disease Resistance/genetics ; Meiosis/genetics ; Plants, Genetically Modified ; },
abstract = {H3K4me3 is a fundamental and highly conserved chromatin mark across eukaryotes, playing a central role in many genome-related processes, including transcription, maintenance of cell identity, DNA damage repair, and meiotic recombination. However, identifying the causal function of H3K4me3 in these diverse pathways remains a challenge, and we lack the tools to manipulate it for agricultural benefit. Here we use the CRISPR-based SunTag system to direct H3K4me3 methyltransferases in the model plant, Arabidopsis thaliana. Targeting of SunTag-SDG2 activates the expression of the endogenous reporter gene, FWA. We show that SunTag-SDG2 can be employed to increase pathogen resistance by targeting the H3K4me3-dependent disease resistance gene, SNC1. Meiotic crossover recombination rates impose a limit on the speed with which new traits can be transferred to elite crop varieties. We demonstrate that targeting of SunTag-SDG2 to low recombining centromeric regions can significantly stimulate proximal crossover formation. Finally, we reveal that the effect is not specific to SDG2 and is likely dependent on the H3K4me3 mark itself, as the orthogonal mammalian-derived H3K4me3 methyltransferase, PRDM9, produces a similar effect on gene expression with reduced off-target potential. Overall, our study supports an instructive role for H3K4me3 in transcription and meiotic recombination and opens the door to precise modulation of important agricultural traits.},
}
@article {pmid41174223,
year = {2025},
author = {Liao, Y and Yan, J and Kong, IY and Li, Z and Ding, W and Clark, S and Maestri, D and Yoshida, T and Giulino-Roth, L and Gewurz, BE},
title = {Lysine-specific histone demethylase complex restricts Epstein-Barr virus lytic reactivation.},
journal = {Nature microbiology},
volume = {10},
number = {12},
pages = {3290-3304},
pmid = {41174223},
issn = {2058-5276},
support = {R01 AI164709/AI/NIAID NIH HHS/United States ; U01CA275301//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01DE033907//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 DE033907/DE/NIDCR NIH HHS/United States ; U01 CA275301/CA/NCI NIH HHS/United States ; P01CA269043//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01CA228700//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R01 CA228700/CA/NCI NIH HHS/United States ; R01AI164709//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; P01 CA269043/CA/NCI NIH HHS/United States ; },
mesh = {*Herpesvirus 4, Human/physiology/genetics ; Humans ; *Histone Demethylases/metabolism/genetics ; *Virus Activation ; Animals ; Virus Latency ; Mice ; Epstein-Barr Virus Infections/virology ; Co-Repressor Proteins/metabolism/genetics ; Histones/metabolism ; Lysine/metabolism ; Trans-Activators/metabolism/genetics ; Cell Line, Tumor ; Methylation ; Burkitt Lymphoma/virology ; CRISPR-Cas Systems ; Host-Pathogen Interactions ; Nerve Tissue Proteins ; },
abstract = {Epstein-Barr virus (EBV) infects >95% of adults and contributes to several human cancers. EBV can remain latent where viral lytic genes are silenced, precluding the use of antiviral agents such as ganciclovir. Little is known about the host factors involved in EBV latency. Here we performed a human genome-wide CRISPR-Cas9 screen in Burkitt lymphoma B cells, which identified lysine-specific histone demethylase 1 (LSD1) and its corepressors REST corepressor 1 (CoREST) and zinc finger protein 217 (ZNF217) as critical for EBV latency. Gene knockout or LSD1 inhibition triggered EBV reactivation, and the latter sensitized cells to ganciclovir cytotoxicity, including in murine tumour xenografts. Mechanistically, ZNF217 recruits LSD1 and CoREST to form a complex that binds a specific DNA motif associated with regions implicated in EBV reactivation. It removes histone 3 lysine 4 (H3K4) methylation marks and restricts host DNA looping. Alternatively, the H3K4 lysine methyltransferase 2D supports EBV lytic reactivation. Our results highlight H3K4 methylation as a major EBV lytic switch regulator and therapeutic target.},
}
@article {pmid41174319,
year = {2026},
author = {Fatmi, MQ and Nadeem, A and Abbasov, M and Sajjad, M},
title = {Computational Methods to Engineer Cas Proteins for Efficient Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2979},
number = {},
pages = {279-300},
pmid = {41174319},
issn = {1940-6029},
mesh = {*Gene Editing/methods ; Molecular Dynamics Simulation ; *CRISPR-Cas Systems/genetics ; *Protein Engineering/methods ; *Computational Biology/methods ; Mutation ; *CRISPR-Associated Proteins/genetics/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) system has revolutionized genome editing through programmable, sequence-specific deoxyribonucleic acid (DNA) targeting. Yet, its broader application remains limited by off-target effects and context-dependent efficiency. To address these challenges, we present an integrated computational protocol with easy-to-do steps for researchers to guide the rational design of CRISPR/Cas variants with improved stability and specificity. The integrated workflow begins with coevolutionary coupling analysis to identify conserved and covarying residues critical for function. These residues are then evaluated for energetically favorable substitutions through mutant stability prediction, followed by network centrality analysis to evaluate the impact of mutations on intramolecular communication pathways, preserving key allosteric interactions. Finally, molecular dynamics (MD) simulations validate the structural integrity and dynamic behavior of the selected variants. Network analysis and molecular dynamics (MD) simulations are applied iteratively, allowing insights from MD to refine network-based evaluations and vice versa. This multiscale strategy offers a streamlined and systematic approach for engineering optimized Cas proteins for genome editing applications.},
}
@article {pmid41174869,
year = {2025},
author = {Li, S and Zhang, B and Ma, P and Zhang, Y and Hu, Z and Wu, X and Chen, Q and Zhao, Y},
title = {The 13-lipoxygenase GmLOX6 is involved in JA biosynthesis and serves as a positive regulator of salt stress tolerance in soybean.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {3},
pages = {e70550},
pmid = {41174869},
issn = {1365-313X},
support = {2023ZD04036//Biological Breeding-National Science and Technology Major Project/ ; 2021YFD1201104-02-02//National Key Research and Development Program of China/ ; 2023YFD2300101//National Key Research and Development Program of China/ ; 31971899//National Natural Science Foundation of China/ ; 32272072//National Natural Science Foundation of China/ ; 32272093//National Natural Science Foundation of China/ ; U23A20192//National Natural Science Foundation of China/ ; },
mesh = {*Glycine max/genetics/physiology/enzymology/metabolism ; *Lipoxygenase/metabolism/genetics ; *Oxylipins/metabolism ; *Cyclopentanes/metabolism ; *Salt Tolerance/genetics/physiology ; *Plant Proteins/metabolism/genetics ; Salt Stress ; Gene Expression Regulation, Plant ; Reactive Oxygen Species/metabolism ; Plant Growth Regulators/metabolism ; Plants, Genetically Modified ; alpha-Linolenic Acid/metabolism ; CRISPR-Cas Systems ; },
abstract = {Salinity represents a major abiotic stressor that significantly impairs soybean growth and yield. Although jasmonic acid (JA) has been firmly established as a key regulator of plant defense against salt stress, the precise functions of lipoxygenase (LOX) genes responsible for initiating JA biosynthesis remain poorly defined. Here, a comprehensive genome-wide analysis of the soybean LOX gene family was performed, and a detailed functional characterization of GmLOX6 was carried out. Subcellular localization confirmed that GmLOX6 is targeted to chloroplasts, while enzymatic assays demonstrated that it acts as a 13-LOX enzyme with a strong preference for α-linolenic acid as substrate. To clarify its role under salt stress, we generated both overexpression and CRISPR/Cas9-mediated knockout lines of soybean. Phenotypic and molecular evaluations revealed that GmLOX6 facilitates JA production under salt stress, thereby contributing to enhanced JA accumulation. This elevation in JA levels was associated with improved salt tolerance through multiple physiological adaptations, including the activation of antioxidant enzymes for the detoxification of reactive oxygen species (ROS), enhanced Na[+] extrusion to preserve ionic balance, and reinforced membrane stability. Moreover, GmRWP-RK11 was identified as a transcriptional repressor of GmLOX6. Functional disruption of GmRWP-RK11 via CRISPR/Cas9 conferred greater salt tolerance, further supporting its negative regulatory role. Collectively, these findings uncover a novel regulatory axis in which GmLOX6-mediated JA biosynthesis enhances soybean resistance to salinity through modulation of ROS homeostasis and Na[+] transport. These insights provide an expanded understanding of the transcriptional and biochemical mechanisms underpinning JA-driven stress adaptation in soybean.},
}
@article {pmid41174881,
year = {2026},
author = {Hierl, M and Bischof, J and Liemberger, B and Kocher, T and Hainzl, S and Gruner, S and Trattner, L and Reichl, V and Klausegger, A and Aussel, C and Ammann, S and Raab, R and Andrieux, G and Wolf, M and Hochmann, S and Ebner, P and Wally, V and Zauner, R and Guttmann-Gruber, C and Gratz, IK and Hofbauer, JP and Cathomen, T and Strunk, D and Bauer, JW and Koller, U},
title = {Prime editing as a promising therapeutic strategy for junctional epidermolysis bullosa.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {2},
pages = {817-831},
pmid = {41174881},
issn = {1525-0024},
mesh = {*Epidermolysis Bullosa, Junctional/therapy/genetics ; Humans ; *Gene Editing/methods ; Collagen Type XVII ; *Non-Fibrillar Collagens/genetics/metabolism ; Keratinocytes/metabolism ; Animals ; Mice ; *Genetic Therapy/methods ; *Autoantigens/genetics/metabolism ; Disease Models, Animal ; CRISPR-Cas Systems ; },
abstract = {Gene therapies offer new possibilities for the precise correction of monogenic disorders. Here, we present the first prime editing (PE)-based gene repair strategy for pathogenic COL17A1 variants that cause junctional epidermolysis bullosa (JEB). Type XVII collagen (C17), encoded by COL17A1, plays a critical role in skin aging, regeneration, and the maintenance of epidermal stem cell integrity. Treatment of primary human JEB keratinocytes with PE mRNAs resulted in COL17A1 editing efficiencies of up to 60% in bulk-treated cells, leading to the restoration of full-length, accurately shed C17. Chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing analysis of gene-edited JEB keratinocytes confirmed the absence of unintended chromosomal rearrangements at potential off-target sites and only minimal on-target aberrations. Remarkably, in a xenograft model, in which C17[+] cells represented only 55.9% of the input population, COL17A1-corrected cells populated 92.2% of the basal keratinocyte layer in the resulting skin grafts after 6 weeks. These observations highlight a potential selective advantage imparted by C17 restoration, in line with its canonical role in anchoring hemidesmosomes to the basement membrane and preserving the structural integrity of the interfollicular epidermal stem cell niche. Based on our results, we envision PE as an efficient and safe option to restore gene function in EB and other genodermatoses.},
}
@article {pmid41175344,
year = {2025},
author = {Shamloo, S and Schloßhauer, JL and Tiwari, S and Denise Fischer, K and Almolla, O and Ghebrechristos, Y and Kratzenberg, L and Bejoy, AM and Aifantis, I and Boccalatte, F and Wang, E and Imig, J},
title = {RNA binding of GAPDH controls transcript stability and protein translation in acute myeloid leukemia.},
journal = {RNA biology},
volume = {22},
number = {1},
pages = {1-23},
pmid = {41175344},
issn = {1555-8584},
support = {R01 HL159175/HL/NHLBI NIH HHS/United States ; R01 CA242020/CA/NCI NIH HHS/United States ; R01 CA271455/CA/NCI NIH HHS/United States ; R01 CA228135/CA/NCI NIH HHS/United States ; P30 CA034196/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/metabolism/pathology ; *Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism/genetics ; *Protein Biosynthesis ; *RNA, Messenger/genetics/metabolism ; RNA-Binding Proteins/metabolism/genetics ; 5' Untranslated Regions ; Cell Line, Tumor ; *RNA Stability ; Protein Binding ; Gene Expression Regulation, Leukemic ; Cell Proliferation ; Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) ; },
abstract = {Dysregulation of RNA binding proteins (RBPs) is a hallmark in cancerous cells. In acute myeloid leukaemia (AML) RBPs are key regulators of tumour proliferation. While classical RBPs have defined RNA binding domains, RNA recognition and function in AML by non-canonical RBPs (ncRBPs) remain unclear. Given the inherent complexity of targeting AML broadly, our goal was to uncover potential ncRBP candidates critical for AML survival using a CRISPR/Cas-based screening. We identified the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a pro-proliferative factor in AML cells. Based on cross-linking and immunoprecipitation (CLIP), we are defining the global targetome, detecting novel RNA targets mainly located within 5'UTRs, including GAPDH, RPL13a, and PKM. The knockdown of GAPDH unveiled genetic pathways related to ribosome biogenesis, translation initiation, and regulation. Moreover, we demonstrated a stabilizing effect through GAPDH binding to target transcripts including its own mRNA. The present findings provide new insights on the RNA functions and characteristics of GAPDH in AML.},
}
@article {pmid41175692,
year = {2026},
author = {Hao, X and Qin, C and He, C and Yuan, Q and Yang, P and Wen, Y and Wu, Y and Cui, R and Zhang, Y and Chen, W and Liu, Y and Ramadan, S and Ying, Y and Li, D and Xu, L},
title = {In situ detection of bacteria from skin interstitial fluid via CRISPR microneedles: An amplification-free platform for point-of-care diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118123},
doi = {10.1016/j.bios.2025.118123},
pmid = {41175692},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Humans ; *Skin/microbiology ; *Extracellular Fluid/microbiology ; CRISPR-Cas Systems ; Gold/chemistry ; Graphite/chemistry ; Limit of Detection ; *DNA, Bacterial/genetics/isolation &amp; purification ; Wearable Electronic Devices ; Needles ; Point-of-Care Systems ; *Staphylococcal Infections/microbiology/diagnosis ; },
abstract = {We report a CRISPR/Cas12a-functionalized microneedle (MN) biosensor for in situ, amplification-free detection of pathogenic bacteria directly from skin interstitial fluid. The platform integrates conductive poly(styrene)/gold/graphene oxide MNs with a minimized electrochemical signal transducer, enabling real-time and highly specific sensing of bacterial DNA through CRISPR-mediated cleavage of ferrocene-labeled ssDNA reporters. Using Staphylococcus aureus as a model target, this strategy achieves direct detection limits of 0.69 pM in ex situ assays and 6.3 pM in situ, without the need for target amplification. The sensor also detects bacterial loads as low as 4.27 × 10[5] CFU/mL in interstitial fluid, which is below typical clinical thresholds. Engineered for biocompatibility and minimal invasiveness, the MNs effectively access interstitial fluid, capture targets and produce quantifiable electrochemical signals within 1 h. Paired with a portable reader and smartphone interface, this wearable system offers a user-friendly tool for point-of-care diagnostics and personalized infectious disease monitoring.},
}
@article {pmid41175865,
year = {2025},
author = {Ahrens-Nicklas, RC and Musunuru, K},
title = {How to create personalized gene editing platforms: Next steps toward interventional genetics.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2826-2829},
doi = {10.1016/j.ajhg.2025.10.006},
pmid = {41175865},
issn = {1537-6605},
support = {U01 TR005355/TR/NCATS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods/trends ; *Precision Medicine/methods/trends ; *Genetic Therapy/methods/trends ; CRISPR-Cas Systems/genetics ; },
abstract = {How do we go from a single individual receiving a personalized gene-editing therapy to a future of "interventional genetics" in which such therapies are the standard of care? First and foremost: regulatory innovation.},
}
@article {pmid41176802,
year = {2025},
author = {Kalinina, NO and Spechenkova, NA and Taliansky, ME},
title = {Erratum to: Biotechnological Approaches to Plant Antiviral Resistance: CRISPR-Cas or RNA Interference?.},
journal = {Biochemistry. Biokhimiia},
volume = {90},
number = {10},
pages = {1450},
doi = {10.1134/S0006297925100013},
pmid = {41176802},
issn = {1608-3040},
}
@article {pmid41176953,
year = {2026},
author = {Lankireddy, SV and Lekkala, S and Khadgi, A and Sripathi, VR and Janga, MR},
title = {Molecular biology of Cotton Leafroll Dwarf Virus (CLRDV) and potential application of CRISPR-Cas technology for developing virus-resistant cotton.},
journal = {Virology},
volume = {614},
number = {},
pages = {110730},
doi = {10.1016/j.virol.2025.110730},
pmid = {41176953},
issn = {1096-0341},
mesh = {*Gossypium/virology/genetics ; *Plant Diseases/virology ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; Aphids/virology ; Animals ; },
abstract = {Cotton leafroll dwarf virus (CLRDV) poses an increasing threat to global cotton production. Transmitted by the cotton aphid (Aphis gossypii) in a persistent, circulative manner, CLRDV exhibits a wide geographical distribution, with documented presence in South America, Africa, Asia, and the USA. Infection can result in either cotton blue disease (CBD) in South America or cotton leafroll dwarf disease (CLRDD) in the USA, both of which are associated with CLRDV. The considerable genetic diversity and frequent recombination events within CLRDV populations contribute to this symptom variability and complicate both diagnosis and management. While resistant cultivars have reduced disease impact in South America, these lines remain susceptible to emerging US strains, underscoring the urgent need for region-specific resistance breeding. Current molecular diagnostics rely on RT-PCR, but there is a need for rapid, field-deployable detection tools. Recent advances, such as CRISPR-Cas13a based SHERLOCK assays, offer sensitive and specific detection of CLRDV, with potential for on-site applications. Efficient screening techniques, supported by next-generation sequencing and transcriptomics, are essential for identifying novel resistance sources and elucidating virus-host interactions. CRISPR-based genome editing holds significant promise, as demonstrated in other crops. Targeted disruption of host susceptibility genes using CRISPR-Cas9, or direct degradation of viral genomes with RNA-targeting systems such as Cas12/Cas13, could offer durable, broad-spectrum resistance. By integrating molecular virology, high-throughput genomics, and precision gene editing, this review outlines a roadmap for translating these advances into sustainable, field-level solutions for CLRDV management and long-term cotton productivity.},
}
@article {pmid41177179,
year = {2026},
author = {Bacci, L and Pollutri, D and Ripa, IJ and D'Andrea, M and Marchand, V and Motorin, Y and Hesse, AM and Couté, Y and Filipek, K and Penzo, M},
title = {Ribosomal protein L5 (RPL5/uL18) I60V mutation is associated to increased translation and modulates drug sensitivity in T-cell acute lymphoblastic leukemia cells.},
journal = {Biochemical pharmacology},
volume = {243},
number = {Pt 1},
pages = {117497},
doi = {10.1016/j.bcp.2025.117497},
pmid = {41177179},
issn = {1873-2968},
mesh = {Humans ; *Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics/drug therapy/metabolism ; *Ribosomal Proteins/genetics/metabolism ; Jurkat Cells ; *Protein Biosynthesis/drug effects/physiology/genetics ; *Mutation ; *Antineoplastic Agents/pharmacology ; *Drug Resistance, Neoplasm/genetics/drug effects ; Dose-Response Relationship, Drug ; Cell Proliferation/drug effects ; },
abstract = {Somatic mutations in ribosomal proteins (RPs), including RPL5, have been reported in approximately 10 % of pediatric patients with T-cell acute lymphoblastic leukemia (T-ALL). In cancer, the incorporation of mutant RPs into ribosomes often disrupts canonical ribosome function, thereby contributing to disease development. In this study, we aimed to characterize the effects of the RPL5-I60V mutation in the context of T-ALL, focusing on its impact on translation and cellular responses to a panel of compounds in vitro. Using CRISPR-Cas9, we generated a homozygous knock-in mutant in Jurkat cells and investigated its effects on ribosome biogenesis. We observed both quantitative and qualitative alterations in the production of the large ribosomal subunit. Ribosomes containing the mutant RPL5 protein exhibited intrinsically increased protein synthesis activity, which correlated with enhanced cellular proliferation. We then evaluated the response of these mutant cells to a panel of compounds targeting protein synthesis at various levels-including an MNK1 inhibitor, metformin, silvestrol, homoharringtonine, anisomycin, resveratrol, and hygromycin B-as well as cytarabine, a chemotherapeutic agent commonly used in T-ALL treatment. Our results showed that the RPL5-I60V mutation confers increased sensitivity to most of these compounds, with the exception of hygromycin B. This study advances our understanding of how oncoribosomes contribute to cancer pathogenesis and highlights the therapeutic potential of directly or indirectly targeting altered ribosomes, offering insights for the development of personalized treatment strategies.},
}
@article {pmid41177337,
year = {2025},
author = {Liu, B and Li, Y and Yang, Z and Wu, J and Jiang, Y and Zhao, L and Ge, J},
title = {A rapid and visual detection for canine Adenovirus-2 using CRISPR-Cas13a-based SHERLOCK technology.},
journal = {Journal of microbiological methods},
volume = {239},
number = {},
pages = {107314},
doi = {10.1016/j.mimet.2025.107314},
pmid = {41177337},
issn = {1872-8359},
mesh = {Animals ; Dogs ; *CRISPR-Cas Systems/genetics ; *Adenoviridae Infections/diagnosis/veterinary/virology ; *Adenoviruses, Canine/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *Dog Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Canine adenovirus type 2 (CAdV-2) is an important pathogen causing infectious tracheobronchitis (ITB) and viral enteritis in puppies, often exacerbating clinical symptoms through co-infection with other viruses. However, existing diagnostic methods for CAdV-2 exhibit notable limitations. Specifically, they are time-consuming, require additional nucleic acid purification steps, depend on expensive detection equipment, and necessitate operation by professional personnel. Collectively, these limitations prevent the achievement of rapid and accurate CAdV-2 detection in resource-limited settings. In this study, we established a novel CAdV-2 detection method by integrating CRISPR/Cas13a collateral cleavage activity with HUDSON rapid nucleic acid extraction, recombinase-aided amplification (RAA), and a lateral flow strip. This isothermal assay allows for visual, naked-eye result interpretation and achieves a sensitivity of 10[2] copies/μL as read by lateral flow strips (corresponding to approximately 750 copies per reaction). It showed excellent specificity with no cross-reactivity observed against five other major canine viruses. When tested on 20 clinical samples, the assay demonstrated a 95 % concordance rate with the conventional simplex PCR results. The entire detection process is simple to perform, requires only basic equipment, and delivers results within 90 min. The developed CRISPR/Cas13a-based detection method exhibits significant application potential for CAdV-2 detection. This study develops a CRISPR/Cas13a-based point-of-care diagnostic tool for CAdV-2, delivering rapid, sensitive, and visual detection that significantly facilitates field-based pathogen surveillance and control efforts, while advancing the application of CRISPR diagnostics in veterinary infectious diseases.},
}
@article {pmid41177343,
year = {2025},
author = {Rahimian, M and Panahi, B},
title = {Genomic insights into Erwinia amylovora prophages: Diversity, defense strategies, and phage-host coevolution.},
journal = {Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases},
volume = {136},
number = {},
pages = {105846},
doi = {10.1016/j.meegid.2025.105846},
pmid = {41177343},
issn = {1567-7257},
mesh = {*Erwinia amylovora/virology/genetics ; *Prophages/genetics ; Phylogeny ; Genomics/methods ; Plant Diseases/microbiology ; *Genome, Viral ; Host-Pathogen Interactions ; Genome, Bacterial ; },
abstract = {Erwinia amylovora, the causative agent of fire blight in Rosaceae plants like apple and pear, is a major agricultural pathogen responsible for significant economic losses. This gram-negative phytopathogen has the potential to acquire antibiotic resistance and virulence genes, a situation that becomes more severe and restricts treatment options. Using an in silico approach, we analyzed 268 E. amylovora genomes and identified seven high-quality temperate prophages, all classified under Caudoviricetes, with average genome sizes of 44.2 kbp and 51 % GC content. These prophages exhibited unique genomic features, including tRNA genes (Ph-Ea644), anti-defense systems like ardc (Ph-Ea6-96), and regulatory/lysis genes (Ph-EaFC01). Comparative genomics and phylogenetic analyses grouped them into five clades, with Ph-Ea4-96, Ph-Ea3-97, and Ph-Ea2-97 being genetically identical. Functional annotation revealed streptomycin resistance genes and a CAZyme (GH23) in Ph-Ea7-3, virulence factors (e.g., alginate biosynthesis proteins), and six auxiliary metabolic genes (AMGs) linked to metabolic adaptation. Additionally, Ph-Ea644 encoded a cell wall-binding receptor protein. The prophages also carried defense systems (Gabija, CBASS) and 31 anti-CRISPR proteins (ACRs), suggesting evasion of host immunity. CRISPR-Cas analysis indicated fewer arrays and spacers in prophage-containing strains, underscoring CRISPR's role in lysogeny resistance. These findings highlight the genomic plasticity of E. amylovora prophages, their interactions with bacterial defenses, and their potential influence on pathogen evolution. This study enhances understanding of temperate phages in agricultural pathogens and underscores challenges in phage-based biocontrol strategies.},
}
@article {pmid41178318,
year = {2026},
author = {Leung, CY and Wimmer, EA and Ahmed, HMM},
title = {Synthetic biology approaches to generate temperature-sensitive alleles for the Sterile Insect Technique.},
journal = {Insect science},
volume = {33},
number = {2},
pages = {517-532},
pmid = {41178318},
issn = {1744-7917},
support = {ResearchcooperationLowerSaxony-Israel//Nieders&#xe4;chsisches Ministerium f&#xfc;r Wissenschaft und Kultur/ ; Project D44003//International Atomic Energy Agency/ ; },
mesh = {Animals ; *Synthetic Biology/methods ; Alleles ; Temperature ; Male ; *Pest Control, Biological/methods ; Female ; *Insecta/genetics/physiology ; },
abstract = {The Sterile Insect Technique (SIT) is an environmentally friendly, sustainable pest control approach, which uses large-scale releases of sterile insects to suppress or eradicate target populations through infertile matings. The efficiency of SIT is enhanced by male-only releases requiring genetic sexing strains (GSSs) that are classically based on selectable recessive visible markers or temperature-sensitive lethal (tsl) mutations and a rescue by a wild-type allele translocated to the male-determining chromosome. The transfer of identified or designed temperature-sensitive alleles might allow the generation of neoclassical GSSs in additional SIT target species. By using precise genome-editing tools, such as CRISPR/Cas, the creation of specific mutations in target genes and the integration of a wild-type copy is feasible without the introduction of foreign DNA. This might ease regulation of neoclassical GSSs, since they are not considered transgenic. However, integration and expression of genes at male-determining loci or chromosomes is not reliably established. Therefore, additional strategies to link temperature-sensitive phenotypes to female development are required, which could be achieved by targeting genes involved in dosage compensation or sex determination. To create temperature-sensitive alleles, rational protein design using advanced modeling and prediction tools to evaluate and tailor the effect of mutations on protein stability and temperature sensitivity can be used. In addition, emerging synthetic biology strategies such as temperature-inducible N-degrons or temperature-sensitive inteins provide powerful tools to generate temperature sensitivity. Such approaches should enable conditional control over proteins causing female lethality or sex conversion and therefore promise straightforward generic approaches to generate GSSs for male-only production in SIT target species.},
}
@article {pmid41178406,
year = {2025},
author = {Pei, C and Yan, B and Wang, Y and Chen, T and Du, K and Ma, L and Wang, J},
title = {TdT/Cas12a cascade amplification biosensor for sensitive ALP activity detection.},
journal = {The Analyst},
volume = {150},
number = {23},
pages = {5330-5337},
doi = {10.1039/d5an00938c},
pmid = {41178406},
issn = {1364-5528},
mesh = {Humans ; *Biosensing Techniques/methods ; *Alkaline Phosphatase/metabolism/analysis ; HeLa Cells ; CRISPR-Cas Systems ; Limit of Detection ; *DNA Nucleotidylexotransferase/metabolism/chemistry ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {This study presents a novel biosensor based on TdT and CRISPR-Cas12a, which integrates the catalytic activity of terminal deoxynucleotidyl transferase (TdT) with the trans-cleavage property of CRISPR-Cas12a to achieve ultra-sensitive biomolecular detection. The biosensor exhibited a broad linear detection range from 0 to 0.2 U L[-1] and a remarkably low detection limit of 1.7 × 10[-3] U L[-1], demonstrating high specificity and sensitivity. In practical validation, the biosensor successfully quantified alkaline phosphatase (ALP) activity in both cervical cancer cells and HeLa cell lysates, even at a dilution factor of up to 10[6]-fold. Its sensitivity allowed precise detection at the single-cell level. This technology offers a robust, simple, and cost-effective platform for cancer diagnosis, treatment monitoring, and enzyme inhibitor screening, while maintaining excellent detection performance in complex biological samples. This breakthrough establishes a foundation for serological tumor screening and early disease diagnosis, while also opening new avenues for enhanced cancer management and clinical translation, indicating significant potential in translational medicine.},
}
@article {pmid41178730,
year = {2025},
author = {Avaro, AS and Santiago, JG},
title = {Engineering guidelines for CRISPR diagnostics.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {95},
pages = {18756-18772},
doi = {10.1039/d5cc04206b},
pmid = {41178730},
issn = {1364-548X},
mesh = {*CRISPR-Cas Systems/genetics ; Kinetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; },
abstract = {This Feature Article reviews engineering guidelines for the design of CRISPR assays, including experimentally validated theoretical models and recommendations for experimental research practice and reporting. First, the state of the art of CRISPR kinetics studies is reviewed. Then presented is a summary of the existence and persistence of widespread gross errors in reports of kinetic rate constants of CRISPR-Cas enzymes, as well as the fact that many CRISPR studies provide insufficient data to check for consistency or assess calibration. Proper experimental procedures including signal calibration are critical to the assessment, design, and future development of CRISPR kinetics assays and CRISPR diagnostics. This review then presents guidelines for the calibration of fluorescence-based CRISPR assays. Fluorescence is the most common detection modality, and incorrect calibration is implicated in high-profile, gross errors in the field. Also presented is a review of enzymatic kinetic rates and reporter molecule degradation as the major factor limiting CRISPR assay sensitivity. Lastly, progress in, and criticism of, microfluidic applications of CRISPR assays is summarized.},
}
@article {pmid41182029,
year = {2025},
author = {Ashworth, KE and Zhang, J and D'Amata, C and Héon, E and Ballios, BG},
title = {USH2A-Mutated Human Retinal Organoids Model Rod-Cone Dystrophy.},
journal = {Investigative ophthalmology &amp; visual science},
volume = {66},
number = {14},
pages = {2},
pmid = {41182029},
issn = {1552-5783},
mesh = {Humans ; *Organoids/pathology/metabolism ; Induced Pluripotent Stem Cells/pathology/metabolism ; *Mutation ; *Extracellular Matrix Proteins/genetics/metabolism ; *Cone-Rod Dystrophies/genetics/pathology/metabolism ; Retinal Rod Photoreceptor Cells/pathology ; *Retinitis Pigmentosa/genetics/pathology ; CRISPR-Cas Systems ; },
abstract = {PURPOSE: USH2A mutations are the leading cause of autosomal recessive retinitis pigmentosa (RP), a progressive blinding disease marked by photoreceptor degeneration. Animal models fail to recapitulate the features of USH2A RP seen in humans, and its earliest pathogenic events remain unknown. Here, we established a human model of USH2A RP using retinal organoids derived from patient induced pluripotent stem cells and CRISPR-Cas9-engineered isogenic-USH2A-/- induced pluripotent stem cells.<br><br>METHODS: We assessed organoids for cellular, molecular, and morphological defects using serial live imaging and whole organoid and fixed section analyses.<br><br>RESULTS: Both patient-derived and isogenic-USH2A-/- organoids showed preferential rod photoreceptor loss followed by widespread degeneration, consistent with the clinical phenotype. Additionally, isogenic-USH2A-/- organoids showed early defects in proliferation and structure.<br><br>CONCLUSIONS: Our findings suggest that molecular changes precede overt photoreceptor loss in USH2A RP, and pathogenesis may begin before clinical symptoms emerge. By defining early and late disease features, we provide new insight on the developmental origins of USH2A RP to guide therapeutic strategies.},
}
@article {pmid41182611,
year = {2026},
author = {Chiurillo, MA and Ahmed, M and González, C and Rosón, JN and Das, A and Lander, N},
title = {Cloning-Free Genome Editing by CRISPR/T7RNAP/Cas9 in Trypanosoma cruzi.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2982},
number = {},
pages = {59-76},
pmid = {41182611},
issn = {1940-6029},
support = {23IPA1054779/AHA/American Heart Association-American Stroke Association/United States ; R00 AI137322/AI/NIAID NIH HHS/United States ; R21 AI178573/AI/NIAID NIH HHS/United States ; },
mesh = {*Trypanosoma cruzi/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *DNA-Directed RNA Polymerases/genetics ; *Viral Proteins/genetics ; Cloning, Molecular ; Humans ; *Genome, Protozoan ; Gene Knockout Techniques ; },
abstract = {The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 technology for genome editing in this organism. Initially, the system was successfully used for gene knockout and endogenous C-terminal tagging in T. cruzi. Recently, an updated version of this technology has been used for gene complementation, site-directed mutagenesis, and N-terminal tagging in trypanosomatids. This cloning-free strategy, called CRISPR/T7RNAP/Cas9, is extremely useful for identifying essential genes when null mutants are not viable. Mutant cell lines obtained by this new system have been used for the functional characterization of proteins in different developmental stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter, we describe the methodology to achieve genome editing by CRISPR/T7RNAP/Cas9 in T. cruzi. Our method involves the generation of T. cruzi epimastigotes that constitutively express the T7 RNA polymerase (T7RNAP) and SpCas9, and their co-transfection with an sgRNA template and donor DNA(s) as polymerase chain reaction (PCR) products. Using this strategy, we have generated genetically modified parasites in 2-3 weeks without the need for gene cloning, cell sorting, or having to perform several transfection attempts to verify the sgRNA efficiency for targeting the gene of interest. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines, and endogenous (N- or C-terminal) tagging in T. cruzi.},
}
@article {pmid41182905,
year = {2025},
author = {Lamb, CH and Riesle-Sbarbaro, S and Prescott, JB and Te Velthuis, AJW and Myhrvold, C and Nilsson-Payant, BE},
title = {Amplification-free detection of zoonotic viruses using Cas13 and multiple CRISPR RNAs.},
journal = {The Journal of general virology},
volume = {106},
number = {11},
pages = {},
pmid = {41182905},
issn = {1465-2099},
support = {DP2 AI175474/AI/NIAID NIH HHS/United States ; R01 AI182281/AI/NIAID NIH HHS/United States ; T32 GM007388/GM/NIGMS NIH HHS/United States ; UL1 TR003017/TR/NCATS NIH HHS/United States ; },
mesh = {Animals ; *RNA, Viral/genetics ; Humans ; *Influenza A virus/genetics/isolation &amp; purification ; Sensitivity and Specificity ; *Orthohantavirus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; Clustered Regularly Interspaced Short Palindromic Repeats ; *Zoonoses/virology/diagnosis ; },
abstract = {Zoonotic viruses such as hantaviruses and influenza A viruses present a threat to humans and livestock. There is thus a need for methods that are rapid, sensitive and relatively cheap to detect infections with these pathogens early. Here, we use an amplification-free clustered regularly interspaced short palindromic repeats-associated protein 13 (CRISPR-Cas13)-based assay, which is simple, cheap and field-deployable, to detect the presence or absence of genomic hantavirus or influenza A virus RNA. In addition, we evaluate whether the use of multiple CRISPR RNAs (crRNAs) can improve the sensitivity of this amplification-free method. We demonstrate that for the hantaviruses Tula virus (TULV) and Andes virus (ANDV), a combination of two or three crRNAs provides the best sensitivity for detecting viral RNA, whereas for influenza virus RNA detection, additional crRNAs provide no consistent benefit. We also show that the amplification-free method can be used to detect TULV and ANDV RNA in tissue culture infection samples, ANDV from hamster lung samples and influenza A virus RNA in clinical nasopharyngeal swabs. In clinical samples, the Cas13 assay has an 85% agreement with RT-qPCR for identifying a positive sample. Overall, these findings indicate that amplification-free CRISPR-Cas13 detection of viral RNA has potential as a tool for rapidly detecting zoonotic virus infections.},
}
@article {pmid41182907,
year = {2025},
author = {Pantoja-Alonso, MA and Camas-Reyes, JA and Cano-Segura, R and Cárdenas-Aquino, MDR and Martínez-Antonio, A},
title = {A comprehensive review of genomic-scale genetic engineering as a strategy to improve bacterial productivity.},
journal = {Microbiology (Reading, England)},
volume = {171},
number = {11},
pages = {},
pmid = {41182907},
issn = {1465-2080},
mesh = {*Bacteria/genetics/metabolism ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; *Genome, Bacterial ; Genomics/methods ; Gene Editing ; Metabolic Engineering/methods ; Synthetic Biology/methods ; },
abstract = {Bacterial genome engineering has evolved to provide increasingly precise, robust and rapid tools, driving the development and optimization of bacterial production of numerous compounds. The field has progressed from early random mutagenesis methods, labour-intensive and inefficient, to rational and multiplexed strategies enabled by advances in genomics and synthetic biology. Among these tools, CRISPR/Cas has stood out for its versatility and its ability to achieve precision levels ranging from 50% to 90%, compared to the 10-40% obtained with earlier techniques, thereby enabling remarkable improvements in bacterial productivity. Nevertheless, like its predecessors, it still demands continuous refinement to reach full maturity. In this context, the present review addresses the lack of a unified overview by summarizing historical milestones and practical applications of genomic engineering tools in bacteria. It integrates diverse approaches to provide a comprehensive perspective on the evolution and prospects of these fundamental biotechnological tools.},
}
@article {pmid41183015,
year = {2025},
author = {Keuthen, H and Pozhydaieva, N and Höfer, K},
title = {Precise Phage Mutagenesis with NgTET-Assisted CRISPR-Cas Systems.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69022},
pmid = {41183015},
issn = {1940-087X},
mesh = {*CRISPR-Cas Systems/genetics ; *Bacteriophages/genetics ; *Mixed Function Oxygenases/genetics/metabolism ; Mutagenesis ; DNA, Viral/genetics/chemistry ; *Mutagenesis, Site-Directed/methods ; },
abstract = {Bacteriophages, viruses that specifically target their bacterial hosts, hold significant potential for biotechnology and medicine, especially in combating multidrug-resistant infections. However, the molecular mechanisms underlying phage infection remain largely underexplored. Precise, site-specific mutagenesis of phages is a powerful tool to elucidate gene functions and phage-host interactions. However, a major challenge in phage genome mutagenesis is the presence of phage DNA modifications that interfere with conventional genome editing tools like CRISPR-Cas. While CRISPR-Cas systems have been used successfully for targeted mutagenesis in various organisms, their effectiveness in phage mutagenesis is often limited by DNA modifications such as cytosine glycosylation. To overcome this barrier, we developed an efficient method that temporarily reduces the abundance of phage DNA modifications, enabling efficient CRISPR-Cas targeting and precise mutation introduction into phage genomes. Specifically, we use the Ten Eleven Translocation (TET) methylcytosine dioxygenase from Naegleria gruberi (NgTET), which iteratively demodifies methylated and hydroxymethylated cytosines in DNA. By oxidizing hydroxymethylated cytosines within phage DNA, NgTET prevents subsequent cytosine modification like glycosylation and significantly enhances the efficiency of Cas-mediated DNA cleavage. In conclusion, the scarless and precise genome-editing approach presented here enables the efficient introduction of point mutations while maintaining the native gene architecture in phage genomes. By preserving intact transcriptional and translational frameworks, this method minimizes unintended disruptions to complex regulatory networks. This is particularly important for investigating essential or multifunctional phage proteins. The ability to generate targeted genetic modifications without introducing extraneous sequences significantly expands the experimental toolkit for phage biology. This strategy not only facilitates detailed functional studies but also enhances the potential for rational engineering of phages for therapeutic and biotechnological applications.},
}
@article {pmid41183022,
year = {2025},
author = {Rajamani, S and Vilchez, L and Cracovia, N and Dule, D and Vata, A and Landaverde, S and Iyengar, A and Dubrovsky, EB},
title = {Mitochondrial-specific perturbation of Drosophila RNase Z in neurons leads to motor impairments, disrupted learning and neurodegeneration.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011938},
pmid = {41183022},
issn = {1553-7404},
mesh = {Animals ; *Mitochondria/genetics/metabolism/pathology ; *Neurons/metabolism/pathology ; *Drosophila Proteins/genetics/metabolism ; Drosophila melanogaster/genetics ; *Endoribonucleases/genetics/metabolism ; Mutation ; Learning/physiology ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology ; Animals, Genetically Modified ; Reactive Oxygen Species/metabolism ; },
abstract = {Clinical studies have linked a rare form of neurological disorder to the highly conserved RNase Z gene, which encodes an endoribonuclease responsible for the processing of nuclear and mitochondrial primary tRNA transcripts. Patients harboring mutant variants of this gene exhibit a spectrum of neurological dysfunction; however, no studies to date have established the causality of RNase Z-linked neuropathology. We employed CRISPR/Cas9 technology to create flies with a neuron-specific knockout of the RNase Z gene, which is rescued with transgenes encoding a wild-type or a mutant copy of RNase Z. Neuronal activity of RNase Z is vital, as mutants display striking morphological abnormalities in central and peripheral neurons, along with attenuated motor circuit function and associative learning performance. Neuron-specific mutations of RNase Z also led to mitochondrial fragmentation and elevated ROS production. By employing the rescue transgene encoding RNase Z devoid of a mitochondrial targeting signal (MTS), we segregated the mitochondrial activity of RNase Z from that in other compartments, allowing us to assess organelle-specific contributions to disease etiology and progression. We found that mutating mitochondrial RNase Z was sufficient to induce the neuropathology in flies, as they recapitulate the salient phenotypes observed in the pan-neuronal mutants. Collectively, our study validates the pathogenicity of mutant RNase Z and establishes mitochondrial-specific contributions to neuropathology.},
}
@article {pmid41183412,
year = {2026},
author = {Liu, Y and Li, H and Wang, J and Liu, J and Zhou, X},
title = {Dual-acting CRISPR/Cas12a system enhanced hydrogel fluorescent aptasensor for one-pot detection of tetracycline in water.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118181},
doi = {10.1016/j.bios.2025.118181},
pmid = {41183412},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Tetracycline/analysis/isolation &amp; purification ; *Aptamers, Nucleotide/chemistry ; *CRISPR-Cas Systems/genetics ; Hydrogels/chemistry ; Limit of Detection ; *Water Pollutants, Chemical/analysis/isolation &amp; purification ; *Anti-Bacterial Agents/analysis ; Water/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid yet ultrasensitive and accurate detection of tetracycline (TC) in aquatic environments subjected to composite pollution remains challenging. CRISPR-mediated biosensors have been extensively studied to achieve ultrasensitive detection. However, these biosensors have always been coupled with a time-consuming nucleic acid amplification process to improve the sensitivity, which may cause erratic signal due to the sophisticated biochemical reaction cascades, hence leading to inaccurate results. To address this issue, we proposed a novel amplification-free fluorescent aptasensor based on polyacrylamide-deoxyribonucleic acid hydrogel integrated with a dual-acting CRISPR/Cas12a system for ultrasensitive and rapid detection of TC in water. With this well-designed dual-acting CRISPR/Cas12a system, a single aptamer-target specific molecular binding event can synchronously initiate pairing between two distinct CRISPR RNA and their target nucleic acid modified on the hydrogel, enabling dual activation of Cas12a. The aptasensor instantly emitted quantifiable strong fluorescence due to the efficient cleavage of reporter probes by the twofold activated Cas12a, demonstrating a TC detection limit of 0.035 μg/L, with approximately 10.6-fold and 5.6-fold sensitivity improvement over the two corresponding single-crRNA systems. The entire detection process can be accomplished in one pot within 10 min. The one-step hydrogel aptasensor shows superior resistance to matrix interference over the conventional solution-phase system, achieving satisfactory recovery percentages (92 %-105 %) for TC in different water matrices. This study offers a new perspective on CRISPR/Cas12a biosensor design and advances the environmental antibiotic monitoring field.},
}
@article {pmid41183413,
year = {2026},
author = {Chen, H and Zeng, Z and Wei, Y and Huang, H and Negahdary, M and Han, X and Lin, Y and He, L and Song, F and Wan, Y},
title = {Dynamic biosensing enables amplifier-collateral-cleavage enhancement for pathogen diagnostic.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118158},
doi = {10.1016/j.bios.2025.118158},
pmid = {41183413},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *DNA/genetics ; },
abstract = {While CRISPR-Cas system and dynamic DNA nanotechnology have been extensively applied to mainstream biomedical domains, including gene editing, biochemical analysis, and molecular imaging-current approaches remain constrained by limitations in addressing increasingly nuanced and specialized experimental scenarios. Here, we report that CRISPR-CasΦ possesses a unique collateral-cleavage blockade characteristic: CasΦ is unable to recognize the "TTN" sequence in the loop region at the 3' end of stem-loop DNA, resulting in the blockade of collateral-cleavage activity. Leveraging this discovery, we successfully designed and customized two back-end signal amplifiers for biosensing by integrating dynamic DNA sensing studies of CasΦ. Based on these two specialized stem-loop amplifiers, we further developed the Amplifier-collateral-cleavage enhancement (ACE) method, achieving exponential signal amplification. Clinical validation using 112 urine samples demonstrated that ACE exhibits 98.8 % sensitivity and 90 % specificity. These findings highlight the potential of CasΦ dynamic sensing and establish a bridge for future integration of dynamic DNA technology and CRISPR systems.},
}
@article {pmid41183414,
year = {2026},
author = {Luo, Q and Zheng, C and Huang, Y and Liang, L and Gong, Y and Zhang, J and Tang, Q and Zhang, K and Liao, X},
title = {Polydopamine-stabilized CsPbBr3 enables toxicity-reduced ECL detection of MMP-2 via CRISPR/Cas12a trans-cleavage.},
journal = {Biosensors &amp; bioelectronics},
volume = {293},
number = {},
pages = {118193},
doi = {10.1016/j.bios.2025.118193},
pmid = {41183414},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; *Indoles/chemistry ; *Polymers/chemistry ; *Matrix Metalloproteinase 2/isolation &amp; purification/chemistry/blood ; Gold/chemistry ; Electrochemical Techniques/methods ; CRISPR-Cas Systems ; Humans ; Limit of Detection ; Luminescent Measurements/methods ; Lead/chemistry ; Titanium/chemistry ; },
abstract = {Lead-halide perovskites offer bright electrochemiluminescence (ECL) but suffer from aqueous instability and Pb[2+] safety concerns. We construct a CsPbBr3@PDA-Au nanointerface that couples polydopamine (PDA) passivation with Au-assisted charge transfer, integrated into a peptide-to-CRISPR/Cas12a amplification scheme for ultrasensitive detection of matrix metalloproteinase-2 (MMP-2). PDA conformally coats CsPbBr3, suppressing trap-mediated quenching and mitigating Pb[2+] release, while providing catechol/amine anchors for a tetrahedral DNA scaffold bearing a ferrocene-labeled hairpin. Upon MMP-2 cleavage of a GPLG↓VRG peptide-DNA chimera, the released strand activates Cas12a trans-cleavage, removing the proximal quencher and switching the interface from "off" to "on." Under unified processing (baseline subtraction and renormalization), the platform achieves a 5.6 aM detection limit with high selectivity over non-target proteases and excellent inter-device reproducibility (triplicate error bars reported). TEM/HRTEM visualize uniform nanocubes; XPS/FTIR evidence PDA catechol/quinone/amine coordination; XRD confirms phase-pure CsPbBr3; and corrected CV/EIS analyses verify accelerated interfacial kinetics after Au decoration. The sensor retains ∼98 % intra-day and ≥91 % 7-day signal, supporting operational robustness. By integrating interfacial stabilization, catalytic electron transfer, and molecular amplification, this modular design advances eco-conscious, perovskite-based ECL diagnostics for low-abundance protease monitoring and broader clinical bioanalysis.},
}
@article {pmid41184108,
year = {2026},
author = {Abir, AH and Benz, J and Frey, B and Bruns, H and Krönke, G and Gaipl, US and Schober, K and Mougiakakos, D and Mielenz, D},
title = {Glycolytic flux sustains human Th1 identity and effector function via STAT1 glycosylation.},
journal = {Life science alliance},
volume = {9},
number = {1},
pages = {},
pmid = {41184108},
issn = {2575-1077},
mesh = {Humans ; *Glycolysis ; Glycosylation ; *STAT1 Transcription Factor/metabolism/genetics ; *Th1 Cells/metabolism/immunology ; Interferon-gamma/metabolism ; Phosphorylation ; CRISPR-Cas Systems ; Cell Differentiation ; Gene Editing ; },
abstract = {T helper (Th) cell lineages are linked to metabolism, but precise mechanisms in human Th1 cells remain unclear. We addressed this question by in vitro stimulation and CRISPR/Cas9-mediated gene editing. Metabolic profiling revealed enhanced glycolytic activity in Th1 versus non-polarized cells, evidenced by increased extracellular acidification rate, ATP production via glycolysis, lactate secretion, NADH abundance, and elevated glycolysis-dependent anabolic activity. Inhibition of glycolysis reduced IFNγ production and STAT1 phosphorylation independent of JAK1/2 or SHP2 activity and STAT1 abundance, implicating glycolysis directly in sustaining STAT1-mediated Th1 functionality. O-glycosylation of STAT1 via O-glycosyltransferase was pivotal in modulating STAT1 activity. Pharmaceutical O-glycosyltransfer-ase inhibition prevented Th1 differentiation as well as STAT1 O-glycosylation. CRISPR/Cas9 mediated mutation of the O-glycosylation Ser499 and Thr510 sites diminished STAT1 Ser727 phosphorylation and IFNγ synthesis. Together, this study highlights glycolysis as key regulator of human Th1 cell identity and effector function, with STAT1 O-glycosylation selectively maintaining Th1 effector capacity. This mechanism could be explored to safeguard Th1 cells.},
}
@article {pmid41184277,
year = {2025},
author = {Nguyen, C and Omotayo, AI and Sanz Juste, S and Feng, X and López Del Amo, V},
title = {A temperature-sensitive CRISPR-Cas12a system for sterile insect technique.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9677},
pmid = {41184277},
issn = {2041-1723},
support = {R01 AI187493/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Male ; Female ; Temperature ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals, Genetically Modified ; *Infertility, Male/genetics ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; Gene Editing/methods ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {The sterile insect technique (SIT) reduces population numbers by releasing sterile males that produce non-viable progeny. Specifically, CRISPR/Cas9-based precision-guided SIT (pgSIT) generates sterile males through genetic crosses of two transgenic lines: a Cas9 strain and a guide RNA (gRNA) strain targeting male sterility and female viability or infertility. However, pgSIT requires separate maintenance of the two lines and sorting to obtain sterile males, creating possible challenges for scaling. To overcome this, we propose using Cas12a nuclease, which is inoperative at lower temperatures but active at higher temperatures. Here, we develop a Cas12a-based pgSIT system involving a single strain containing both the Cas12a nuclease and gRNAs to induce male sterility and female lethality. This strain can be maintained as a mixed stock of both sexes and only activated by increasing temperature, producing sterile males after just one generation. By reducing the challenges that arise with maintaining two separate lines, this system could offer a scalable alternative for vector control in combating vector-borne diseases.},
}
@article {pmid41184307,
year = {2025},
author = {White, N and Chalk, JA and Hu, YT and Pins, SM and Joseph, CR and Antoniou, P and Wimberger, S and Svensson, S and Caetano-Silva, SP and Mudde, ACA and Rai, R and Selvaraj, S and Feist, WN and Romito, M and Sienski, G and Nitsch, R and Booth, C and Santilli, G and Cavazza, A and Porteus, MH and Maresca, M and Thrasher, AJ and Turchiano, G},
title = {Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9571},
pmid = {41184307},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; 217112/Z/19/Z//Wellcome Trust (Wellcome)/ ; },
mesh = {Humans ; *DNA Repair/genetics ; *T-Lymphocytes/metabolism ; Gene Editing/methods ; DNA Breaks, Double-Stranded ; CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism ; *Endonucleases/metabolism/genetics ; Mutation ; },
abstract = {DNA repair mechanisms in human primary cells, including error-free repair, and, recurrent nuclease cleavage events, remain largely uncharacterised. We elucidate gene-editing related repair processes using Cleavage and Lesion Evaluation via Absolute Real-time dPCR (CLEAR-time dPCR), an ensemble of multiplexed dPCR assays that quantifies genome integrity at targeted sites. Utilising CLEAR-time dPCR we track active DSBs, small indels, large deletions, and other aberrations in absolute terms in clinically relevant edited cells, including HSPCs, iPSCs, and T-cells. By quantifying up to 90% of loci with unresolved DSBs, CLEAR-time dPCR reveals biases inherent to conventional mutation screening assays. Furthermore, we accurately quantify DNA repair precision, revealing prevalent scarless repair after blunt and staggered end DSBs and recurrent nucleases cleavage. This work provides one of the most precise analyses of DNA repair and mutation dynamics, paving the way for mechanistic studies to advance gene therapy, designer editors, and small molecule discovery.},
}
@article {pmid41184760,
year = {2025},
author = {Guo, F and Wang, D and Wu, H and Zhao, X and Xu, F and Cui, Y and Shao, Y and Zhang, Y and Bu, W and Wen, T},
title = {Genomic characteristics, antimicrobial resistance profiles and virulence factors of Gallibacterium anatis isolates from layer chickens in Northern China.},
journal = {BMC microbiology},
volume = {25},
number = {1},
pages = {706},
pmid = {41184760},
issn = {1471-2180},
support = {31802193//National Natural Science Foundation of China/ ; XMS202505//the R &amp; D Foundation of Institute of Animal Husbandry and Veterinary Medicine/ ; KJCX20230427//the Special Program on Science and Technology Innovation Capacity Building of BAAFS/ ; },
mesh = {Animals ; *Chickens/microbiology ; China ; *Virulence Factors/genetics ; *Poultry Diseases/microbiology ; Phylogeny ; *Pasteurellaceae/genetics/drug effects/pathogenicity/isolation &amp; purification/classification ; *Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; *Pasteurellaceae Infections/veterinary/microbiology ; Drug Resistance, Multiple, Bacterial/genetics ; Virulence ; Genomics ; *Drug Resistance, Bacterial ; },
abstract = {BACKGROUND: Gallibacterium anatis is an emerging pathogen causing substantial economic losses in global poultry production. Despite its growing clinical significance, the genomic basis of pathogenicity and antimicrobial resistance in this species remains poorly understood, particularly in China.<br><br>RESULTS: We sequenced and analyzed five clinical G. anatis isolates from Chinese layer chickens alongside 31 global strains, their genomes ranged from 2.25 to 2.81&#xa0;Mb with 39.8% average GC content. Phylogenetic analysis revealed that Chinese isolates cluster according to historical breeding stock importation patterns, reflecting international trade influences on pathogen distribution. Contemporary isolates showed extensive multidrug resistance compared to the antimicrobial-sensitive historical strain F149, with resistance profiles correlating directly with documented antibiotic usage in Chinese poultry production. Virulence analysis identified universal conservation of the RTX toxin system (97-100% prevalence) across all strains, contrasting sharply with variable distribution of other factors including fimbriae (30-35%) and secretion systems (50-60%). All strains harbored CRISPR-Cas systems, predominantly types I and III, indicating strain-specific phage defense adaptations.<br><br>CONCLUSIONS: The RTX toxin system represents a core virulence mechanism and potential universal vaccine target for G. anatis. The rapid evolution from antimicrobial-sensitive to extensively resistant phenotypes demonstrates how global poultry trade accelerates both pathogen spread and resistance development. These findings provide molecular insights for targeted interventions against an increasingly problematic poultry pathogen.},
}
@article {pmid41185939,
year = {2025},
author = {Kong, H and Liu, X and Xia, K and Guan, Y and Zhang, J and Zhang, Y and Liu, B and Ge, Z and Li, Q and Zuo, X and Wang, L and Li, J and Zhu, Y and Fan, C},
title = {Magnetically Driven High-Speed Rolling Nanoclusters for Enhanced CRISPR/Cas9 Genome Editing.},
journal = {ACS applied materials &amp; interfaces},
volume = {17},
number = {45},
pages = {61707-61717},
doi = {10.1021/acsami.5c15498},
pmid = {41185939},
issn = {1944-8252},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Magnetite Nanoparticles/chemistry ; Plasmids/genetics/chemistry/metabolism ; Jurkat Cells ; Protein Serine-Threonine Kinases/genetics ; Cell Cycle Proteins/genetics ; Viscosity ; },
abstract = {Despite the tremendous potential of the CRISPR/Cas9 gene-editing technology in precision therapeutics, intracellular delivery remains a major challenge. High cytoplasmic viscosity and lysosomal entrapment significantly impair the cytosolic transport and gene-editing efficiency. In this study, we demonstrate that both the size and magnetic responsiveness of Fe3O4 nanoclusters can be finely tuned by modulating ionic strength, enabling their rapid propulsion under external magnetic fields. Leveraging this property, we develop magnetic nanoparticle cluster nanorobots (MagCbots) of approximately 200 nm in size by electrostatically assembling Fe3O4 nanoclusters with CRISPR-Cas9 plasmids. Under magnetic actuation, MagCbots exhibit rapid rotation in highly viscous intracellular environments, achieving a linear velocity of ∼0.41 μm/s. MagCbots reduce intracellular viscosity by approximately 50% and enhance lysosomal escape efficiency by 3-fold compared to nonactuated counterparts. Their porous architecture not only offers high payload capacity but also protects plasmid DNA from enzymatic degradation. Notably, MagCbots enable efficient genome editing of both PD1 and PLK1 genes across various cell lines including hard-to-transfect Jurkat T cells. This magnetically driven nanorobot platform presents a promising strategy for active intracellular delivery and holds significant potential for advancing gene therapy and related biomedical applications.},
}
@article {pmid41186962,
year = {2026},
author = {Misceo, D and Terkelsen, T and Bøen Keim, SM and Bjørnstad, PM and Strand, ME and Orszagh, VC and Jensen, UB and Frengen, E},
title = {CRISPR Activation Reveals the Spliceogenicity of an Intronic NEB Variant in Fetuses With Arthrogryposis Multiplex Congenita 6.},
journal = {Clinical genetics},
volume = {109},
number = {4},
pages = {772-777},
pmid = {41186962},
issn = {1399-0004},
support = {//Norwegian National Advisory Unit on Rare Disorders/ ; //Legatet til Henrik Homans Minde/ ; },
mesh = {Humans ; *Arthrogryposis/genetics/diagnosis/pathology ; *Introns/genetics ; Female ; Fetus/pathology ; Pregnancy ; *RNA Splicing/genetics ; *CRISPR-Cas Systems/genetics ; Pedigree ; Male ; Frameshift Mutation ; RNA Splice Sites/genetics ; },
abstract = {Whole-genome sequencing identifies intronic variants whose pathogenicity can be predicted with tools like SpliceAI. However, an actionable classification of such variants may require RNA-based validation, which can be limited by low expression in clinically accessible tissues. We report two fetuses from one family with Arthrogryposis multiplex congenita 6 (AMC6 [OMIM # 619334]) and biallelic NEB variants: a paternally inherited likely pathogenic frameshift variant, Chr2(GRCh38):g.151579391del; NM_001164508.2:c.16653del; NP_001157980.2:p.(Asp5552ThrfsTer5), and a maternally inherited intronic variant of uncertain clinical significance, Chr2(GRCh38):g.151496267G>A; NM_001164508.2:c.24486+9C>T; NP_001157980.2:p.(?). Because NEB is poorly expressed in fibroblasts, we used CRISPR activation to induce its expression in fibroblasts from the heterozygous mother. RNA-sequencing subsequently confirmed that the intronic variant generated a novel splice donor site associated with inferred loss of splicing at the canonical donor site. After NMD-inhibition, we could thus identify 45.5% of NEB transcripts with a 7 bp exon extension, predicted to result in a protein-coding frameshift. The intronic variant was classified as likely pathogenic, allowing a genetic diagnosis.},
}
@article {pmid41186998,
year = {2025},
author = {Wang, W and Liu, H and Yang, Z and Wang, L and Li, W and He, H and Chen, S and Xu, M and Lu, Y},
title = {Enhancement of Spinosyn Production by Integrating a Static and Dynamic CRISPRi-Mediated Metabolic Switch in Saccharopolyspora spinosa.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {46},
pages = {29717-29728},
doi = {10.1021/acs.jafc.5c08276},
pmid = {41186998},
issn = {1520-5118},
mesh = {*Saccharopolyspora/genetics/metabolism/growth &amp; development ; *Macrolides/metabolism ; *Bacterial Proteins/metabolism/genetics ; Metabolic Engineering/methods ; CRISPR-Cas Systems ; Multigene Family ; Gene Expression Regulation, Bacterial ; *Insecticides/metabolism ; },
abstract = {Spinosyns, polyketide-derived macrolide insecticides produced by Saccharopolyspora spinosa, have broad market prospects. However, their large-scale production is still challenged by the low titers of industrial strains. Here, we developed an integrated static and dynamic CRISPRi strategy to redirect metabolic flux toward spinosyn overproduction without affecting cell growth. Initially, static CRISPRi-mediated repression of four genes (gltA1, fabH3, fabH4, and glgC) in three key nodes of primary metabolism boosted spinosyn titers by 19-55% without growth impairment. Simultaneous repression of these genes led to a further increase of spinosyn production. However, static repression of two other genes (pyc or gltA2) severely hindered bacterial growth and reduced spinosyn biosynthesis. To address this, two growth phase-dependent promoters from the spinosyn biosynthetic gene cluster (BGC) were characterized and employed for dynamic repression of pyc and gltA2, resulting in increased spinosyn titers by 44-68% while maintaining robust growth. Finally, combined static and dynamic repression of these six targets simultaneously achieved over a 2-fold increase of spinosyn production. Collectively, integrated static and dynamic CRISPRi-based reprogramming of primary metabolic pathways in S. spinosa achieved a balance between growth and secondary metabolism, thereby leading to spinosyn overproduction. This strategy has broad potential applicability in other actinomycetes for natural product overproduction.},
}
@article {pmid41187082,
year = {2025},
author = {Avaro, AS and Mirjalili, S and Griffiths, AD and Santiago, JG},
title = {Microfluidic networks using isotachophoresis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {45},
pages = {e2511724122},
pmid = {41187082},
issn = {1091-6490},
support = {//Swedish e-Science Research Centre (SeRC)/ ; //Kungliga Tekniska H&#xf6;gskolan (KTH)/ ; },
abstract = {The development of microfluidic technologies has enabled chemical and biological analysis systems with increased functionality, complexity, and parallelization. These functionalities often drive the creation and control of complex and dynamic fluidic architectures. Introduced here is a class of microfluidic network based on isotachophoresis (ITP), an electrokinetic process that can extract and purify samples, selectively transport, mix, and aliquot (split) samples in a system with no moving parts. Presented is a theoretical framework to describe these networks. The framework relies on the coupling between a one-dimensional description of ITP and two-dimensional, transient graphs to describe the dynamic evolution of ITP networks. We leverage this framework to create numerical simulations of branched ITP circuits. We build, control, and experimentally study a variety of ITP networks. These systems automatically split and merge ITP zones, enabling complex sample manipulation with minimal external control. The model captures the experimentally observed sample dynamics. We demonstrate an example system where an ITP network is used to control and quantify parallel CRISPR-Cas enzymatic reactions. The methods described here are generally applicable to highly complex topologies and may offer a basis for easily reconfigurable, electric field-driven microfluidic systems. Networks generally offer broad potential for automated chemical and biochemical analysis and lab-on-a-chip integration.},
}
@article {pmid41187216,
year = {2025},
author = {Treaster, MJ and McCann, J and Solovei, KS and Palmieri, RJ and White, MA},
title = {A Y-linked duplication of anti-Mullerian hormone is the sex determination gene in threespine stickleback.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011932},
pmid = {41187216},
issn = {1553-7404},
support = {R01 GM147312/GM/NIGMS NIH HHS/United States ; T32 GM142623/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Smegmamorpha/genetics ; Male ; *Anti-Mullerian Hormone/genetics ; *Sex Determination Processes/genetics ; Female ; *Y Chromosome/genetics ; *Gene Duplication ; CRISPR-Cas Systems ; Evolution, Molecular ; Sex Differentiation/genetics ; },
abstract = {Many taxa have independently evolved genetic sex determination where a single gene located on a sex chromosome controls gonadal differentiation. The gene anti-Mullerian hormone (amh) has convergently evolved as a sex determination gene in numerous vertebrate species, but how this gene has repeatedly evolved this novel function is not well understood. In the threespine stickleback (Gasterosteus aculeatus), amh was duplicated onto the Y chromosome (amhy) ~22 million years ago. To determine whether amhy is the primary sex determination gene, we used CRISPR/Cas9 and transgenesis to show that amhy is necessary and sufficient for male sex determination, consistent with the function of a primary sex determination gene. We find that amhy contributes to a higher total dosage of amh early in development and likely contributes to differential germ cell proliferation key to sex determination. The creation of sex-reversed lines also allowed us to investigate the genetic basis of secondary sex characteristics. Threespine stickleback have striking differences in behavior and morphology between sexes. Here we show one of the classic traits important for reproductive success, blue male nuptial coloration, is controlled by both sex-linked genetic factors as well as hormonal factors independent of sex chromosome genotype. This research establishes stickleback as a model to investigate how amh regulates gonadal development and how this gene repeatedly evolves novel function in sex determination. Analogous to the "Four Core Genotypes" model in house mice, sex-reversed threespine stickleback offer a new vertebrate model for investigating the separate contributions of gonadal sex and sex chromosomes to sexual dimorphism.},
}
@article {pmid41187596,
year = {2026},
author = {Doherty-Boyd, WS and Tsimbouri, PM and Jayawarna, V and Walker, M and Taqi, AF and Mahon, N and Meek, D and Young, P and Miller, A and West, A and Salmeron-Sanchez, M and Dalby, MJ and Donnelly, H},
title = {Synthetic peptide hydrogels as a model of the bone marrow niche demonstrate efficacy of a combined CRISPR-CAR T-cell therapy for acute myeloid leukaemia.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123803},
doi = {10.1016/j.biomaterials.2025.123803},
pmid = {41187596},
issn = {1878-5905},
mesh = {Humans ; *Leukemia, Myeloid, Acute/therapy/pathology ; *Hydrogels/chemistry ; *Peptides/chemistry ; *CRISPR-Cas Systems ; *Bone Marrow/pathology ; Mesenchymal Stem Cells/cytology ; Gene Editing ; *Receptors, Chimeric Antigen ; T-Lymphocytes ; *Immunotherapy, Adoptive/methods ; },
abstract = {Leukaemias, driven by mutations in haematopoietic stem cells (HSCs), rely on interactions with the bone marrow (BM) niche and other cell populations such as mesenchymal stromal cells (MSCs) for growth and survival. While chimeric antigen receptor (CAR) T-cell therapy shows promise for other haematological malignancies, its application to acute myeloid leukaemia (AML) is hindered by tumour heterogeneity and off-target toxicity. Combining CRISPR-Cas9 gene editing with CAR T-cell therapy has potential for selectively targeting AML cells while sparing healthy tissue. However, validating the efficacy of these treatments prior to clinical trial is hampered by the differences between humans and animal models typically used for pre-clinical testing. Furthermore, traditional in vitro models fail to replicate the complexity of the BM niche and often overestimate treatments' efficacy. Here, we present a bioengineered human-cell containing endosteal BM niche model combining a fibronectin-presenting polymeric surface and a synthetic peptide hydrogel (PeptiGel) that mimics native BM tissue's mechanical properties. This platform supports niche phenotypes in MSCs and HSCs and enables the evaluation of combined CRISPR-CAR T-cell therapy, demonstrating potential as a preclinical human model for testing novel therapies.},
}
@article {pmid41187652,
year = {2025},
author = {Masser-Mitchell, BE and McLoughlin, HS},
title = {Generation of an ITPR1[+/-] and isogenic control induced pluripotent stem cell line for SCA15/16 model development.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103858},
doi = {10.1016/j.scr.2025.103858},
pmid = {41187652},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Spinocerebellar Ataxias/pathology/genetics/metabolism ; *Inositol 1,4,5-Trisphosphate Receptors/genetics/metabolism ; Cell Line ; Cell Differentiation ; CRISPR-Cas Systems ; },
abstract = {Spinocerebellar ataxia type 15/16 (SCA15/16) is a rare neurodegenerative disorder caused by heterozygous deletions of ITPR1, leading to haploinsufficiency of the encoded endoplasmic reticulum membrane calcium channel. Patients present with progressive gait disturbances, abnormal eye movements, difficulties with speech and swallowing, and tremors associated with atrophy of the cerebellum. Using CRISPR/Cas9 technology, we generated ITPR1[+/-] and isogenic control induced pluripotent stem cell (iPSC) lines from PGP1 iPSCs for SCA15/16 model development. The clones were genotyped, karyotyped, and assessed for pluripotency and differentiation potential.},
}
@article {pmid41188277,
year = {2025},
author = {Yeh, TY and Chiu, IM and Huang, YJ and Wu, SC and Hsieh, CC and Sung, HH and Hwu, WL and Wang, GJ and Ju, JC},
title = {Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {38648},
pmid = {41188277},
issn = {2045-2322},
support = {108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; 108-2313-B-039-002//Ministry of Science and Technology, Taiwan/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Mice ; *Blastocyst/metabolism/cytology ; *Kidney/metabolism/embryology ; Mice, Knockout ; Female ; Gene Targeting ; RNA, Guide, CRISPR-Cas Systems/genetics ; Green Fluorescent Proteins/genetics/metabolism ; Gene Editing ; },
abstract = {Blastocyst complementation has been reported to produce exogenous mouse organs including the pancreas, lungs, and kidneys, but the complemented kidneys still failed to rescue the host animals. In the present study, we generated mouse complemented kidneys through a two-step procedure: using CRISPR/Cas9 ribonucleoproteins (RNP) to knockout Osr1 alleles, followed by injecting mouse embryonic stem (ES) cells that express enhanced green fluorescent protein (EGFP). When two different sgRNAs targeting the exon 2 of Osr1 were microinjected into the pronucleus of a mouse zygote, 34% of the embryos had deletions on both alleles, and these Osr1-knockouts died with no mesonephric duct development shown by histochemical staining. With three sgRNA injections, the knockout efficiencies increased, and mesonephric duct development with EGFP-positive cells was observed in ES cell-injected E12.5 embryos. Most of the ES cell-injected Osr1-knockout embryos degenerated from E13.5 to E15.5. Four of the 264 ES cell-injected embryos were born alive and survived to the second day, with strong EGFP signals observed in both the kidneys and the heart. Therefore, complementation of the Osr1-knockout blastocyst is a potential method to produce exogenous kidneys, although further modification is still needed to increase the efficiency.},
}
@article {pmid41188812,
year = {2025},
author = {Hussain, T and Awasthi, S and Shahid, F and Yi, SS and Sahni, N and Aldaz, CM},
title = {Therapeutic potential of PRMT1 as a critical survival dependency target in multiple myeloma.},
journal = {BMC cancer},
volume = {25},
number = {1},
pages = {1704},
pmid = {41188812},
issn = {1471-2407},
support = {R35GM133658/GM/NIGMS NIH HHS/United States ; R35 GM137836/GM/NIGMS NIH HHS/United States ; 7016-18//Leukemia and Lymphoma Society/ ; R35 GM133658/GM/NIGMS NIH HHS/United States ; R35GM137836/NH/NIH HHS/United States ; },
mesh = {*Protein-Arginine N-Methyltransferases/antagonists &amp; inhibitors/genetics/metabolism ; Humans ; *Multiple Myeloma/drug therapy/genetics/pathology/metabolism ; Cell Survival/drug effects ; Cell Line, Tumor ; *Repressor Proteins/antagonists &amp; inhibitors/genetics/metabolism ; Arginine/metabolism ; Gene Expression Regulation, Neoplastic/drug effects ; CRISPR-Cas Systems ; Cell Cycle/drug effects ; DNA Damage ; },
abstract = {Multiple myeloma (MM) is a neoplasm of antibody-producing plasma cells and is the second most prevalent hematological malignancy worldwide. Development of drug resistance and disease relapse significantly impede the success of MM treatment, highlighting the critical need to discover novel therapeutic targets. In a custom CRISPR/Cas9 screen targeting 197 DNA damage response-related genes, Protein Arginine N-Methyltransferase 1 (PRMT1) emerged as a top hit, revealing it as a potential therapeutic vulnerability and survival dependency in MM cells. PRMT1, a major Type I PRMT enzyme, catalyzes the asymmetric transfer of methyl groups to arginine residues, influencing gene transcription and protein function through post-translational modification. Dysregulation or overexpression of PRMT1 has been observed in various malignancies including MM and is linked to chemoresistance. Treatment with the Type I PRMT inhibitor GSK3368715 resulted in a dose-dependent reduction in cell survival across a panel of MM cell lines. This was accompanied by reduced levels of asymmetric dimethylation of arginine (ADMA) and increased arginine monomethylation (MMA) in MM cells. Cell cycle analysis revealed an accumulation of cells in the G0/G1 phase and a reduction in the S phase upon GSK3368715 treatment. Additionally, PRMT1 inhibition led to a significant downregulation of genes involved in cell proliferation, DNA replication, and DNA damage response (DDR), likely inducing genomic instability and impairing tumor growth. This was supported by Reverse Phase Protein Array (RPPA) analyses, which revealed a significant reduction in levels of proteins associated with cell cycle regulation and DDR pathways. Overall, our findings indicate that MM cells critically depend on PRMT1 for survival, highlighting the therapeutic potential of PRMT1 inhibition in treating MM.},
}
@article {pmid41190715,
year = {2026},
author = {Simon, MK and Yuan, L and Che, P and Day, K and Jones, T and Godwin, ID and Koltunow, AMG and Albertsen, MC},
title = {Induction of Synthetic Apomixis in Two Sorghum Hybrids Enables Seed Yield and Genotype Preservation Over Multiple Generations.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1712-1724},
pmid = {41190715},
issn = {1467-7652},
support = {INV-002955/GATES/Gates Foundation/United States ; OPP1076280//Bill and Melinda Gates Foundation/ ; //Corteva Agriscience/ ; INV-002955/GATES/Gates Foundation/United States ; },
mesh = {*Sorghum/genetics/physiology ; *Apomixis/genetics ; *Seeds/genetics/growth &amp; development/physiology ; Genotype ; Hybridization, Genetic ; CRISPR-Cas Systems ; Plant Breeding/methods ; Meiosis/genetics ; Parthenogenesis/genetics ; Plant Proteins/genetics/metabolism ; },
abstract = {Induction of apomixis, or clonal reproduction through seed, could economise commercial hybrid seed production and enable smallholder farmers to save and sow hybrid seed. Here, we demonstrate the synthetic induction of apomixis in two sorghum hybrids and show that the clonal hybrid seed can be maintained across multiple seed generations. This was achieved through the combination of avoidance of meiosis and induced parthenogenesis. Meiotic avoidance was generated by CRISPR/Cas9 knockout of the sorghum meiosis genes Spo11, Rec8, OsdL1 and OsdL3. Parthenogenesis was induced in the resultant diploid egg cell by the expression of the Cenchrus ASGR-BBML2 gene coding sequence. Two different strategies were used to combine these components to induce synthetic apomixis in two sorghum hybrids. Each hybrid used Tx623 as a female parent and either Tx430 or the African landrace Macia as a male parent. Seed yields in both apomictic hybrids were consistent and stable for multiple generations following self-pollination but reduced relative to the sexual hybrids. Sorghum contains two copies of the Osd1 gene that function in meiotic non-reduction. CRISPR/Cas9 knockout of both OsdL1 and OsdL3 loci was sufficient to produce clonal hybrid progeny in conjunction with the other components, but this led to a reduction in seed set. By contrast, a single in-frame edited allele of either OsdL1 or OsdL3 significantly improved seed set of clonal hybrid progeny. Fine-tuning OsdL activity appears to be essential to optimising fertility; however, additional improvements are required to unlock the agronomic potential of synthetically induced apomictic sorghum in the field.},
}
@article {pmid41190788,
year = {2025},
author = {Orzechowski, M and Hoikkala, V and Chi, H and McMahon, S and Gloster, T and White, MF},
title = {A viral SAVED protein with ring nuclease activity degrades the CRISPR second messenger cA4.},
journal = {The Biochemical journal},
volume = {482},
number = {22},
pages = {1707-1719},
pmid = {41190788},
issn = {1470-8728},
mesh = {*Viral Proteins/metabolism/genetics/chemistry ; *Adenine Nucleotides/metabolism ; *CRISPR-Cas Systems ; *Second Messenger Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Oligoribonucleotides ; },
abstract = {Type III CRISPR systems typically generate cyclic oligoadenylate second messengers such as cyclic tetra-adenylate (cA4) on detection of foreign RNA. These activate ancillary effector proteins which elicit a diverse range of immune responses. The Calp (CRISPR associated Lon protease) system elicits a transcriptional response to infection when CalpL (Calp Lon protease) binds cA4 in its SAVED (SMODS associated and fused to various effectors domain) sensor domain, resulting in filament formation and activation of the Lon protease domain, which cleaves the anti-Sigma factor CalpT, releasing the CalpS (Calp Sigma factor) for transcriptional remodelling. Here, we show that thermophilic viruses have appropriated the SAVED domain of CalpL as an anti-CRISPR, AcrIII-2 (second anti-CRISPR of type III systems), which they use to degrade cA4. AcrIII-2 dimers sandwich cA4, degrading it in a shared active site to short linear products, using a mechanism highly reminiscent of CalpL. This results in inhibition of a range of cA4 activated effectors in vitro. This is the first example of a virally encoded SAVED domain with ring nuclease activity, highlighting the complex interplay between viruses and cellular defences.},
}
@article {pmid41191093,
year = {2025},
author = {Sun, Y and Han, H and Zhao, R and Liu, X and Liu, Z and Liang, J and Wang, B and Zhao, B and Pang, Y and Xiao, R},
title = {CRISPR-assisted MoS2@TQD nanosheets-based lateral flow assay for dual-mode and sensitive diagnosis of SARS-CoV-2.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {792},
pmid = {41191093},
issn = {1436-5073},
mesh = {Humans ; *SARS-CoV-2/isolation &amp; purification/genetics ; *COVID-19/diagnosis ; *Molybdenum/chemistry ; *Quantum Dots/chemistry ; Limit of Detection ; *Disulfides/chemistry ; Colorimetry/methods ; CRISPR-Cas Systems ; Nanostructures/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; COVID-19 Testing/methods ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) assembled lateral flow assay (LFA) have been developed for rapid virus diagnosis, however, the single-mode visual half-quantitative characteristics limit their applications in accurate quantitative diagnosis. Herein, a CRISPR assisted MoS2@TQD (MoS2 nanosheet assembled ternary quantum dot) based colorimetric and fluorescent dual-mode LFA was fabricated for the ultrasensitive and flexible detection of the SARS-CoV-2. Compared with single QDs, MoS2@TQD can provide stable and superior colorimetric/fluorescent signals in various bio-samples. RT-RAA/CRISPR-Cas13 was adopted to couple with MoS2@TQD so that the detection sensitivity can be further enhanced. Based on the dual-mode LFA, the colorimetric visual detection limit was 400 copies/mL, and for fluorescence quantification mode, as low as 250 copies/mL can be obtained. For clinic diagnosis, 35 SARS-CoV-2 positive throat swab samples and 18 negative samples were detected by this LFA, 100% accuracy and 100% consistency with quantitative polymerase chain reaction (qPCR) results can be obtained. It indicated that MoS2@TQD-based dual-mode LFA can be a rapid, sensitive, and accurate detection platform for SARS-CoV-2 and other infectious pathogens in the point-of-care diagnosis.},
}
@article {pmid41191214,
year = {2025},
author = {Choi, S and Lee, JM and Kim, KK},
title = {Biomolecular condensates: molecular structure, biological functions, diseases, and therapeutic targets.},
journal = {Molecular biomedicine},
volume = {6},
number = {1},
pages = {99},
pmid = {41191214},
issn = {2662-8651},
support = {RS-2025-00513647//National Research Foundation of Korea/ ; 20240678//Bisa Research Grant of Keimyung University/ ; },
mesh = {Humans ; *Biomolecular Condensates/chemistry/metabolism ; Animals ; Neurodegenerative Diseases/metabolism ; },
abstract = {Cells constantly encounter environmental and physiological fluctuations that challenge homeostasis and threaten viability. In response to these cues, specific proteins and nucleic acids engage in multivalent interactions and undergo phase separation to form membraneless assemblies known as biomolecular condensates. Nuclear condensates include paraspeckles, nuclear speckles, and Cajal bodies, while cytoplasmic condensates include stress granules, processing bodies, RNA transport granules, U-bodies, and Balbiani bodies. These assemblies regulate transcription, splicing fidelity, RNA stability, translational reprogramming, and integration of signaling pathways, thereby serving as dynamic platforms for metabolic regulation and physiological adaptation. However, dysregulation of these condensates has been increasingly recognized as a central pathogenic mechanism in neurodegenerative diseases, cancers, and viral infections, contributing to toxic protein aggregation, nucleic acid dysregulation, and aberrant cell survival signaling. This review provides a comprehensive synthesis of the molecular mechanisms governing condensation, delineates the diverse types and functions of major biomolecular condensates, and examines therapeutic approaches based on their pathophysiological relevance to disease development and progression. Furthermore, we highlight the cutting-edge technologies, including CRISPR/Cas-based imaging, optogenetic manipulation, and AI-driven phase separation prediction tools, which enable the real-time monitoring and precision targeting of cytoplasmic biomolecular condensates. These insights underscore the emerging potential of biomolecular condensates as both biomarkers and therapeutic targets, paving the way for precision medicine approaches in condensate-associated diseases.},
}
@article {pmid41191401,
year = {2025},
author = {Guo, Y and Xiong, Y and Tong, W and Huang, X and Xiong, Y},
title = {CRISPR/Cas12a-Based Dynamic Light Scattering Assay for Nucleic Acid Amplification-Free and Ultrasensitive Detection of Listeria monocytogenes.},
journal = {Analytical chemistry},
volume = {97},
number = {45},
pages = {25232-25244},
doi = {10.1021/acs.analchem.5c04740},
pmid = {41191401},
issn = {1520-6882},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Dynamic Light Scattering/methods ; Limit of Detection ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Nucleic Acid Amplification Techniques ; *Biosensing Techniques/methods ; Particle Size ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive signal transduction systems are essential for developing rapid, robust, and sensitive nucleic acid tests using the amplification-free CRISPR-Cas12a system. In this study, we introduce a novel platform termed CRISPR-DART (dynamic light scattering Assisted Rapid Test), which combines CRISPR-Cas12a with a dynamic light scattering (DLS) signal readout. We systematically evaluated the effects of nanoparticle size (20, 60, and 100 nm) and morphology (spherical, cubic, and flower-like) on DLS sensing performance. Results demonstrate that larger and more structurally complex nanoparticles significantly enhance scattering intensity, allowing stable DLS signals at lower concentrations and improving sensitivity for detecting low-abundance nucleic acid targets. By utilizing the high specificity and trans-cleavage activity of CRISPR-Cas12a, target-triggered cleavage of single-stranded DNA linkers modulates nanoparticle aggregation, enabling quantitative nucleic acid detection. Notably, CRISPR-DART based on 100 nm gold nanoflowers with large size and complex morphology achieves a detection limit (LOD) of 32 aM, an improvement of 5 orders of magnitude over conventional CRISPR-Cas12a assays using fluorophore-quencher as signal output. Furthermore, the amplification-free CRISPR-DART platform also achieves a LOD of 92 CFU/mL for Listeria monocytogenes in food samples, and after a brief preincubation step, successfully detects 1 CFU in 25 g of food sample. In summary, the CRISPR-DART platform provides a straightforward, highly sensitive, and specific tool for rapid on-site diagnostics and food safety monitoring.},
}
@article {pmid41191525,
year = {2026},
author = {Wang, K and Wang, J and Feng, R and Dudnyk, K and Kim, YJ and Lim, JYS and Lee, M and Zhang, Y and Gao, X and Cheng, Y and Orkin, SH and Zhou, J and Weiss, MJ and Yue, F and Xu, J},
title = {Silencing of BCL11A by disrupting enhancer-dependent epigenetic insulation.},
journal = {Blood},
volume = {147},
number = {13},
pages = {1470-1484},
pmid = {41191525},
issn = {1528-0020},
mesh = {Humans ; *Enhancer Elements, Genetic ; *Epigenesis, Genetic ; *Gene Silencing ; CRISPR-Cas Systems ; Erythroid Cells/metabolism ; Repressor Proteins ; *Nuclear Proteins/genetics/metabolism ; *Carrier Proteins/genetics/metabolism ; Fetal Hemoglobin/genetics/metabolism ; Chromatin/genetics/metabolism ; },
abstract = {The transcription factor BCL11A is a genetically and clinically validated regulator of the fetal-to-adult hemoglobin switch in human erythroid cells. CRISPR editing of an intronic enhancer within the BCL11A gene reactivates fetal hemoglobin (HbF) in adult erythroid cells, serving as the first CRISPR-based therapy for β-hemoglobinopathies. However, the molecular basis for the remarkable efficacy of CRISPR-mediated enhancer ablation remains elusive. Here, we describe a new genome architecture, an enhancer-dependent chromatin rosette, that is essential for epigenetic insulation and the developmentally regulated, hematopoietic lineage-specific expression of BCL11A. CRISPR-mediated disruption of the BCL11A erythroid enhancer impairs transcription of enhancer-driven RNAs and NIPBL-dependent cohesin loading, leading to the destabilization of the rosette structure, loss of chromatin insulation, and epigenetic silencing of BCL11A. Moreover, targeted depletion of enhancer RNAs using antisense oligonucleotides silences BCL11A by disrupting epigenetic insulation, causing HbF reactivation in adult erythroid cells. These findings uncover an essential role for enhancer-driven epigenetic insulation in transcriptional control, presenting a new strategy for the therapeutic targeting of BCL11A.},
}
@article {pmid41191919,
year = {2026},
author = {Liu, C and Xu, F and Wu, Y and Li, J and Ni, M and Xia, S and Chen, L and Zhao, H and Yu, M and Zhou, Y and Zhang, M and Li, J and Wu, X and Huang, Y and Zhu, T and Chen, X},
title = {Genome-wide CRISPR-Cas9 screening identifies CLK1 inhibition as a strategy to restore PARP inhibitor sensitivity via ERCC1 isoform switching.},
journal = {Protein &amp; cell},
volume = {17},
number = {3},
pages = {248-262},
pmid = {41191919},
issn = {1674-8018},
support = {82403208//National Natural Science Foundation of China/ ; 82303660//National Natural Science Foundation of China/ ; 82272898//National Natural Science Foundation of China/ ; 82272682//National Natural Science Foundation of China/ ; 82503636//National Natural Science Foundation of China/ ; SACA-AX202215//Shanghai Anti-Cancer Association/ ; 22YF1408100//Science and Technology Commission of Shanghai Municipality/ ; 16411950200//Science and Technology Commission of Shanghai Municipality/ ; KW1711//Science and Technology Commission of Shanghai Municipality/ ; 17411963000//Science and Technology Commission of Shanghai Municipality/ ; 22YF1409100//Science and Technology Commission of Shanghai Municipality/ ; CORP-252-1//Chinese Anti-Cancer Association-Hengrui PARP Inhibitor Cancer Research Fund/ ; 2025ZD0545600//Noncommunicable Chronic Diseases-National Science and Technology Major/ ; LGF21H160008//Social Development Project of Public Welfare Technology Research in Zhejiang Province/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; Female ; *Protein-Tyrosine Kinases/genetics/metabolism ; *Endonucleases/metabolism/genetics ; *Poly(ADP-ribose) Polymerase Inhibitors/pharmacology ; *Ovarian Neoplasms/drug therapy/genetics/pathology/metabolism ; Animals ; *DNA-Binding Proteins/metabolism/genetics ; Cell Line, Tumor ; *Protein Serine-Threonine Kinases/antagonists &amp; inhibitors/genetics/metabolism ; Mice ; Phthalazines/pharmacology ; Piperazines/pharmacology ; Drug Resistance, Neoplasm/drug effects ; *Carcinoma, Ovarian Epithelial/drug therapy/genetics/pathology/metabolism ; Protein Isoforms/metabolism/genetics ; Apoptosis/drug effects ; },
abstract = {Epithelial ovarian cancer (EOC) is an aggressive malignancy with limited therapeutic options. Poly(ADP-ribose) polymerase inhibitors (PARPi) have shown remarkable efficacy, especially in BRCA-mutant patients, and are approved as maintenance therapy to prevent recurrence after initial response to chemotherapy. However, the development of PARPi resistance poses a major clinical challenge. This study utilized a whole-genome CRISPR-Cas9 genetic screening to identify genes associated with PARPi sensitivity upon knockout. Based on the screening and validated through further experiments, we confirmed that CLK1 knockdown is synthetically lethal with PARPi in ovarian cancer. The combination of the PARPi Olaparib and CLK1 inhibitor TG003 exhibited potent anti-proliferative effects both in vitro and in vivo. Mechanistically, CLK1 inhibition downregulated the functional ERCC1-202 isoform, resulting in enhanced DNA damage and apoptosis. Our findings reveal a novel mechanism underlying PARPi sensitivity and suggest that targeting CLK1 in combination with PARPi may represent a promising therapeutic strategy for PARPi-resistant ovarian cancer.},
}
@article {pmid41192264,
year = {2025},
author = {Raabe, J and Fuchs, S and Augustin, C and Hammerschmidt, A and Krämer, E and Orthey, E and Ehler, E and Cuello, F},
title = {Generation of a NRAP-overexpressing mutant from a human iPSC line.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103863},
doi = {10.1016/j.scr.2025.103863},
pmid = {41192264},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Myocytes, Cardiac/metabolism/cytology ; Cell Differentiation ; Cell Line ; *Mutation ; CRISPR-Cas Systems ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; },
abstract = {Cardiomyopathies are a major contributor to cardiovascular mortality and are frequently linked to abnormalities in intercalated discs, which coordinate mechanical and electrical signaling between cardiomyocytes. The Nebulin-Related Anchoring Protein (NRAP), a key component of these structures, is essential for myofibril formation and force transmission. In various cardiac diseases such as cardiomyopathies with differing genetic mutations, NRAP protein abundance is increased, yet the functional consequences of this expression change remain insufficiently characterized. To investigate the outcome of NRAP-overexpression (NRAP-OE) on cardiac development and disease, we established a human induced pluripotent stem cell (hiPSC) line with stable and specific NRAP-OE in cardiomyocytes using CRISPR-Cas9-based genome editing. The resulting line was rigorously validated for chromosomal integrity, pluripotency markers, absence of off-target effects and mycoplasma contamination, as well as its capacity for trilineage differentiation. This NRAP-OE model offers a novel platform for investigating how increased NRAP levels influence cardiomyocyte structure and function, and may provide insight into its role in the pathogenesis of cardiomyopathy.},
}
@article {pmid41192497,
year = {2026},
author = {Wang, X and Chen, Y and Hu, Y and Luo, S and Wang, S and Mao, B and Peng, C and Chen, C and Pan, W and Yan, H and Liao, J and Zhou, Q and Li, W},
title = {Dual-target CRISPR-Cas12 diagnostics based on asymmetrically chemical-modified DNA probe.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {53},
number = {4},
pages = {732-743},
doi = {10.1016/j.jgg.2025.10.007},
pmid = {41192497},
issn = {1673-8527},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *DNA Probes/genetics/chemistry ; Nucleic Acid Amplification Techniques/methods ; Human papillomavirus 16/genetics/isolation &amp; purification ; Human papillomavirus 18/genetics/isolation &amp; purification ; Influenza A virus/genetics/isolation &amp; purification ; Papillomavirus Infections/diagnosis/virology ; },
abstract = {CRISPR-based nucleic acid detection technologies have revolutionized infectious disease detection and environmental monitoring by leveraging RNA-DNA complementarity to enable rapid, precise, and cost-effective detection of targets. However, achieving multitarget detection in one tube still presents challenges that necessitate further research. Here, we develop a nucleic acid detection module based on the CRISPR-Cas12i system. Importantly, we find that Cas12i and AapCas12b exhibit opposite trans-cleavage preferences for asymmetrically phosphorothioate-modified single-strand DNA probes, enabling the development of an effective dual-target nucleic acid detection platform by combining these two Cas12 nucleases in one tube. Moreover, this dual-target detection platform exhibits high specificity and sensitivity in genotyping the nucleic acid targets of human papillomavirus (HPV) 16 and HPV18, as well as Influenza A virus (FluA) and Respiratory syncytial virus. Notably, combined with loop-mediated isothermal amplification, this platform achieves high detection rates for clinical samples (18/18 FluA and 18/18 GAPDH internal reference detection rate). Taken together, these results can broaden the application of CRISPR-based Cas12 proteins for multi-target nucleic acid detection in one tube.},
}
@article {pmid41193657,
year = {2025},
author = {Li, H and Melnyk, JE and Fu, BXH and Shrestha, R and Zhang, M and Sjöström, M and Feng, S and Anderson, JA and Han, W and Chesner, LN and Shin, HJ and Farsh, T and Suarez, HJ and Nath, S and Chou, J and Das, R and Egusa, EA and Calvert, M and Kishishita, A and Barpanda, A and Zhu, J and Maheshwari, A and Chen, WS and Alshalalfa, M and Winters, A and Hua, JT and Liu, T and Davicioni, E and Wiita, AP and Stohr, BA and Siddiqui, J and Huang, B and Small, EJ and Shokat, KM and Nelson, PS and Quigley, DA and Wasmuth, EV and Gilbert, LA and Feng, FY},
title = {Genome-scale CRISPR screens identify PTGES3 as a direct modulator of androgen receptor function in advanced prostate cancer.},
journal = {Nature genetics},
volume = {57},
number = {12},
pages = {3027-3038},
pmid = {41193657},
issn = {1546-1718},
support = {YI//Prostate Cancer Foundation (PCF)/ ; P50 CA097186/CA/NCI NIH HHS/United States ; R01GM131641//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; 2018-00382//Vetenskapsr&#xe5;det (Swedish Research Council)/ ; R01 CA221969/CA/NCI NIH HHS/United States ; 1R01CA244550//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; PC230420//Prostate Cancer Foundation (PCF)/ ; young investigator//Prostate Cancer Foundation (PCF)/ ; R01 GM131641/GM/NIGMS NIH HHS/United States ; 1R01CA227025//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; DP2 CA239597/CA/NCI NIH HHS/United States ; P50 CA186786/CA/NCI NIH HHS/United States ; 1F32CA236347-01//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 GM124334/GM/NIGMS NIH HHS/United States ; 1R01CA230516-01//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01GM124334//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; W81XWH-20-1-0136//U.S. Department of Defense (United States Department of Defense)/ ; P50CA097186 PNW Prostate Cancer SPORE Career Enhancement Program//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; P41 GM103481/GM/NIGMS NIH HHS/United States ; P50 CA275741/CA/NCI NIH HHS/United States ; R01 CA227025/CA/NCI NIH HHS/United States ; CA230516-02S1//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R00 CA204602/CA/NCI NIH HHS/United States ; CA204602//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; P30 CA015704/CA/NCI NIH HHS/United States ; V2024-016//V Foundation for Cancer Research (V Foundation)/ ; K99 CA204602/CA/NCI NIH HHS/United States ; HT9425-23-1-0462//U.S. Department of Defense (United States Department of Defense)/ ; R01 CA290875/CA/NCI NIH HHS/United States ; 1R01CA221969-01//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA234715/CA/NCI NIH HHS/United States ; S10 OD016229/OD/NIH HHS/United States ; 17CHAL06//Prostate Cancer Foundation (PCF)/ ; R01 CA230516/CA/NCI NIH HHS/United States ; Young Investigator Award//Prostate Cancer Foundation (PCF)/ ; R01 CA244550/CA/NCI NIH HHS/United States ; R00 GM140264/GM/NIGMS NIH HHS/United States ; 21YOUN12//Prostate Cancer Foundation (PCF)/ ; F32 CA236347/CA/NCI NIH HHS/United States ; Prostate Cancer Program 2021 Pilot Research Awards//UC | UC San Francisco | Department of Medicine, University of California, San Francisco (UCSF Department of Medicine)/ ; P50CA186786//U.S. Department of Health &amp; Human Services | NIH | National Cancer Institute (NCI)/ ; R01 CA266452/CA/NCI NIH HHS/United States ; },
mesh = {Male ; *Receptors, Androgen/genetics/metabolism ; Humans ; *Prostatic Neoplasms/genetics/pathology/metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems/genetics ; Homeodomain Proteins/genetics/metabolism ; GATA2 Transcription Factor/genetics/metabolism ; Drug Resistance, Neoplasm/genetics ; },
abstract = {The androgen receptor (AR) is a critical driver of prostate cancer (PCa). Here, to study regulators of AR protein levels and oncogenic activity, we developed a live-cell quantitative endogenous AR fluorescent reporter. Leveraging this AR reporter, we performed genome-scale CRISPRi flow cytometry sorting screens to systematically identify genes that modulate AR protein levels. We identified and validated known AR protein regulators, including HOXB13 and GATA2, and also unexpected top hits including PTGES3-a poorly characterized gene in PCa. PTGES3 repression resulted in loss of AR protein, cell-cycle arrest and cell death in AR-driven PCa models. Clinically, analysis of PCa data demonstrates that PTGES3 expression is associated with AR-directed therapy resistance. Mechanistically, we show PTGES3 binds directly to AR, regulates AR protein stability and is necessary for AR function in the nucleus at AR target genes. PTGES3 represents a potential therapeutic target for overcoming known mechanisms of resistance to existing AR-directed therapies in PCa.},
}
@article {pmid41193960,
year = {2025},
author = {Van Goethem, MW and Vikram, S and Cowan, DA and Makhalanyane, TP},
title = {Comparative genomics reveals adaptive traits in novel Antarctic lithic cyanobacteria.},
journal = {BMC genomics},
volume = {26},
number = {1},
pages = {994},
pmid = {41193960},
issn = {1471-2164},
abstract = {BACKGROUND: Terrestrial polar cyanobacteria persist at the cold limits of life, enduring a suite of extreme stressors including sub-zero temperatures, frequent freeze–thaw cycles, oligotrophic soils, variable light regimes with long periods of darkness, and desiccation. To survive, cyanobacteria have evolved diverse physiological strategies. A key adaptation among Antarctic terrestrial cyanobacteria is niche colonization: inhabiting the undersides of translucent quartz rocks (hypoliths) and the interior spaces of porous rocks (endoliths), which buffer environmental extremes and sustains the potential for photoautotrophic carbon fixation. However, the full genomic repertoire facilitating their resilience is incomplete.<br><br>RESULTS: We cultivated cyanobacteria from endolithic and hypolithic niches in Victoria Valley, Eastern Antarctica, and recovered four near-complete genomes (100% completeness,&#x2009;<&#x2009;1% contamination). Three hypolithic genomes showed near-identical sequence similarity (whole genome average nucleotide identity&#x2009;=&#x2009;99.98%) and phylogenomic proximity to the genus Coleofasciculus, yet represent a novel species, Coleofasciculus caryii H7-2. The fourth genome, derived from an endolith, showed moderate similarity to Aliterella antarctica (whole genome average nucleotide identity&#x2009;=&#x2009;79.1%), and is proposed as a new species, Aliterella bergstromii E5.1. C. caryii H7-2 possessed a larger genome (~&#x2009;6.1 Mbp) than A. bergstromii E5.1 (~&#x2009;5.4 Mbp). Both genomes encoded complete pathways for carbon fixation via oxygenic photosynthesis (RuBisCO and phosphoribulokinase), extensive phycobilisomes, and multiple photoprotective mechanisms. Predicted optimal growth temperatures were 21.7&#xa0;&#xb0;C and 23.2&#xa0;&#xb0;C, respectively. Shared stress-mitigation genes included those for osmotic, thermal and oxidative (superoxide dismutase) stress response. All genomes contained biosynthetic gene clusters associated with stress-adaptive secondary metabolites, including heterocyst glycolipids, siderophores, phenazines, compounds related to nostopeptolide and merocyclophane. The C. caryii H7-2 genome encoded multiple CRISPR-Cas systems, suggesting adaptive immunity and historical phage exposure. In contrast, A. bergstromii E5.1 harboured a single prophage and an array of 24 plasmids.<br><br>CONCLUSIONS: These finding reveal that the newly-described cyanobacteria possess a rich genomic repertoire of adaptations to withstand Antarctic extremes, emphasizing the resilience and ecological importance of lithobiontic cyanobacteria in polar deserts.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12864-025-12203-7.},
}
@article {pmid41194386,
year = {2025},
author = {Sanchez-Quirante, T and Kužmová, E and Riopedre-Fernandez, M and Golojuch, S and Vopálenský, P and Raindlová, V and El-Sagheer, AH and Brown, T and Hocek, M},
title = {Enzymatic Synthesis of Modified RNA Containing 5-Methyl- or 5-Ethylpyrimidines or Substituted 7-Deazapurines and Influence of the Modifications on Stability, Translation, and CRISPR-Cas9 Cleavage.},
journal = {ACS chemical biology},
volume = {20},
number = {11},
pages = {2755-2767},
pmid = {41194386},
issn = {1554-8937},
mesh = {*CRISPR-Cas Systems ; *Pyrimidines/chemistry/metabolism ; *RNA/chemistry/metabolism ; Humans ; *Purines/chemistry/metabolism ; Protein Biosynthesis ; DNA-Directed RNA Polymerases/metabolism ; Viral Proteins ; },
abstract = {A set of modified 5-methyl- and 5-ethylpyrimidine (uracil and cytosine) and 7-methyl-, 7-ethyl-, and 7-unsubstituted 7-deazapurine (deazaadenine and deazaguanine) ribonucleoside triphosphates was synthesized and used for enzymatic synthesis of base-modified RNA using in vitro transcription (IVT). They all were good substrates for T7 RNA polymerase in the IVT synthesis of model 70-mer RNA, mRNA encoding Renilla luciferase, and 99-mer single-guide RNA (sgRNA). The effect of modifications in the particular RNA on the stability and efficiency in in vitro and in cellulo translation as well as in CRISPR-Cas9 gene cleavage was quantified. In the in vitro translation assay, we observed moderately enhanced luciferase production with 5-methyluracil and -cytosine, while any 7-deazaadenines completely inhibited the translation. Surprisingly, in cellulo experiments showed a significant enhancement of translation with mRNA containing 7-deazaguanine and moderate enhancement with 5-methyl- or 5-ethylcytosine. Most of the modifications had a minimal effect on the efficiency of the gene cleavage in CRISPR-Cas9 except for 7-alkyl-7-deazaadenines that completely inhibited the cleavage. The results are important for further design of potential base-modified RNA therapeutics.},
}
@article {pmid41194485,
year = {2026},
author = {Yang, TT and Zhang, JR and Xie, ZH and Ren, ZL and Zhao, MY and Hu, WJ and Yan, JW and Ni, M},
title = {CRISPR-Cas9-Targeted Nanopore Sequencing for STR Typing.},
journal = {Electrophoresis},
volume = {47},
number = {1},
pages = {57-67},
doi = {10.1002/elps.70051},
pmid = {41194485},
issn = {1522-2683},
support = {2021QY2004//We thank the National Key Research and Development Program of China/ ; 82030058//National Natural Science Foundation of China/ ; },
mesh = {Humans ; *Nanopore Sequencing/methods ; *Microsatellite Repeats/genetics ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; *DNA Fingerprinting/methods ; Pilot Projects ; *Sequence Analysis, DNA/methods ; DNA/genetics ; },
abstract = {CRISPR-Cas9-targeted sequencing can enrich DNA regions of interest by directing the Cas9 protein to bind and cleave specific DNA sequences via single-guide RNA (sgRNA). It is interesting to explore the efficacy of using CRISPR-Cas9-targeted nanopore sequencing (referred to as Cas9-seq), a polymerase chain reaction (PCR)-free workflow, for forensic short tandem repeats (STR) profiling, and to compare it with the amplification-based approach. In this pilot study, we constructed a Cas9-seq method for profiling seven STR loci, including D18S51, FGA, TPOX, D16S539, vWA, CSF1PO, and TH01. With 3 µg DNA inputs from human NA12878 and 293T cell lines, we achieved 643.45- and 468.34-fold enrichment ratios of the sgRNA-targeted regions by using Cas9-seq, respectively. Compared to nanopore sequencing of PCR amplicon products (amplicon-seq) of the ForenSeq DNA Signature Prep kit, the Cas9-seq reads had an ultralow strand bias. However, surprisingly, Cas9-seq did not show advantages in allele balance and had higher noise in the reads. At the seven STR loci for the two samples, both Cas9-seq and amplicon-seq had three genotyping errors. Additionally, there were no false-positive single-nucleotide polymorphisms (SNPs) introduced by Cas9-seq, whereas amplicon-seq produced three. In sum, we conclude that the PCR-free Cas9-seq might not be favorable for forensic STR genotyping.},
}
@article {pmid41195477,
year = {2026},
author = {Lee, SK and Hong, WJ and Kim, EJ and Lee, C and Moon, S and Ha, SH and Jung, KH},
title = {Developing an Efficient System for Hybrid Rice Seed Production Using Partial Male Sterility.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1740-1749},
pmid = {41195477},
issn = {1467-7652},
support = {RS-2024-00322278//Rural Development Administration/ ; RS-2021-NR059380//National Research Foundation of Korea/ ; RS-2021-NR060084//National Research Foundation of Korea/ ; },
mesh = {*Oryza/genetics/physiology/growth &amp; development ; *Seeds/genetics/growth &amp; development ; *Plant Infertility/genetics ; *Plant Breeding/methods ; Pollination/genetics ; Hybridization, Genetic ; CRISPR-Cas Systems ; Pollen/genetics ; Mutation ; },
abstract = {Efficient production of hybrid rice seeds requires male sterility systems to overcome the challenges of self-pollination. In this study, we identified male gametic transfer defect (GTrD)5 and GTrD9 as essential genes for the process of male gamete transmission in rice. Mutations in these genes cause partial male sterility by impairing pollen tube elongation, thereby reducing the fertilisation rate. Using CRISPR/Cas9 technology, we developed gtrd5flo5 and gtrd9flo5 double mutants that combined the gtrd5 and gtrd9 genes responsible for partial male sterility with the floury endosperm (FLO)5 gene, whose defects result in seeds with an opaque endosperm, allowing us to easily differentiate between hybrid and self-pollinated seeds. This two-line hybrid system demonstrated a high rate of hybrid seed production with significantly reduced self-pollination ratios. The hybrid seeds resulted in plants with increased height, panicle size and grain yield compared with those obtained from the parental lines and also displayed heterosis. Unlike the current two-line hybrid systems based on photoperiod- and thermosensitive genic male-sterile lines, our approach is independent of environmental factors, ensuring a stable and reliable hybrid seed production. This novel method simplifies seed production, enhances efficiency and offers a cost-effective and environmentally sustainable solution for hybrid rice breeding.},
}
@article {pmid41195558,
year = {2025},
author = {Mohammadi, R and Sakic, I and Jain, A and Mathur, P and Weiss, T and deMello, AJ and Stavrakis, S and Asghari, M},
title = {Ultra-High-Throughput Viscoelastic Squeezing for Mechanoporation and Efficient Intercellular Delivery.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {51},
pages = {e07981},
doi = {10.1002/smll.202507981},
pmid = {41195558},
issn = {1613-6829},
mesh = {Viscosity ; Humans ; *Elasticity ; *Electroporation/methods ; Microfluidics/methods ; CRISPR-Cas Systems/genetics ; },
abstract = {Cell-based therapies have transformed the treatment landscape for a range of diseases, leveraging both genome modification and cell reprogramming to create targeted treatments. Such therapies rely on the efficient internalization of biomolecules into living cells. Unfortunately, existing cargo delivery methods, such as those based on viral vectors and electroporation, are often compromised by cytotoxicity, poor delivery efficiencies, and low throughput. To overcome these limitations, a viscoelastic squeezing methodology is presented that uses viscoelastic microfluidics to perform mechanoporation in a rapid and contact-free manner. Through the control of the flow rates of a sample stream containing cells and cargo and a surrounding viscoelastic sheath flow, the width of a "virtual channel" formed between the two streams can be regulated. Elastic forces generated within this virtual channel are then used to deform contained cells and internalize user-defined payloads. The effectiveness and utility of the platform are assessed through the delivery of mRNA, plasmid DNA, and clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) ribonucleoprotein complexes into a variety of cell lines. Data confirms that viscoelastic squeezing provides for enhanced delivery efficiencies when compared to conventional poration techniques, whilst maintaining high cell viabilities and throughputs of 20 million cells per minute, and thus represents a powerful tool for cellular engineering.},
}
@article {pmid41197724,
year = {2025},
author = {Ito, C and Yamamoto, M and Yokota, N and Nakamura, N and Shiomi, M and Kizu, M and Ichinose, M and Fujii, S and Fujii, T and Fukunaga, R},
title = {Dissecting stress-activated protein kinase (SAPK)-signaling pathways using multiplex gene knockout HeLa cells.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {12},
pages = {110901},
pmid = {41197724},
issn = {1083-351X},
mesh = {Humans ; HeLa Cells ; Gene Knockout Techniques ; *MAP Kinase Signaling System ; Early Growth Response Protein 1/metabolism/genetics ; Phosphorylation/drug effects ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism/genetics ; *JNK Mitogen-Activated Protein Kinases/metabolism/genetics ; Proto-Oncogene Proteins c-fos/metabolism/genetics ; CRISPR-Cas Systems ; Anisomycin/pharmacology ; },
abstract = {The stress-activated protein kinase (SAPK) family consists of three c-Jun N-terminal kinase (JNK) and four p38 members. To explore the isotype-specific or overlapping roles of SAPK members, HeLa-derived multiplex SAPK-KO cells, such as JNK1/2/3-triple KO, p38α/β/γ/δ-quadruple KO, and JNK1/2/3/p38α/β/γ/δ-septuple KO cells, were generated using the CRISPR-Cas9 method. Also, "sole survivor" (ss)-hextuple KO cells, in which only one of seven SAPK genes remains intact, were generated. Western blot analyses using phospho-specific antibodies for SAPK substrates showed that serum- or anisomycin-induced phosphorylation of MAPKAPK2, MSK1, Mnk1, and CREB (cyclic AMP response element-binding protein)/ATF-1 largely depended on p38, whereas anisomycin-induced phosphorylation of c-Jun/JunD depended on JNK. Similar analyses using the ss-hextuple KO cell lines revealed that JNK1 rather than JNK2 contributed to the c-Jun/JunD phosphorylation, whereas p38α was the primary species phosphorylating the examined p38 substrates. Expression analyses of three typical immediate-early genes, c-Jun, EGR1, and c-Fos, demonstrated that JNK1 and JNK2 are responsible for c-Jun expression induced by interleukin-1β, tumor necrosis factor-α, UV-C, and heat shock (HS), whereas p38 is predominant in EGR1 expression induced by UV and HS and in c-Fos expression induced by the cytokines, UV, and HS. On the other hand, neither JNK nor p38 contributed significantly to the cytokine-induced EGR1 expression, suggesting complicated SAPK-signaling mechanisms that regulate immediate-early gene expression. Together, these results demonstrate the utility of the comprehensive multigene KO and ss-KO strategy in dissecting intracellular signaling pathways consisting of multiple family members.},
}
@article {pmid41198678,
year = {2025},
author = {Nukaga, S and Shiraishi, K and Hamabe, K and Mochizuki, A and Hamaguchi, Y and Ogawa, E and Le, NT and Shimada, Y and Ono, H and Nishinakamura, H and Kobayashi, Y and Hamamoto, J and Ui, A and Araki, M and Sagae, Y and Ohgino, K and Sugihara, K and Endo, S and Miyakoshi, J and Shiraishi, Y and Yasuda, H and Okuno, Y and Yoshida, T and Goto, Y and Ohe, Y and Watanabe, SI and Yatabe, Y and Nishikawa, H and Hamamoto, R and Kohno, T and Nakaoku, T},
title = {Mutation of CMTR2 in Lung Adenocarcinoma Alters RNA Alternative Splicing and Reveals Therapeutic Vulnerabilities.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9754},
pmid = {41198678},
issn = {2041-1723},
support = {JP23ak0101205//Japan Agency for Medical Research and Development (AMED)/ ; JP24ama221233//Japan Agency for Medical Research and Development (AMED)/ ; JP24ck0106905//Japan Agency for Medical Research and Development (AMED)/ ; 25K02539//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 22KJ3158//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 20H00545//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJCR1689//MEXT | Japan Science and Technology Agency (JST)/ ; JPMJCR18Y4//MEXT | Japan Science and Technology Agency (JST)/ ; },
mesh = {Humans ; *Adenocarcinoma of Lung/genetics/drug therapy ; *Lung Neoplasms/genetics/drug therapy/pathology ; *Alternative Splicing/genetics ; *Mutation ; Cell Line, Tumor ; *Methyltransferases/genetics/metabolism ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; },
abstract = {RNA splicing dysregulation has emerged as a hallmark of cancer and a promising therapeutic target; however, its full landscape in human solid cancer remains poorly characterized. To address this, we perform alternative splicing analyses using RNA-sequencing data from 751 lung adenocarcinoma samples from our cohort integrated with 519 samples from The Cancer Genome Atlas. Visualization of splicing patterns using t-distributed stochastic neighbor embedding reveals substantial inter-tumor heterogeneity driven by distinct molecular subtypes and histological differentiation. We identify a unique molecular subtype associated with inactivating mutations in CMTR2, which encodes Cap-specific mRNA (nucleoside-2'-O-)-methyltransferase 2. CMTR2 mutations are observed in 3.8% of cases and are predominantly truncating mutations, which form an isolated cluster within the splicing landscape. Intrinsic and CRISPR-Cas9-engineered CMTR2 mutations disrupt alternative splicing and sensitize cancer cells to sulfonamide-based RNA splicing modulators and immune checkpoint blockade therapy. Retrospective patient data confirm the increased sensitivity of CMTR2-deficient tumors to immune checkpoint blockade therapy. These findings uncover a previously unrecognized RNA splicing deficiency in human cancers and define a molecular subtype of lung adenocarcinoma driven by RNA splicing dysregulation, suggesting targets for therapeutic intervention in lung cancer.},
}
@article {pmid41198952,
year = {2025},
author = {Makarova, KS and Shmakov, SA and Wolf, YI and Mutz, P and Altae-Tran, H and Beisel, CL and Brouns, SJJ and Charpentier, E and Cheng, D and Doudna, J and Haft, DH and Horvath, P and Moineau, S and Mojica, FJM and Pausch, P and Pinilla-Redondo, R and Shah, SA and Siksnys, V and Terns, MP and Tordoff, J and Venclovas, &#x10c; and White, MF and Yakunin, AF and Zhang, F and Garrett, RA and Backofen, R and van der Oost, J and Barrangou, R and Koonin, EV},
title = {An updated evolutionary classification of CRISPR-Cas systems including rare variants.},
journal = {Nature microbiology},
volume = {10},
number = {12},
pages = {3346-3361},
pmid = {41198952},
issn = {2058-5276},
support = {R35 GM118160/GM/NIGMS NIH HHS/United States ; Intramural funds//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*Bacteria/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; *CRISPR-Cas Systems/genetics ; *Evolution, Molecular ; *Genetic Variation ; },
abstract = {The known diversity of CRISPR-Cas systems continues to expand. To encompass new discoveries, here we present an updated evolutionary classification of CRISPR-Cas systems. The updated CRISPR-Cas classification includes 2 classes, 7 types and 46 subtypes, compared with the 6 types and 33 subtypes in our previous survey 5 years ago. In addition, a classification of the cyclic oligoadenylate-dependent signalling pathway in type III systems is presented. We also discuss recently characterized alternative CRISPR-Cas functionalities, notably, type IV variants that cleave the target DNA and type V variants that inhibit the target replication without cleavage. Analysis of the abundance of CRISPR-Cas variants in genomes and metagenomes shows that the previously defined systems are relatively common, whereas the more recently characterized variants are comparatively rare. These low abundance variants comprise the long tail of the CRISPR-Cas distribution in prokaryotes and their viruses, and remain to be characterized experimentally.},
}
@article {pmid41199069,
year = {2025},
author = {Singh, S and Chaudhary, R and Tiwari, S},
title = {CRISPR/Cas9-mediated gene targeting at BBM2 locus demonstrates HDR-assisted precise knock-in in banana cv. Grand Naine.},
journal = {Plant cell reports},
volume = {44},
number = {12},
pages = {265},
pmid = {41199069},
issn = {1432-203X},
support = {BRIC-NABI Core Research Grant//BRIC-National Agri-Food and Biomanufacturing Institute (BRIC-NABI)/ ; BIRAC/Tech Transfer/08/I2/QUT-BBF//Biotechnology Industry Research Assistance Council/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Musa/genetics ; Gene Knock-In Techniques ; Gene Editing ; Green Fluorescent Proteins/genetics/metabolism ; *Plants, Genetically Modified ; Gene Targeting/methods ; Plant Proteins/genetics/metabolism ; Recombinational DNA Repair/genetics ; Sequence Analysis ; Genes, Plant ; },
abstract = {The present study demonstrates the first CRISPR/Cas-mediated precise knock-in of the eGFP gene at the BABYBOOM2 (GN-BBM2) locus in banana cv. Grand Naine, facilitating the detection of editing events in early embryogenic developmental stages. Genome editing has accelerated crop improvement programs by introducing targeted and precise genetic modifications. Among different tools, CRISPR/Cas-based genome editing has been widely used for enabling mutations through double-stranded breaks (DSBs), repaired either by non-homologous end joining (NHEJ) for gene knockouts or homology-directed repair (HDR) to generate knock-in events. While gene knockouts are well established in banana, efficient knock-in remains a major challenge due to low HDR activity, sterility, and the vegetatively propagated nature of banana. In the present study, we report the first successful CRISPR/Cas-based gene knock-in editing in banana by targeting the BABYBOOM2 (BBM2) gene, which encodes a transcription factor involved in somatic embryogenesis. The enhanced green fluorescent protein (eGFP) gene was precisely inserted at the BBM2 locus in banana cv. Grand Naine to enable visual detection during embryogenesis. In vitro validation showed ~ 95% target cleavage efficiency of the selected gRNA. The PCR-based screening and shift-in amplicon size analyses confirmed three edited lines (#3, #11, and #14) harboring eGFP knock-in at the targeted locus. Sequencing of the amplicon from these lines further confirmed the precise knock-in events. Hence, this study establishes a foundation for precise knock-in-based genome modification in banana and opens new avenues for targeted trait improvement in this important clonally propagated crop.},
}
@article {pmid41199512,
year = {2025},
author = {Friess, L and van Sinderen, D and Lee, C},
title = {A CRISPRi Gene Regulation System for Bifidobacteria.},
journal = {Microbial biotechnology},
volume = {18},
number = {11},
pages = {e70260},
pmid = {41199512},
issn = {1751-7915},
support = {883766//Horizon 2020 Framework Programme/ ; SFI/12/RC/2273-412 P1/SFI_/Science Foundation Ireland/Ireland ; SFI/12/RC/2273 P2/SFI_/Science Foundation Ireland/Ireland ; //APC Microbiome Institute/ ; },
mesh = {*CRISPR-Cas Systems ; Streptococcus thermophilus/genetics/enzymology ; *Gene Expression Regulation, Bacterial ; *Bifidobacterium/genetics ; Plasmids ; *Bifidobacterium breve/genetics ; },
abstract = {This work describes the development of a CRISPR interference (CRISPRi) system for targeted gene repression in bifidobacteria. We first validated the CRISPRi-based approach using Bifidobacterium breve strains engineered to express nuclease-dead orthologs of Cas9 and demonstrated that the CRISPR-Cas system from Streptococcus thermophilus is efficient at targeting both reporter and endogenous genes through the use of single guide RNAs corresponding to the gene of interest. We also developed a one-plasmid system for targeted gene repression in bifidobacteria and demonstrated its utility by targeting genes involved in nucleotide metabolism and carbohydrate metabolism in several species of bifidobacteria. Efficient gene repression was achieved across all tested bifidobacterial species without the requirement for extensive optimization of transformation parameters or sequence optimization to avoid restriction modification systems thus removing the key barriers to genetic manipulation in this genus. This CRISPRi system provides a novel approach to functional genomics in bifidobacteria which facilitates future mechanistic studies in these commercially important microbes.},
}
@article {pmid41200999,
year = {2025},
author = {Moreira, R and Nascimento, H and Martins, TMDM and Barbieri, G and Pires, P and Carvalho, LN and Rosa, LR and Almeida, A and Pessuti, CL and Ferrer, H and Gomes, J&#xc1;P and Goulart, E and Raia, S and Belfort, R},
title = {Porcine Corneal Tissue and Xenozoonotic Risks: A Review of the Current Evidence.},
journal = {Xenotransplantation},
volume = {32},
number = {6},
pages = {e70068},
pmid = {41200999},
issn = {1399-3089},
support = {//by FAPESP/ ; },
mesh = {Animals ; *Transplantation, Heterologous/methods/adverse effects ; Swine ; Humans ; *Corneal Transplantation/methods/adverse effects ; *Cornea/microbiology/virology ; Heterografts/microbiology ; },
abstract = {Corneal opacities affect millions worldwide, with corneal transplantation as the primary treatment. However, donor shortages remain a challenge, leaving thousands waiting for transplants. Xenotransplantation using porcine corneas has emerged as a promising alternative due to anatomical and physiological similarities with human corneas. Advances in CRISPR/Cas technology enable genetic modifications to address immune rejection and zoonotic risks. A key concern is xenozoonosis, the transmission of infectious agents from pigs to humans. Pathogens such as porcine endogenous retroviruses (PERVs), porcine cytomegalovirus (PCMV), and bacteria pose potential risks. While PERVs can infect human cells in vitro, no transmission has been documented in vivo. Regulatory bodies, including the WHO and IXA, have established guidelines for monitoring and clinical trials. The first human corneal xenotransplantation trials in South Korea and China are underway. Enhanced biosecurity measures in tissue banks have reduced microbial contamination, improving safety. Corneal xenotransplantation presents lower risks compared to solid organ xenotransplants. With ongoing research, stringent regulations, and improved pathogen-free animal models, this technique could become a viable clinical option. Future human trials will provide crucial insights into its long-term safety and effectiveness.},
}
@article {pmid41201092,
year = {2025},
author = {Wu, Y and Zhong, A and Ramirez, B and Wai, SS and Zhou, T},
title = {Protocol to rapidly verify silent gene reporter in human pluripotent stem cells using CRISPR activation.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104164},
pmid = {41201092},
issn = {2666-1667},
support = {P30 CA008748/CA/NCI NIH HHS/United States ; UM1 HG012654/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Pluripotent Stem Cells/metabolism/cytology ; *Genes, Reporter/genetics ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Transcriptional Activation/genetics ; },
abstract = {Validating human pluripotent stem cell (hPSC) reporters targeting silent genes typically requires inducing gene expression through cell state transitions, which can be time consuming and complex. Here, we present a rapid workflow to verify reporter knockins at unexpressed loci in hPSCs using CRISPR-mediated transcriptional activation (CRISPRa). We detail steps for designing and cloning single-guide RNA (sgRNA), delivery of CRISPRa into reporter cells, and detection of reporter gene. In this protocol, we illustrate this process using KLF17-GFP reporter hPSCs. For complete details on the use and execution of this protocol, please refer to Wu et al.[1].},
}
@article {pmid41201289,
year = {2025},
author = {Liu, T and Su, C and Yan, X and Zhang, Y and Wang, Y and Duan, K},
title = {Creating Hypoallergenic and Low Antinutritional Soybeans through CRISPR/Cas12a-Mediated Multiplex Gene Editing.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {48},
pages = {30922-30932},
doi = {10.1021/acs.jafc.5c08935},
pmid = {41201289},
issn = {1520-5118},
mesh = {*Glycine max/genetics/immunology/chemistry/metabolism ; Animals ; *Gene Editing ; *CRISPR-Cas Systems ; Mice ; *Soybean Proteins/genetics/immunology ; *Plants, Genetically Modified/genetics/immunology/metabolism/chemistry ; *Allergens/immunology/genetics ; Humans ; Food Hypersensitivity/immunology ; Female ; },
abstract = {Soybean protein is a valuable plant-based nutrition source for food. However, allergens and antinutritional factors like Gly Bd 30k (P34), Kunitz trypsin inhibitor (KTI), and soybean agglutinin (LE) pose risks. In this study, we employed the CRISPR/Cas12a system to generate hypoallergenic and low antinutritional soybean lines without agronomic traits penalties. The edited lines exhibited markedly reduced levels and enzymatic activity of trypsin inhibitors, as well as soybean agglutinin content. Feeding mice with these edited soybeans, deficient in P34, KTI, and LE proteins, significantly alleviated allergenic responses. This research provides a promising resource of hypoallergenic and low antinutritional soybeans for applications in food production, animal feed, and immunotherapy.},
}
@article {pmid41201433,
year = {2026},
author = {Jetley, U and Balwani, I and Sharma, P and Miller, IC and Luther, A and Dutta, I and Saravanan, N and Goel, S and Zhang, Q and Zhang, B and Kilic, O and Liu, B and Han, BW and Liu, D and Schultes, B and Prodeus, A and Zhang, Y},
title = {A differentiated and durable allogeneic strategy applicable to cell therapies.},
journal = {Cytotherapy},
volume = {28},
number = {3},
pages = {101991},
doi = {10.1016/j.jcyt.2025.10.001},
pmid = {41201433},
issn = {1477-2566},
mesh = {Humans ; CRISPR-Cas Systems/genetics ; *Cell- and Tissue-Based Therapy/methods ; Gene Editing/methods ; Killer Cells, Natural/immunology ; *T-Lymphocytes/immunology ; Transplantation, Homologous ; Cell Differentiation ; *Immunotherapy, Adoptive/methods ; },
abstract = {BACKGROUND AIMS: Autologous T-cell therapies have shown profound clinical responses; however, their widespread use has been limited primarily as the result of their individualized manufacturing requirements.<br><br>METHODS: To develop a persistent "off-the-shelf" allogeneic (Allo) approach, a multiplex Nme2Cas9-based cytosine base editor was deployed to knockout select human leukocyte antigens (HLA) class I and II alleles (HLA-A, HLA-B and the class II transactivator [CIITA]) while retaining HLA-C to protect from natural killer (NK) cell rejection.<br><br>RESULTS AND CONCLUSION: Matching the residual HLA-C allele from homozygous donors to the host prevented rejection of the donor T cells by allogeneic host T and NK cells. Site-specific integration of a tumor-specific CAR or TCR into the TRAC locus using SpyCas9 nuclease and an adeno-associated virus template allowed for a high localized insertion rate while simultaneously removing the endogenous TCR and preventing graft-versus-host disease. Using an optimized T-cell engineering process involving orthogonal CRISPR/Cas9 cleavage and base editors coupled with lipid nanoparticle delivery, we achieved efficient production of Allo-CAR T cells with high editing rates and cell expansion in a scalable manner. These allogeneic T cells demonstrated comparable functional activity to their autologous counterparts in preclinical assays. Moreover, this gene-editing approach significantly minimized the occurrence of chromosomal aberrations. This promising allogeneic approach also has been applied to induced pluripotent stem cells (iPSCs) with triple edits targeting HLA-A, HLA-B and CIITA (TKO). Pancreatic progenitor cells or cardiomyocytes derived from TKO iPSCs were protected from host peripheral blood mononuclear cell-mediated rejection when matched for HLA-C, suggesting potential applications in regenerative medicine applications.},
}
@article {pmid41201953,
year = {2025},
author = {Fan, J and Huang, L and Chen, J and Jiang, Y and Du, X and Wang, L and Ding, K and Huang, J and Qiu, S and Liu, H and Song, H},
title = {Highly Sensitive Field Detection Technology for Anthrax Based on the CRISPR/Cas13a System.},
journal = {Microbial biotechnology},
volume = {18},
number = {11},
pages = {e70240},
pmid = {41201953},
issn = {1751-7915},
support = {32141003//National Science Foundation of China/ ; 32300080//National Science Foundation of China/ ; },
mesh = {*Bacillus anthracis/genetics/isolation &amp; purification ; *Anthrax/diagnosis/microbiology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Humans ; Point-of-Care Testing ; },
abstract = {In this study, we established a highly sensitive on-site detection technology for Bacillus anthracis. Firstly, we integrated Multiple Enzyme Isothermal Rapid Amplification (MIRA) with the clustered regularly interspaced short palindromic repeats (CRISPR) /associated protein 13a (CRISPR/Cas13a) detection system to develop a highly sensitive CRISPR/Cas13a assay. After testing crRNA selection, MIRA primers, reaction temperature, and CRISPR detection conditions, the CRISPR/Cas13a detection system employing dual crRNAs achieved a detection limit of 1000 copies/mL for B. anthracis. Quantitative analysis was additionally attempted. Compared with other common respiratory pathogens, the assay demonstrated high specificity. In clinically simulated samples, all 20 positive specimens were correctly identified, and all 13 negatives were unambiguously classified as negative. Based on these findings, we established a CRISPR point-of-care testing technology. By developing a CRISPR point-of-care testing device together with a tested lyophilised reagent system, the device achieved a detection limit of 250 copies/mL and delivered results within 30 min. All positive samples were accurately identified, and every negative sample was classified as negative. Consequently, this study presents a highly sensitive and portable technology for on-site detection of B. anthracis. It holds significant value for on-site detection of emerging infectious diseases.},
}
@article {pmid41201956,
year = {2026},
author = {Sinha, R and Patil, RV and Romano, R and Sharma, D and Lee, E and Perriman, R and Takeda, S and Lesch, BJ and Yao, Z and Liu, YL and Cromer, MK and Porteus, MH and Bertaina, A},
title = {Novel humanized loss-of-function NF1 mouse model of juvenile myelomonocytic leukemia.},
journal = {Blood advances},
volume = {10},
number = {3},
pages = {837-849},
pmid = {41201956},
issn = {2473-9537},
mesh = {Animals ; *Leukemia, Myelomonocytic, Juvenile/genetics/pathology/metabolism/therapy ; Mice ; Disease Models, Animal ; Humans ; *Neurofibromin 1/genetics/metabolism ; *Loss of Function Mutation ; Granulocyte-Macrophage Colony-Stimulating Factor ; Hematopoietic Stem Cells/metabolism ; CRISPR-Cas Systems ; },
abstract = {Juvenile myelomonocytic leukemia (JMML) is a fatal pediatric cancer characterized by classical features such as splenomegaly, monocytosis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity, with RAS pathway mutations being the major drivers. Mutations causing loss-of-function of the Neurofibromin 1 gene (NF1LOF) occur in ∼20% of patients with JMML. NF1LOF drives upregulation of RAS/MAPK/PI3K pathways that leads to aggressive proliferation/differentiation of immature myeloid cells. Hematopoietic stem cell transplantation is the only curative option, but relapse occurs in ∼50% of patients, indicating an urgent need for novel and targeted therapeutic strategies. However, low patient sample availability and a lack of reliable disease models have made it difficult to study and treat JMML. Using CRISPR/Cas9, we have generated NF1LOF in human umbilical cord blood-derived hematopoietic stem and progenitor cells (HSPCs). We achieved a high gene knockout rate of ∼89% and concomitant loss of NF1 protein in the modified HSPCs. Importantly, NF1LOF cells displayed marked GM-CSF hypersensitivity in in vitro colony-forming unit assays, mirroring JMML. When transplanted into NSG-SGM3 mice, they caused rapid lethality (median survival of 32 days), myeloid expansion, tissue infiltration (spleen, liver, and lungs), and specific upregulation of RAS/MAPK pathway and STAT5 genes, consistent with patient profiles. This first humanized NF1LOF mouse model recapitulates key JMML features, enabling investigation of disease mechanisms and targeted therapies.},
}
@article {pmid41202069,
year = {2025},
author = {Verkuyl, C and Belotserkovsky, A and Zerbes, T and Williams, D and Krishnan, MR and Zhu, S and Grunnesjӧ, S and Eid, S and Zhang, C and Zhao, W and Xu, L and Lin, E and O'Shea, T and Draper, B and Jungman, A and Most, P and Schmitt-Ulms, G},
title = {Toward an all-in-one recombinant adeno-associated virus vector for functionally ablating the prion gene using CRISPR-Cas technology.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0336578},
pmid = {41202069},
issn = {1932-6203},
mesh = {*Dependovirus/genetics ; *CRISPR-Cas Systems/genetics ; Animals ; *Genetic Vectors/genetics ; Mice ; Humans ; Gene Editing/methods ; Genetic Therapy/methods ; Prion Diseases/genetics/therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; },
abstract = {Any strategy that can selectively and persistently lower the brain levels of the cellular prion protein (PrPC) is expected to extend survival in prion diseases. Recent advances in the virus-mediated delivery of gene therapies prompted us to explore if a recombinant adeno-associated virus (rAAV) vector delivering a CRISPR-Cas-based gene editor can be devised that induces a functional knockout of the prion gene. Whereas the eventual objective is to assess the therapeutic potency of an optimized vector in prion-infected mice, in this proof-of-concept study, we evaluated tools and methods that are suited to achieve this goal. The result of these efforts is a first-generation all-in-one rAAV vector that codes for a prion gene-specific guide RNA and a small Cas9 endonuclease, whose expression is controlled by a truncated neural cell adhesion molecule 1 (NCAM1) promoter that is active in PrPC expressing cells. We also constructed a second rAAV vector coding for a prion gene-specific 'traffic light reporter' (TLR). The TLR can be used to monitor prion gene-editing efficacy by coding for red and green fluorescent proteins separated by a segment of the prion gene that is targeted by the gene editor. For the purification of AAVs, we adopted a robust and scalable rAAV vector assembly pipeline and undertook proof-of-concept prion gene editing experiments in human cells and mice, which to date yielded prion gene editing rates of approximately 20% and 5%, respectively. Finally, we compared brain distributions of rAAV vectors following intrathalamic versus retro-orbital injection, and selected the 9P31 capsid for future studies based on a 7.5-fold higher heterologous gene expression level as compared to the PHP.eB capsid.},
}
@article {pmid41202131,
year = {2025},
author = {Liu, Y and Ban, Y and Gao, D},
title = {Oligo-CALL: A next-generation barcoding platform for studying resistance to targeted therapy.},
journal = {Science advances},
volume = {11},
number = {45},
pages = {eadw9990},
pmid = {41202131},
issn = {2375-2548},
support = {R01 CA244413/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Drug Resistance, Neoplasm/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Lung Neoplasms/genetics/drug therapy/pathology ; Single-Cell Analysis ; Molecular Targeted Therapy ; Proto-Oncogene Proteins p21(ras)/genetics/antagonists &amp; inhibitors ; },
abstract = {Understanding therapy resistance requires deconvolving heterogeneous cell populations and tracking clonal trajectories. While CRISPR-based cellular barcoding is powerful for lineage tracing, many platforms suffer from low efficiency and limited compatibility with single-cell transcriptomics. We developed Oligo-CALL (Oligonucleotide-inducible CRISPR transcriptional activator-Assisted Lineage Labeling), an advanced barcoding system enabling precise lineage tracing, live clone isolation, and seamless integration with single-cell RNA sequencing. Applied to lung cancer cells treated with a KRAS[G12C] inhibitor, Oligo-CALL identified clones consistently enriched posttreatment, supporting a model of predestined resistance. Oligo-CALL achieved >95% efficiency in linking lineage identity to transcriptomes, uncovering diverse clone-specific pathways with underlying resistance. Paired analysis of barcode-matched clones from naïve and resistant populations revealed transient and fixed resistance phenotypes. Notably, DNA repair pathways are recurrently altered in resistant clones, and inhibition of poly(adenosine 5'-diphosphate-ribose) polymerase synergizes with KRAS G12C inhibition to overcome resistance. Together, Oligo-CALL provides a versatile platform for dissecting lineage evolution and molecular dynamics of targeted therapy resistance.},
}
@article {pmid41202215,
year = {2025},
author = {Wang, T and Brogan, DJ and Zawalick, NM and Chen, F and Mkrtchian, Y and Komor, AC and Akbari, OS},
title = {CTRL Enables Gene-Specific RNA Regulation Using CRISPR-Cas7-11.},
journal = {ACS synthetic biology},
volume = {14},
number = {11},
pages = {4597-4605},
doi = {10.1021/acssynbio.5c00658},
pmid = {41202215},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Messenger/genetics/metabolism ; Synthetic Biology/methods ; *Gene Expression Regulation/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Advancements in synthetic biology have enabled the development of precision gene expression technologies for comprehensive investigations of biological and biochemical networks. Here, we describe the development of a refined and innovative tool, CRISPR-Cas Transgenic Repressible eLement (CTRL), which utilizes the direct repeat processing activity of the recently discovered CRISPR-Cas7-11 effector to site-specifically target synthetic mRNA molecules. We demonstrate that CTRL exhibits high efficiency, tunable regulation of expression, and gene-specific repression of mRNA and protein expression. We engineered multiple permutations of the Cas7-11 effector that differ in their ability to reduce gene expression, suggesting flexibility for the application of choice. CTRL is a novel variation on gene repression technology that exhibits broad applicability across multiple model systems.},
}
@article {pmid41202468,
year = {2025},
author = {Zingarelli, F and Nanni, J and Cristiano, G and Zannoni, L and Curti, A},
title = {CRISPR-Cas9 in acute myeloid leukaemia: Current state-of-art and future perspectives.},
journal = {Current opinion in pharmacology},
volume = {85},
number = {},
pages = {102582},
doi = {10.1016/j.coph.2025.102582},
pmid = {41202468},
issn = {1471-4973},
mesh = {Humans ; *Leukemia, Myeloid, Acute/genetics/therapy/diagnosis ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *Genetic Therapy/methods ; },
abstract = {CRISPR-Cas9 gene editing technology has gained attention as a new, reliable and manageable tool for the treatment of previously incurable monogenic diseases. Besides exciting results in this setting, ethical, safety and crucial technical issues have not been fully clarified. More importantly, the role of this potent editing tool in the context of a genetically complex and heterogeneous hematologic malignancy such as acute myeloid leukemia (AML) has not yet been defined to date. In this review we aim to summarize and exploring the ultimate CRISPR-cas9 based strategies for diagnosis, risk stratification and treatment in the context of AML.},
}
@article {pmid41202696,
year = {2025},
author = {Ito, S and Nakamura, K and Murata, K and Nakajima, R and Kanou, M and Koketsu, M and Yamana, K and Yamanouchi, K and Ueda, H},
title = {Generation of Cre/LoxP-mediated extracellular TurboID knock-in rats with CRISPR/Cas9 system.},
journal = {Biochemical and biophysical research communications},
volume = {791},
number = {},
pages = {152898},
doi = {10.1016/j.bbrc.2025.152898},
pmid = {41202696},
issn = {1090-2104},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Gene Knock-In Techniques/methods ; Rats ; *Integrases/metabolism/genetics ; Biotinylation ; Cell Membrane/metabolism ; },
abstract = {The plasma membrane plays a central role in regulating signal transduction from the extracellular environment to the intracellular space and represents a major site of drug targeting. Proximity-dependent biotinylation with biotin ligases such as BioID and its derivatives, including TurboID and AirID, enables identification of novel protein‒protein interactions by fusion to a target protein. In vivo application of biotin ligases for cell membrane analysis has been attempted; however, methods for expressing biotin ligases on the target cell membrane remain largely limited to viral vector delivery. In this study, we inserted a Cre-dependent TurboID expression cassette into the rat genome to express TurboID on the cell surface via the CRISPR/Cas9 system. We generated TurboID knock-in (KI) rats that express TurboID on the cell surface, enabling biotinylation of extracellular proteins. The TurboID KI rats thus provide a valuable model for in vivo analysis of cell-surface molecules and may facilitate identification of novel drug targets or antigens.},
}
@article {pmid41202713,
year = {2026},
author = {Wang, Y and Xiang, L and Su, Z},
title = {CRISPR/Cas9 gene editing in muscle-related genetic disorders: Restoring function and exercise capacity.},
journal = {Tissue &amp; cell},
volume = {98},
number = {},
pages = {103217},
doi = {10.1016/j.tice.2025.103217},
pmid = {41202713},
issn = {1532-3072},
mesh = {Animals ; Humans ; Clinical Trials as Topic ; *CRISPR-Cas Systems ; Exercise Tolerance ; *Genetic Therapy ; *Motor Neuron Disease/therapy ; *Muscular Dystrophies/therapy ; Recovery of Function ; Gene Editing ; },
abstract = {Due to their progressive and debilitating character and lack of curative therapies, muscle-related hereditary illnesses such as muscular dystrophies (MDs), myopathies, and motor neuron diseases need immediate care. A pressing need exists for novel therapeutics that can accurately and persistently correct the underlying mutations in muscle-related genetic disorders, as existing treatments are mostly palliative rather than addressing the underlying genetic cause. Traditional therapies for genetic diseases, such as hereditary myopathies and MDs, are often insufficient; however, the advent of CRISPR/Cas9 technology has altered this trajectory. While other evaluations have compiled the potential of gene editing, this one compiles the latest findings on CRISPR/Cas9 applications for musculoskeletal disorders. With an emphasis on the repair of recessive and dominant-negative mutations, we provide a critical evaluation of the development of new therapeutic vectors and in vivo editing techniques that have gone from conceptual models to preclinical triumphs. More specifically, we address the issues of efficient delivery, off-target effects, and immunological responses specific to muscle tissue, all of which are ongoing challenges. By facilitating targeted correction of pathogenic mutations, CRISPR/Cas9 holds promise for restoring muscle integrity and functional performance, potentially translating into improved exercise capacity. This study offers a prospective view on the near-term therapeutic promise of CRISPR/Cas9 for treating crippling muscle diseases by combining recent advances with an evaluation of the translational route.},
}
@article {pmid41202787,
year = {2026},
author = {Xiao, X and Zhong, Y and Xie, H and Liao, H and Wang, H and Liu, H and Liang, F and Chen, J and Zhong, H and Chen, Z and Yu, L},
title = {A reverse transcription-free CRISPR/Cas12a biosensor for ultrasensitive detection of SARS-CoV-2 variants.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129072},
doi = {10.1016/j.talanta.2025.129072},
pmid = {41202787},
issn = {1873-3573},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *COVID-19/diagnosis/virology ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Mutation ; RNA, Viral/genetics ; Reverse Transcription ; CRISPR-Associated Proteins/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {In this work, we developed a reverse transcription-free self-primer isothermal exponential amplification reaction (RTF-SP-EXPAR) combined with a CRISPR/Cas12a biosensor for the simultaneous detection of single- and double-stranded amplification products of SARS-CoV-2 mutant variants. SP-EXPAR can recognize RNA targets directly, simultaneously generate double-stranded DNA products containing PAM sequences, and single-stranded DNA products without PAM sequences. Both types of SP-EXPAR products can be recognized by the crRNA and produce fluorescent signals, thereby enhancing detection sensitivity. This RTF-SP-EXPAR-CRISPR/Cas12a biosensor enables the detection of SARS-CoV-2 mutations within 1 h, achieving a detection limit of 7.49 aM and a dynamic range of 10 aM to 10 pM. This method shows high specificity in differentiating mutant variants from wild-type sequences. For the detection of 106 clinical samples, this RTF-SP-EXPAR-CRISPR/Cas12a assay demonstrates 100 % sensitivity and 100 % specificity compared with DNA sequencing results. These findings highlight our proposed assay's strong applicability for the application of RNA samples without reverse transcription.},
}
@article {pmid41202983,
year = {2026},
author = {Hocq, R and Chartier, G and Lopes Ferreira, N and Wasels, F},
title = {CRISPR/anti-CRISPR genome editing in Clostridium beijerinckii.},
journal = {Journal of biotechnology},
volume = {409},
number = {},
pages = {165-169},
doi = {10.1016/j.jbiotec.2025.11.002},
pmid = {41202983},
issn = {1873-4863},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Clostridium beijerinckii/genetics/drug effects ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Listeria monocytogenes/genetics ; Genome, Bacterial ; CRISPR-Associated Protein 9/genetics ; },
abstract = {The development of CRISPR technologies has revolutionized genome editing. However, in bacteria, CRISPR-based methods can be difficult to implement due to the cytotoxicity of CRISPR-associated proteins, which often impair or entirely prevent transformation. In this work, we combine inducible expression of classical CRISPR-Cas9 components with the anti-CRISPR protein AcrIIA4 from Listeria monocytogenes to tightly regulate Cas9 activity. Using this approach, we demonstrate efficient and iterative genome editing in the genetically recalcitrant Clostridium beijerinckii DSM 6423. While deletion of upp alone was not sufficient to render the strain sensitive to 5-fluorouracil, the additional deletion of a second gene involved in the uracil salvage pathway conferred resistance to the drug and validated our gene editing strategy. Collectively, our results show that CRISPR/anti-CRISPR systems can overcome a key limitation of CRISPR-based genome editing and may offer a broadly applicable strategy for engineering otherwise intractable bacterial species.},
}
@article {pmid41203276,
year = {2025},
author = {Ding, Y and Guo, W},
title = {[Advances in the application of genome editing technologies in plant pathogenic fungi].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {10},
pages = {3683-3700},
doi = {10.13345/j.cjb.250539},
pmid = {41203276},
issn = {1872-2075},
mesh = {*Gene Editing/methods ; *Fungi/genetics/pathogenicity ; *CRISPR-Cas Systems/genetics ; *Plant Diseases/microbiology ; *Plants/microbiology ; *Genome, Fungal/genetics ; },
abstract = {Filamentous fungi represent an important group of eukaryotic microorganisms with diverse ecological functions and ubiquitous distribution in various ecosystems. Among them, many species are closely associated with agriculture, functioning as major plant pathogens that cause yield losses and produce mycotoxins to compromise both the quality and safety of agricultural products. In recent years, the CRISPR/Cas system has emerged as a powerful and programmable genome editing tool, and it has been extensively applied to the genetic study of plant pathogenic fungi. This technology has greatly facilitated the investigation of pathogenic mechanisms, mycotoxin biosynthetic pathways and key gene functions, antifungal resistance, and rapid pathogen detection. This review summarizes the development of CRISPR/Cas systems and the key strategies for their application in plant pathogenic fungi and makes an outlook on the practical deployment. With the continuous advancement of gene editing technologies, emerging fungal-adapted editing systems hold great promise for advancing functional genomics and enabling innovations in disease-resistant breeding and sustainable crop protection.},
}
@article {pmid41203292,
year = {2025},
author = {Yu, D and Duan, C},
title = {[Epigenetics and precise crop breeding for resistance].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {10},
pages = {3918-3938},
doi = {10.13345/j.cjb.250513},
pmid = {41203292},
issn = {1872-2075},
mesh = {*Epigenesis, Genetic/genetics ; *Crops, Agricultural/genetics ; *Plant Breeding/methods ; DNA Methylation/genetics ; Gene Editing ; *Disease Resistance/genetics ; CRISPR-Cas Systems ; },
abstract = {Epigenetics refers to a heritable phenomenon that dynamically modulates gene expression without altering the DNA sequence, through molecular mechanisms such as DNA methylation, histone modification, non-coding RNA, chromatin remodeling, and RNA modifications. In plants, these modifications are extensively involved in key biological processes, including flowering time, gametogenesis, stress responses, and immune defenses. Over the past few decades, the research on epigenetics has gradually shifted from fundamental studies primarily conducted in Arabidopsis thaliana to investigations in various crop species such as rice and tomato. This transition has revealed the multifaceted roles of epigenetic regulation in shaping agronomic traits. This review integrates current knowledge of epigenetic regulatory mechanisms and their functions in plant responses to both biotic and abiotic stresses. Epigenetic editing tools such as CRISPR-dCas9 enable targeted DNA methylation or histone acetylation. Emerging transformation technologies, including magnetic nanoparticles and virus-based delivery systems, have the potential to overcome the bottlenecks of plant regeneration, offering new possibilities for precise epigenetic editing. In future agriculture, it is essential to further elucidate multi-layered epigenetic regulatory mechanisms at the single-cell level, develop efficient delivery systems, and leverage artificial intelligence to advance the application of epigenetic breeding for sustainable agricultural development.},
}
@article {pmid41203361,
year = {2025},
author = {Xia, N and Gupta, N},
title = {Function and therapeutic potential of transketolase enzymes in parasitic protists.},
journal = {Methods in enzymology},
volume = {722},
number = {},
pages = {97-115},
doi = {10.1016/bs.mie.2025.07.010},
pmid = {41203361},
issn = {1557-7988},
mesh = {*Toxoplasma/enzymology/genetics/pathogenicity ; *Transketolase/genetics/metabolism ; Animals ; Mice ; *Protozoan Proteins/genetics/metabolism ; *Toxoplasmosis/parasitology/therapy ; CRISPR-Cas Systems ; Recombinant Proteins/genetics/metabolism ; Gene Editing ; Humans ; },
abstract = {Transketolase (TKT), the rate-limiting enzyme in the non-oxidative branch of the pentose phosphate pathway (PPP), catalyzes the reversible transfer of glycolaldehyde moieties between sugar phosphates. Here, we present wide-ranging methods to study TKT function in a clinically-relevant and globally-prevalent model intracellular pathogen, Toxoplasma gondii. The experimental framework utilizes heterologous expression of recombinant TgTKT proteins in a bacterial system, anti-TgTKT antibody production, CRISPR/Cas9-assisted genome editing to generate TgTKT-null mutants, and in-depth phenotyping, including [13]C-based metabolomic analysis to determine the carbon flux in the acutely-infectious tachyzoite stage of T. gondii. The phenotypic studies comprise the parasite proliferation in vitro and virulence assessment in a murine model of acute toxoplasmosis. The data suggest TgTKT as a metabolic rheostat regulating nucleotide biosynthesis and enabling adaptation of this highly promiscuous parasite in different nutritional environments. Last but not least, the work presents the therapeutic potential of the TgTKT-null mutant as a metabolically-attenuated vaccine, highlights the evolutionary divergence of TgTKT proteins, and provides a robust foundation to investigate TgTKT orthologs in other pathogenic protozoan organisms.},
}
@article {pmid41203686,
year = {2025},
author = {Sun, Y and Qu, K and Corsi, GI and Anthon, C and Pan, X and Xiang, X and Jensen, LJ and Lin, L and Luo, Y and Gorodkin, J},
title = {Deep learning models simultaneously trained on multiple datasets improve base-editing activity prediction.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9821},
pmid = {41203686},
issn = {2041-1723},
support = {NNF21OC0068988//Novo Nordisk Fonden (Novo Nordisk Foundation)/ ; },
mesh = {*Deep Learning ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Neural Networks, Computer ; Humans ; Software ; },
abstract = {CRISPR-derived base editors (BE) enable precise single nucleotide substitution without introducing double-stranded DNA breaks. Apart from the base editing enzymes, efficient base editing strongly depends on both the CRISPR guide RNA (gRNA) efficiency and the edited position. Here, we show that the accuracy of BE gRNA design can be significantly improved by generating more data and by introducing deep neural networks trained on multiple different datasets simultaneously. Generating ~20,000 gRNAs for A•T to G•C and C•G to T•A conversions, we present such deep learning models, which also allow users to do dataset-aware predictions. The methods are available online and as stand-alone software.},
}
@article {pmid41203870,
year = {2025},
author = {Chen, X and Xiao, L and Wang, Q and Zhou, L and Xu, Y and Zhou, C and Dai, M and Wang, F and Xu, H and Luo, D and Sirois, P and Li, K and Liao, D and Zhang, J},
title = {Coloring target and off-target effects of genetically modified nucleases by blue &amp; white colony assays.},
journal = {Biotechnology letters},
volume = {47},
number = {6},
pages = {128},
pmid = {41203870},
issn = {1573-6776},
support = {SKY2021047//the Suzhou Science and Technology Planning Project/ ; ND2022B04//Program of Clinical Research Center of Neurological Disease/ ; NS2021016//Funda&#xe7;&#xe3;o de Apoio ao Desenvolvimento do Ensino, Ci&#xea;ncia e Tecnologia do Estado de Mato Grosso do Sul/ ; },
mesh = {*Gene Editing/methods ; Escherichia coli/genetics ; *CRISPR-Cas Systems ; *Endonucleases/genetics/metabolism ; Humans ; beta-Galactosidase/genetics/metabolism ; },
abstract = {More sensitive evaluation of the off-target effects of gene editing nucleases is crucial for human gene therapy. Here we report chromogenic assays designed for sensitive evaluation of gene editing activities using CRISPR/Cas9 test system. Based on beta-galactosidase alpha complementation, qualitative and quantitative evaluations of the target and off-target effects of CRISPR/Cas9 were well established through the color alteration of the E.coli colonies. In addition to target effect analysis, these new assays provide extremely sensitive and efficient tool to profile the off-target effects with one or more bases mismatched between the targets and the gRNAs. Moreover, these assays allow the identification of gene editing effects for off-targets with one base mismatched PAM sites.},
}
@article {pmid41205588,
year = {2025},
author = {Kumar, KRR},
title = {Plant genome editing goes viral: balancing innovation and biosafety.},
journal = {Trends in biotechnology},
volume = {43},
number = {11},
pages = {2684-2685},
doi = {10.1016/j.tibtech.2025.09.006},
pmid = {41205588},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *Genome, Plant/genetics ; *Plants, Genetically Modified/genetics ; Genetic Vectors/genetics ; CRISPR-Cas Systems ; Containment of Biohazards ; },
abstract = {Weiss and colleagues demonstrated a breakthrough in transgene-free heritable genome editing using viral vectors. While promising for controlled laboratory applications, the open-field use envisaged by Sajjad and colleagues raises ecological and biosafety concerns. Rigorous risk assessment is essential to harness innovation responsibly while safeguarding biodiversity and public trust.},
}
@article {pmid41205600,
year = {2025},
author = {Saini, M and Castro-Giner, F and Hotz, A and Sznurkowska, MK and Nüesch, M and Cuenot, FM and Budinjas, S and Bilfeld, G and Ildız, ES and Strittmatter, K and Krol, I and Diamantopoulou, Z and Paasinen-Sohns, A and Waldmeier, M and Cássio, R and Kreutzer, S and Kontarakis, Z and Gvozdenovic, A and Aceto, N},
title = {StealTHY: An immunogen-free CRISPR platform to expose concealed metastasis regulators in immunocompetent models.},
journal = {Cell},
volume = {188},
number = {26},
pages = {7591-7609.e32},
doi = {10.1016/j.cell.2025.10.007},
pmid = {41205600},
issn = {1097-4172},
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; Humans ; *Neoplasm Metastasis/genetics ; Mice ; Cell Line, Tumor ; Immunocompetence ; *Neoplasms/genetics/immunology/pathology ; Mice, Inbred C57BL ; Female ; },
abstract = {CRISPR screens have become standard gene discovery platforms in various contexts, including cancer. Yet commonly available CRISPR-Cas9 tools are increasingly recognized as unfit for in vivo investigations in immunocompetent contexts, due to broad immunogenicity of bacterial nucleases and reporters. Here, we show how conventional CRISPR screens in tumor grafts are systematically jeopardized by immunoediting in syngeneic and humanized immunocompetent hosts, resulting in iatrogenic clonal dropouts and ultimately compromising target identification. To resolve this, we present StealTHY, an immunogen-free CRISPR platform compatible with virtually all immunocompetent designs, enabling preservation of clonal architecture and exposing previously concealed cancer vulnerabilities. Among these, we identify the AMH-AMHR2 axis as a formerly unappreciated metastasis target. Thus, with StealTHY, we provide a new resource to expand the applicability of CRISPR screens to immunocompetent models, including humanized tumor grafts, revealing metastasis regulators of therapeutic relevance.},
}
@article {pmid41205603,
year = {2025},
author = {Regan, SB and Medhi, D and Xu, Y and White, TB and Jiang, YZ and Kim, JE and Wang, SC and Deng, Q and Jia, S and Baasan, D and Connelly, JP and Chang, TC and Pruett-Miller, SM and Jasin, M},
title = {Megabase-scale loss of heterozygosity provoked by CRISPR-Cas9 DNA double-strand breaks.},
journal = {Molecular cell},
volume = {85},
number = {22},
pages = {4119-4137.e10},
pmid = {41205603},
issn = {1097-4164},
support = {F31 CA268775/CA/NCI NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R35 CA253174/CA/NCI NIH HHS/United States ; T32 HD060600/HD/NICHD NIH HHS/United States ; },
mesh = {*DNA Breaks, Double-Stranded ; Humans ; *CRISPR-Cas Systems ; Animals ; *Loss of Heterozygosity ; Mice ; DNA End-Joining Repair ; Gene Editing/methods ; Flow Cytometry ; CRISPR-Associated Protein 9/genetics/metabolism ; Epithelial Cells/metabolism ; },
abstract = {Harnessing DNA double-strand breaks (DSBs) is a powerful approach for gene editing, but it may provoke loss of heterozygosity (LOH), a common feature of tumor genomes. To interrogate this risk, we developed a flow cytometry-based system (Flo-LOH), detecting LOH in ∼5% of mouse embryonic and human epithelial cells following a DSB. Inhibition of both non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) massively increases LOH, although the dependence on individual pathways differs in the two cell types. Multiple mechanisms lead to LOH, including chromosome truncations with de novo telomere addition and whole chromosome loss. LOH spans megabases distal from the DSB but also frequently tens of megabases centromere-proximal, which can arise from breakage-fusion-bridge events. Unlike DSBs, Cas9 nicks and adenine base editing did not noticeably impact LOH. The capacity for large-scale LOH must therefore be considered when using DSB-based gene editing, especially in conjunction with end-joining inhibition.},
}
@article {pmid41205654,
year = {2025},
author = {Yu, L and Niu, M and Dong, Z and Dong, X and Han, Y and An, J and Jiang, T and Chen, Y and Feng, Y and Sun, Y and Li, H},
title = {High-efficiency inhibition of human adenovirus type 55 replication by CRISPR-Cas12a.},
journal = {The Journal of infection},
volume = {91},
number = {5},
pages = {106650},
doi = {10.1016/j.jinf.2025.106650},
pmid = {41205654},
issn = {1532-2742},
mesh = {*CRISPR-Cas Systems ; *Virus Replication ; *Adenoviruses, Human/classification ; Humans ; *Adenovirus Infections, Human/therapy/virology ; A549 Cells ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {OBJECTIVES: Human adenovirus 55 (HAdV-55), a highly pathogenic double-stranded DNA virus, presents a serious global public health challenge due to its rapid transmission and complex pathogenesis. Current antiviral treatment options for HAdV-55 are limited, with no specific antiviral drugs available. The CRISPR-Cas system, capable of precisely targeting viral genomes, has emerged as a novel approach for antiviral therapy. This study aimed to leverage targeted DNA cleavage activity of the CRISPR-Cas12a system to develop a therapeutic strategy for effectively inhibiting HAdV-55 replication.<br><br>METHODS: We developed a rapid and efficient screening platform for identifying antiviral targets by integrating CRISPR-Cas12a fluorescence detection technology with bioinformatics analysis. Using this platform, we systematically screened 194 candidate targets against HAdV-55.<br><br>RESULTS: The E1B-crRNA6-Cas12a system was identified, demonstrating a highly potent antiviral activity with 99.17% inhibitory efficiency and a selectivity index (SI) of 2482.80. This target significantly outperformed the clinical broad-spectrum anti-adenovirus drug cidofovir in both inhibitory efficacy and duration.<br><br>CONCLUSIONS: This study not only holds promise for providing safe and highly effective antiviral candidate targets for HAdV-55 therapy but also, through the construction of an interdisciplinary technical platform, is expected to enhance the translational potential of CRISPR antiviral technology for preclinical applications.},
}
@article {pmid41205860,
year = {2026},
author = {Yunus, IS and Carruthers, DN and Chen, Y and Gin, JW and Baidoo, EEK and Petzold, CJ and Garcia Martin, H and Adams, PD and Mukhopadhyay, A and Lee, TS},
title = {Predictive CRISPR-mediated gene downregulation for enhanced production of sustainable aviation fuel precursor in Pseudomonas putida.},
journal = {Metabolic engineering},
volume = {94},
number = {},
pages = {67-76},
doi = {10.1016/j.ymben.2025.11.007},
pmid = {41205860},
issn = {1096-7184},
mesh = {*Pseudomonas putida/genetics/metabolism ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; *Down-Regulation ; *Bacterial Proteins/genetics/biosynthesis/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR interference (CRISPRi) has emerged as a valuable tool for redirecting metabolic flux to enhance bioproduction. However, its application is often constrained by two challenges: (i) rationally identifying effective gene targets for downregulation and (ii) efficiently constructing multiplexed CRISPRi systems. In this study, we address both challenges by integrating a computational prioritization tool with a versatile assembly method for building multiplexed CRISPRi systems. FluxRETAP (Flux-Reaction Target Prioritization) accurately identified gene targets whose knockdown led to substantial increase of isoprenol titers in Pseudomonas putida KT2440, outperforming a conventional non-computational, pathway-guided target selection. The highest isoprenol titer of nearly 1.5 g/L was achieved by knocking down PP_4118 (a gene encoding α-ketoglutarate dehydrogenase). The use of VAMMPIRE (Versatile Assembly Method for MultiPlexing CRISPRi-mediated downREgulation) enabled accurate assembly of CRISPRi constructs containing up to five sgRNA arrays, reducing context dependency and achieving uniform, position-independent gene downregulation. The integration of FluxRETAP and VAMMPIRE has the potential to advance metabolic engineering by rapidly identifying CRISPRi-mediated knockdowns and knockdown combinations that enhance bioproduction titers, with potential applicability to other microbial systems.},
}
@article {pmid41205968,
year = {2025},
author = {Zhang, Z and Chen, H and Tang, K and Xu, J and Jiang, L and Ning, Y},
title = {Triple-helix molecular switch-based aptasensor integrating exonuclease I-assisted target recycling and CRISPR-Cas13a-mediated signal amplification for fluorescence detection of adenosine triphosphate.},
journal = {International journal of biological macromolecules},
volume = {333},
number = {Pt 1},
pages = {148753},
doi = {10.1016/j.ijbiomac.2025.148753},
pmid = {41205968},
issn = {1879-0003},
mesh = {*Adenosine Triphosphate/analysis ; *Exodeoxyribonucleases/metabolism ; *Aptamers, Nucleotide/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; Methicillin-Resistant Staphylococcus aureus ; Graphite/chemistry ; Fluorescence ; Limit of Detection ; Spectrometry, Fluorescence ; },
abstract = {It is essential to develop new methods for detecting adenosine triphosphate (ATP), as it plays crucial roles in the regulating numerous biological processes and its levels are significantly correlated with various diseases. This study developed a fluorescent aptasensor for ATP detection utilizing a triple-helix molecular switch (THMS) based on exonuclease I (Exo I)-assisted target recycling and CRISPR-Cas13a-mediated signal amplification. Without the target, all carboxyfluorescein (fluorescence-active molecule; FAM)-labeled probes remain close to the graphene oxide (GO) substrate, resulting in the fluorescence quenching of FAM. In contrast, the target initiates THMS opening, which triggers the simultaneous target recycling and signal amplification. The iterative reactions lead to FAM accumulation, and the fluorescence can be assayed at excitation/emission wavelengths of 480/514 nm, respectively. The target was detected at concentrations from 1 fM to 1 nM with a detection limit of 0.54 fM. The aptasensor showed satisfactory performance for evaluating ATP in methicillin-resistant Staphylococcus aureus and studying drug action mechanisms, highlighting its potential for clinical diagnostics and screening antibacterial agents.},
}
@article {pmid41206041,
year = {2025},
author = {Liu, Q and Pan, TT and Wang, LJ and Zhang, CY},
title = {Mirror-synchronized asymmetric CRISPR nanoswitch for single-molecule profiling of multiple circRNAs in different stages of breast cancer.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206041},
issn = {1362-4962},
support = {22474019//National Natural Science Foundation of China/ ; },
mesh = {*RNA, Circular/genetics ; *Breast Neoplasms/genetics/pathology ; Humans ; *CRISPR-Cas Systems/genetics ; Female ; *RNA/genetics ; CRISPR-Associated Proteins/genetics/metabolism ; Single Molecule Imaging/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs characterized by their covalently closed circular structures. They have been implicated in significant transcriptional and post-transcriptional regulation of gene expression. Here, we present a one-pot method for the detection of circRNAs based on engineered DNA hairpins and CRISPR-Cas12a signal amplification, which involves signal pre-amplification via coupled probe-mediated hairpin amplification of two palindromic hairpins and Cas12a signal generation via trans-cleavage. We demonstrate that this platform is sensitive (detection limit of 1.07 aM), specific (capable of single-mismatch discrimination), and fast (reaction time of 25 min) and can be used to detect different circRNAs from RNase R-treated RNA (both in vitro and in clinically relevant samples, including correct classification of disease progression). This method enables single-molecule profiling and can be extended to detect other types of nucleic acids.},
}
@article {pmid41206045,
year = {2025},
author = {Nakamura, J and Shiraishi, M and Yamamoto, J and Suzuki, K},
title = {Development of programmable RNA imaging with RNA-guided GFP via click chemistry.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206045},
issn = {1362-4962},
support = {25K21779//The Uehara Memorial Foundation and the Japan Society/ ; JPMJSP2138//JST/ ; //Japan Society for the Promotion of Science/ ; },
mesh = {*Click Chemistry/methods ; *Green Fluorescent Proteins/genetics/chemistry/metabolism ; Humans ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; CRISPR-Cas Systems ; *RNA ; Cyclooctanes/chemistry ; HEK293 Cells ; *Molecular Imaging/methods ; Azides/chemistry ; },
abstract = {The CRISPR-Cas system revolutionized molecular biology by guiding Cas proteins to target nucleic acid sequences using customizable guide RNAs, offering unparalleled precision and versatility. Inspired by this innovation, we developed RNA-guided green fluorescent protein (RGG), a simple and programmable platform for targeting nucleic acid. Using a streamlined click chemistry approach, known for its high efficiency and specificity, we conjugated dibenzocyclooctyne (DBCO)-modified guide nucleic acids, designed to complement target sequences, with azide-exposed proteins to construct RGG. Systematic optimization identified 30-nt RNA with 3'-DBCO modifications as the most effective configuration for RGG, enabling precise visualization of nuclear-localized RNAs, including NEAT1 and Satellite III RNA, in living cells. This establishes RGG as a customizable and efficient system for RNA imaging and molecular analysis, underscoring the potential of direct conjugation between guide nucleic acids and proteins to enable precise nucleic acid recognition and dynamic molecular modification in living cells.},
}
@article {pmid41206049,
year = {2025},
author = {Wu, JN and Li, C and Liu, Z and Li, X and Wang, J and Lou, X and Xia, F and Dai, J and Wu, T},
title = {Photocontrolled dissociation and toehold-mediated strand displacement-based synergistic regulation of CRISPR-Cas12a.},
journal = {Nucleic acids research},
volume = {53},
number = {20},
pages = {},
pmid = {41206049},
issn = {1362-4962},
support = {22&#x2009;474&#x2009;045//National Natural Science Foundation of China/ ; 82&#x2009;172&#x2009;372//National Natural Science Foundation of China/ ; HUST: 2024JYCXJ010//Central Universities/ ; S202410487577//Undergraduates of Hubei Province/ ; },
mesh = {*CRISPR-Cas Systems/radiation effects/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; *Gene Editing/methods ; Ultraviolet Rays ; *Endodeoxyribonucleases/metabolism/genetics ; DNA/chemistry/genetics/metabolism ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {The trans-cleavage activity of the CRISPR-Cas system holds broad potential across diverse fields, yet precise spatiotemporal regulation remains challenging due to the predominantly single-direction control strategies available. Here, we present a bidirectional, multi-round modulation strategy for CRISPR-Cas12a trans-cleavage activity, utilizing toehold-mediated strand displacement and photocontrolled dissociation. This approach enables dynamic transitions between on and off states: Cas12a activity is activated by an activator, inhibited by a photosensitive blocker, and reactivated via UV light. We further integrated this system with DNA cryptography, establishing a hierarchical temporal authorization system that enhanced cryptographic security. The method supported multi-round modulation, achieving restoration of 95.4% activity after multiple cycles in the on state while maintaining suppression to 12.4% in the off state. This precise control strategy provides a versatile tool for spatiotemporal regulation in CRISPR-based applications, with significant implications for advanced gene editing, diagnostics, and bioengineering.},
}
@article {pmid41206491,
year = {2026},
author = {Landesman, WJ and Hudson, TR and Bedore, SE and Suarez, MC and Hayden, MS},
title = {A CRISPR/LbCas12a system for Borrelia burgdorferi sensu stricto detection in blacklegged ticks.},
journal = {Journal of medical entomology},
volume = {63},
number = {1},
pages = {},
pmid = {41206491},
issn = {1938-2928},
support = {P20 GM103449/GM/NIGMS NIH HHS/United States ; P20GM103449//Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health/ ; U01CK000661/ACL/ACL HHS/United States ; U01 CK000661/CK/NCEZID CDC HHS/United States ; /CC/CDC HHS/United States ; U01CK000661/ACL/ACL HHS/United States ; /CC/CDC HHS/United States ; },
mesh = {Animals ; *Borrelia burgdorferi/isolation &amp; purification/genetics ; *Ixodes/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; Vermont ; },
abstract = {CRISPR/Cas systems have the potential to revolutionize DNA detection of vector-borne pathogens with highly specific and user-friendly assays. One such system, named DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), uses a guide RNA (gRNA) and Cas enzyme to bind to and cut DNA targets. Following cutting, Cas12a exhibits non-specific collateral cleavage of single-stranded DNA (ssDNA). A ssDNA reporter in the reaction allows the trans-cleavage activity to be harnessed as an amplified output signal upon recognition of the target by the Cas12a/gRNA complex. We developed a DETECTR assay to detect Borrelia burgdorferi sensu stricto, the primary Lyme disease pathogen in the United States, in blacklegged ticks (Ixodes scapularis) collected from forests in southern Vermont. We compared DETECTR to gel electrophoresis of PCR-amplified products and used quantitative real-time PCR (qPCR) of a different B. burgdorferi primer set for independent confirmation. We found that 123/125 of the samples had identical results for DETECTR and gel electrophoresis. Both assays identified the same 33 B. burgdorferi-positive samples and the same 90 B. burgdorferi-negative samples. On a subset of eight samples, we tested DETECTR using lateral flow test strips and obtained identical results to those obtained with the fluorescence-based DETECTR. The sensitivity of DETECTR was lower than qPCR, which detected nine additional B. burgdorferi-positive samples. When qPCR is not available, the DETECTR assay offers a robust alternative to gel electrophoresis that is more user-friendly and requires less time. Due to the highly specific nature of the assay, DETECTR provides additional confidence that a B. burgdorferi target is present.},
}
@article {pmid41206864,
year = {2025},
author = {Docherty, JAD and Cook, R and Kiu, R and Dyball, X and Brown, TL and Kujawska, M and Smith, RL and Phillips, S and Watt, R and Telatin, A and Tiwari, SK and Hall, LJ and Adriaenssens, EM},
title = {Diverse defense systems and prophages in human-associated Bifidobacterium species reveal coevolutionary "arms race" dynamics.},
journal = {Cell reports},
volume = {44},
number = {11},
pages = {116542},
doi = {10.1016/j.celrep.2025.116542},
pmid = {41206864},
issn = {2211-1247},
mesh = {*Prophages/genetics/physiology ; *Bifidobacterium/virology/genetics ; Humans ; Genome, Bacterial ; CRISPR-Cas Systems/genetics ; Gastrointestinal Microbiome/genetics ; Bacteriophages/genetics ; },
abstract = {Bacteria of the genus Bifidobacterium are pivotal for human health, especially in early life, where they dominate the gut microbiome in healthy infants. Bacteriophages, as drivers of gut bacterial composition, can affect bifidobacterial abundance. Here, we use a bioinformatics approach to explore direct interactions between human-associated Bifidobacterium spp. and prophages, as evidenced by their genomes. Analysis of 1,086 bifidobacterial genomes reveals the presence of complex systems that prevent viral invasion, with 34 defense systems and 56 subtypes detected, including several different CRISPR-Cas systems. CRISPR spacers target almost three-quarters of bifidobacteria-derived prophages, indicating dynamic interactions. At least one prophage is present in ∼67% of strains, with phages exhibiting high genomic diversity and evidence of historical recombination. These prophages encode various defense and anti-defense systems, such as anti-CRISPR genes and restriction-modification mechanisms. Overall, this investigation reveals that coevolutionary "arms race" dynamics drive genomic diversity in both bifidobacteria and their phages.},
}
@article {pmid41207276,
year = {2026},
author = {Luo, N and Li, X and Tian, S and Li, X and Zou, L},
title = {Label-free fluorescent aptasensor for sensitive detection of oxytetracycline based on CRISPR/Cas12a and G-quadruplex DNAzyme with rhodamine B reporting.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 2},
pages = {127141},
doi = {10.1016/j.saa.2025.127141},
pmid = {41207276},
issn = {1873-3557},
mesh = {*Rhodamines/chemistry ; *Oxytetracycline/analysis ; *G-Quadruplexes ; *Aptamers, Nucleotide/chemistry ; Limit of Detection ; *Biosensing Techniques/methods ; *DNA, Catalytic/chemistry/metabolism ; Milk/chemistry ; *CRISPR-Cas Systems ; Spectrometry, Fluorescence/methods ; Animals ; Fluorescent Dyes/chemistry ; *Anti-Bacterial Agents/analysis ; *Endodeoxyribonucleases/metabolism ; Rivers/chemistry ; *CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; },
abstract = {Oxytetracycline (OTC), a widely used broad-spectrum antibiotic, frequently persists in food and the environment due to overuse, posing risks of resistance, allergy, and toxicity. The World Health Organization has set strict limits for OTC residues, highlighting the need for rapid and reliable detection. Conventional assays, though accurate, remain costly and technically demanding, underscoring demand for simpler alternatives. We report a label-free fluorescent aptasensor integrating CRISPR/Cas12a with G-quadruplex (G4) DNAzyme catalysis, using rhodamine B (RhB) as a reporter. In the absence of OTC, the aptamer binds the activator strand, allowing G4 DNAzyme formation that quenches RhB fluorescence. OTC binding liberates the activator, triggering Cas12a cleavage of G4 DNA, thereby preventing DNAzyme formation and preserving fluorescence. This switch provides efficient signal amplification with low background. The assay achieved a detection limit of 0.3 nM and enabled accurate quantification of OTC in river water and milk samples. This platform offers a cost-effective, label-free strategy for antibiotic residue monitoring, eliminating dependence on nanomaterials or external labeling. Its validated sensitivity and robustness in real samples underscore its potential for practical applications in food safety and environmental surveillance.},
}
@article {pmid41208113,
year = {2025},
author = {Hermann, MD and Fernandez Perez, N and Yeroslaviz, A and Murray, PJ},
title = {Normal macrophage signaling and gene expression in Rosa26 Cas9-expressing mice.},
journal = {ImmunoHorizons},
volume = {9},
number = {11},
pages = {},
pmid = {41208113},
issn = {2573-7732},
support = {//Deutsche Forschungsgemeinschaft/ ; //Max-Planck-Gesellschaft/ ; },
mesh = {Animals ; Mice ; *Macrophages/metabolism/immunology ; *Signal Transduction/genetics ; CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; *RNA, Untranslated/genetics ; Mice, Inbred C57BL ; Toll-Like Receptor 3/metabolism ; Mice, Transgenic ; Toll-Like Receptor 4/metabolism ; },
abstract = {Cas9-expression from the Rosa26 "safe harbor" locus are widely used for gene manipulation and Crispr-based screening. Recently, experimental evidence suggested that macrophages isolated from Rosa26-Cas9 mice may have signaling differences compared to control mice in terms of TRIF signaling downstream of TLR3 and TLR4. As we frequently use the Rosa26-Cas9 mice made by Feng Zhang (Cas9-FZ, Jackson Laboratory stock No. 026179), arguably the strain with the widest distribution and utilization, we were motivated to test macrophage signaling in these mice under our conventional conditions. We used different macrophage polarization and signaling conditions combined with RNA sequencing and measurement of TLR signaling by immunoblotting. Our results suggest that the Cas9-FZ mice bear no obvious defects in any commonly used macrophage signaling pathway. We document the differences in our macrophage culture techniques compared to Raychowdhury et al., which may aid in how individual laboratories use Cas9-expressing macrophages, especially for focused or genome-wide screening.},
}
@article {pmid41208252,
year = {2026},
author = {Patterson, FM and Nguyen Tran, MT and Guinan, T and Mohd Khalid, MKN and Kc, R and Fairfax, KA and Liu, GS and Cook, AL and Hung, SS and Hewitt, AW},
title = {Generalizable features of pegRNA design for prime editing of inherited retinal diseases.},
journal = {Ophthalmic genetics},
volume = {47},
number = {1},
pages = {59-66},
doi = {10.1080/13816810.2025.2576786},
pmid = {41208252},
issn = {1744-5094},
mesh = {*Gene Editing/methods ; Humans ; *Retinal Diseases/genetics/therapy ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND AND OBJECTIVES: The variety of ocular cell types involved in inherited retinal disease (IRD) necessitates the use of gene editing therapeutics which have generalizable components. In our study, we investigate the generalizable characteristics of non-engineered pegRNA design (PE2) for efficient, proof-in-principle gene correction of over 21 genes implicated in IRDs and associated syndromes. We use a single-transgene oligopool approach, comprising approximately 12,000 uniquely barcoded pegRNAs that target a synthetically integrated, 50 bp sequence motif, which faithfully recapitulate the disease context of their various counterpart IRDs. Using this approach, we perform a high throughput, pooled analysis of pegRNA characteristics across non- and ocular cell types to propose a cell-line agnostic set of pegRNA design guidelines.<br><br>RESULTS: Briefly, we find that non-engineered pegRNA 3' extensions should mediate substitution-type edits and that the desired edit should be placed within five nucleotides upstream of the nick site induced by the Cas-endonuclease. Further, PBS and RTT lengths of at least 12 and 14 nucleotides, respectively, should be used and each non-engineered pegRNA 3' extension should obviate an initial templating cytosine nucleotide.<br><br>CONCLUSION: We establish a set of recommendations for the generalizable design of the non-engineered pegRNA 3' extension for the correction of several IRDs, enabling overall simplification of design parameters for PE2-based systems.},
}
@article {pmid41208769,
year = {2025},
author = {Luo, D and Wang, N and Major-Monfried, H and Ralls, J and Rha, S and Maitland, SA and Ponnienselvan, K and Yamada, M and Bauer, DE and Wolfe, SA and Kentsis, A},
title = {Modular Platform for Therapeutic Drug Delivery Using Trifunctional Bio-Orthogonal Macromolecular Conjugates.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {11},
pages = {2472-2486},
doi = {10.1021/acs.bioconjchem.5c00440},
pmid = {41208769},
issn = {1520-4812},
mesh = {Humans ; *Cell-Penetrating Peptides/chemistry ; *Drug Delivery Systems/methods ; *Macromolecular Substances/chemistry ; Gene Editing ; Cell Line, Tumor ; Peptidomimetics/chemistry/pharmacology ; Ligands ; CRISPR-Cas Systems ; },
abstract = {Targeted delivery of macromolecular therapeutics holds great promise for overcoming the limitations of conventional small molecules, enabling the modulation of protein-protein interactions and precise genome editing. However, efficient, safe, and cell type-specific delivery remains a major challenge. To address this, we developed a modular platform for synthesizing heterotrifunctional bio-orthogonal macromolecular conjugates (BMCs) by engineering diverse combinations of targeting ligands, cell-penetrating peptides (CPPs), and bioactive cargos. We optimized facile bioconjugation chemistries to generate BMCs with improved yields, structural integrity, and activity. Modular BMCs accommodate diverse components, including antibodies and receptor ligands for targeting, CPPs for intracellular trafficking, and optical probes, therapeutic peptidomimetics, and CRISPR-Cas9 nuclease as cargo to confer specific biological activities. We assayed their utility across multiple applications: BMCs with fluorescently labeled cargo revealed endosomal escape and intracellular accumulation; peptidomimetic MYB transcription factor inhibitor BMCs exhibited potent antileukemic activity against acute myeloid leukemia cells; and Cas9 BMCs achieved rapid delivery and cell type-specific gene editing in human cells. The BMC approach enables the customizable delivery of functional macromolecules, nominating BMCs as a broadly applicable platform for biomedical applications.},
}
@article {pmid41209346,
year = {2025},
author = {Wei, Y and Sun, J and Zhu, R},
title = {CRISPR-epigenetic crosstalk: From bidirectional regulation to therapeutic potential.},
journal = {Computational and structural biotechnology journal},
volume = {27},
number = {},
pages = {4496-4504},
pmid = {41209346},
issn = {2001-0370},
abstract = {Recent advances in epigenetics have elucidated the pivotal roles of epigenetic modifications in genomic regulation and disease pathogenesis. Concurrently, CRISPR-based technologies have transcended conventional gene-editing applications and have emerged as powerful tools for target gene screening, chromatin imaging, and epigenetic modulation. Notably, epigenetic landscapes substantially influence the CRISPR editing efficiency, whereas CRISPR itself can reshape epigenetic states, forming a dynamic CRISPR-Epigenetics Regulatory Circuit. This review systematically examines the bidirectional interplay between CRISPR systems and epigenetic modifications, emphasizing their collective impact on genome-editing precision, disease progression, and therapeutic development. Existing studies have predominantly focused on the application of CRISPR in epigenetic modifications or the impact of epigenetic landscapes on CRISPR, exhibiting unidirectional characteristics. However, accumulating evidence suggests a bidirectional interaction between the two. Here, a transformative "CRISPR-Epigenetics Regulatory Circuit" model is synthesized and presented, supported by three pivotal breakthroughs: demonstrating CRISPR as an active epigenetic programmer, synthesizing the epigenetic preconditioning therapeutic paradigm, and elucidating the first predictive mathematical model (EPIGuide). Further exploration of this circuit is expected to enhance CRISPR performance, optimize sgRNA design via epigenetic predictive models, and pioneer sequential epigenetic or gene editing therapies.},
}
@article {pmid41210586,
year = {2025},
author = {Joo, JH and Lee, S and Kim, KP},
title = {Precision gene editing: The power of CRISPR-Cas in modern genetics.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102733},
pmid = {41210586},
issn = {2162-2531},
abstract = {Gene editing has transformed molecular biology by enabling precise modifications to genomic DNA across a wide variety of organisms. Gene editing technologies make it possible to add, remove, or modify specific DNA sequences, with a range of applications including gene knockouts, therapeutic gene correction, and the design of targeted genetic traits. These techniques depend on two main DNA repair mechanisms: homology-directed repair (HDR), which facilitates precise changes to the genome, and non-homologous end joining (NHEJ), which often results in mutations such as deletions or frameshift errors. Among the diverse gene-editing platforms, the CRISPR-Cas system has emerged as the most extensively employed, owing to its simplicity, low cost, and efficiency. This review presents the evolution of gene-editing technologies, with a particular emphasis on the CRISPR-Cas system and its expanding applications in genetics, biotechnology, agriculture, and medicine. Furthermore, advanced gene editing approaches are discussed, offering an overview of emerging trends.},
}
@article {pmid41211185,
year = {2025},
author = {Allmang, S and Witzel, HR and Hausen, A and Marquard, S and Eckert, C and Marnet, N and Hörner, N and Mayer, P and Heinrich, S and Dang, H and Roth, W and Gaida, MM},
title = {Aberrant Expression of A Disintegrin and Metalloproteinase With Thrombospondin Motifs 13 (ADAMTS13) in Pancreatic Cancer Leads to Dichotomic Functions.},
journal = {MedComm},
volume = {6},
number = {11},
pages = {e70462},
pmid = {41211185},
issn = {2688-2663},
abstract = {Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers characterized by highly invasive growth into the surrounding peripancreatic fat tissue, where tumor cells can directly interact with adipocytes. Due to poor response to the currently available (radio)chemotherapies, there is an urgent need for advanced therapy concepts. The present study shows that ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), a key factor in blood coagulation, is significantly overexpressed in human PDAC. Immunohistochemical analysis revealed that ADAMTS13 expression is associated with prolonged survival and negatively correlated with vascular density. In vitro and in vivo experiments demonstrate its partial induction by leptin. Mechanistically, CRISPR/Cas-mediated ADAMTS13 knockout in PDAC cells resulted in reduced migration and invasion. In an avian xenograft tumor model, ADAMTS13 loss led to increased vascularization, decreased vascular length, and diminished tumor growth, accompanied by reduced expression of multiple key angiogenic and angioplastic factors. Furthermore, loss of ADAMTS13 was associated with decreased expression of mesenchymal markers. In conclusion, we identified an aberrant expression and alternative function of ADAMTS13 in PDAC linked to tumor progression, plasticity, and angiogenesis, partly induced by the peripancreatic fat tissue, making this metalloproteinase an interesting target for personalized therapies.},
}
@article {pmid41211267,
year = {2025},
author = {Ramesh, B and Fakoya, AO},
title = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas Unleashed: Transforming Gene Editing With Breakthroughs, Applications, and Ethical Dilemmas.},
journal = {Cureus},
volume = {17},
number = {11},
pages = {e95908},
pmid = {41211267},
issn = {2168-8184},
abstract = {The most significant breakthrough in gene editing is the advent of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. This innovative technology enables scientists to insert or delete genes using specific enzymes, facilitating modifications to genomes that can influence an organism's phenotype. The Cas9 enzyme is the most widely used within the CRISPR framework and has already received approval for treating sickle cell disease, with many other applications likely to follow. As this rapidly evolving field continues to advance, it holds great promise for addressing genetic disorders and diseases. This article will explore the various enzymes available in the CRISPR system, the range of diseases and conditions that could be treated using this technology, alternative gene therapy methods, and the ethical considerations surrounding its use.},
}
@article {pmid41211856,
year = {2026},
author = {Kelly, AA and Fulda, M and Aden, M and Abreu, IN and Feussner, K and Feussner, I},
title = {Reducing the Sinapine Levels of Camelina sativa Seeds Through Targeted Genome Editing of REF1.},
journal = {Plant biotechnology journal},
volume = {24},
number = {3},
pages = {1839-1865},
pmid = {41211856},
issn = {1467-7652},
support = {INST 186/1434-1//Deutsche Forschungsgemeinschaft/ ; ZUK 45/2010//Deutsche Forschungsgemeinschaft/ ; 031B0343A//Bundesministerium f&#xfc;r Bildung und Forschung/ ; },
mesh = {*Seeds/metabolism/genetics ; *Gene Editing/methods ; *Choline/analogs &amp; derivatives/metabolism ; *Brassicaceae/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Plants, Genetically Modified ; CRISPR-Cas Systems ; },
abstract = {Sinapine (O-sinapoyl choline) is the major phenolic metabolite typically found in the oil-rich seeds of Brassicaceae such as Camelina sativa and Brassica napus. It imparts a bitter taste to the seeds as a defence mechanism against herbivores, but it also renders them less palatable to livestock. To improve Camelina flour for human consumption or as animal feed, we reduced sinapine content through CRISPR/Cas9-based genome editing of REF1 (REDUCED EPIDERMAL FLUORESCENCE1), which encodes the NADP[+]-dependent coniferaldehyde/sinapaldehyde dehydrogenase (CALDH/SALDH), a key enzyme for sinapine biosynthesis in Arabidopsis thaliana and B. napus. Inactivation of all three homoeologues found in C. sativa lowered the sinapine content in seeds by an overall 56% in two cultivars indicating the presence of a REF1-independent pathway for sinapine biosynthesis. Most importantly however, crucial agronomic seed traits such as total lipid or protein content of the seeds, seed weight or germination were not affected. Hence, the ref1 mutant lines produced here provide a valuable trait, that can be combined with other traits through gene stacking to obtain crops with significantly improved product quality. Furthermore, metabolite fingerprinting by ultra-performance liquid chromatography-electrospray ionisation-quadrupole time-of-flight mass spectrometry of ref1 mutant lines revealed a contrasting phenylpropanoid profile in seeds and leaves, indicating that REF1 oxidises sinapaldehyde to sinapate in seeds and coniferyl aldehyde to ferulate in leaves. In contrast to Arabidopsis however, Camelina accumulates no comparable levels of sinapoyl malate, but substantial amounts of chlorogenic acid, of which an additional chlorogenic acid isomer distinguishes the two different Camelina cultivars as a metabolite marker.},
}
@article {pmid41211945,
year = {2025},
author = {Laffin, LJ and Nicholls, SJ and Scott, RS and Clifton, PM and Baker, J and Sarraju, A and Singh, S and Wang, Q and Wolski, K and Xu, H and Nielsen, J and Patel, N and Duran, JM and Nissen, SE},
title = {Phase 1 Trial of CRISPR-Cas9 Gene Editing Targeting ANGPTL3.},
journal = {The New England journal of medicine},
volume = {393},
number = {21},
pages = {2119-2130},
doi = {10.1056/NEJMoa2511778},
pmid = {41211945},
issn = {1533-4406},
mesh = {Adult ; Aged ; Female ; Humans ; Male ; Middle Aged ; *Angiopoietin-Like Protein 3/blood/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Dyslipidemias/blood/drug therapy/genetics ; *Gene Editing/methods ; Hypercholesterolemia/blood/drug therapy/genetics ; Hypertriglyceridemia/blood/drug therapy/genetics ; Loss of Function Mutation ; *RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/adverse effects/genetics ; Infusions, Intravenous ; Treatment Outcome ; },
abstract = {BACKGROUND: Angiopoietin-like protein 3 (ANGPTL3) inhibits lipoprotein and endothelial lipases. ANGPTL3 loss-of-function genetic variants are associated with decreased levels of low-density lipoprotein cholesterol and triglycerides and a decreased lifetime risk of atherosclerotic cardiovascular disease.<br><br>METHODS: We conducted an ascending-dose phase 1 trial to assess the safety and efficacy of CTX310, a lipid-nanoparticle-encapsulated clustered regularly interspaced short palindromic repeats-Cas9 endonuclease (CRISPR-Cas9) messenger RNA (mRNA) and guide RNA targeting hepatic ANGPTL3 to induce a loss-of-function mutation. Adults who had uncontrolled hypercholesterolemia, hypertriglyceridemia, or mixed dyslipidemia and were receiving maximally tolerated lipid-lowering therapy received a single intravenous dose of CTX310 (0.1, 0.3, 0.6, 0.7, or 0.8 mg per kilogram of body weight). The primary end point was adverse events, including dose-limiting toxic effects.<br><br>RESULTS: A total of 15 participants received CTX310 and had at least 60 days of follow-up. No dose-limiting toxic effects related to CTX310 occurred. Serious adverse events occurred in two participants (13%): one participant had a spinal disk herniation, and the other died suddenly 179 days after treatment with the 0.1-mg-per-kilogram dose. Infusion-related reactions were reported in three participants (20%), and one participant (7%) who had elevated levels of aminotransferases at baseline had a transient elevation in aminotransferases to between three times and five times as high as those at baseline, peaking on day 4 and returning to baseline by day 14. The mean percent change in ANGPTL3 level was 9.6% (range, -21.8 to 71.2) with the dose of 0.1 mg per kilogram, 9.4% (range, -25.0 to 63.9) with 0.3 mg per kilogram, -32.7% (range, -51.4 to -19.4) with 0.6 mg per kilogram, -79.7% (range, -86.8 to -72.5) with 0.7 mg per kilogram, and -73.2% (range, -89.0 to -66.9) with 0.8 mg per kilogram.<br><br>CONCLUSIONS: Editing of ANGPTL3 was associated with few adverse events and resulted in reductions from baseline in ANGPTL3 levels. (Funded by CRISPR Therapeutics; Australia New Zealand Clinical Trials Registry number, ACTRN12623000809639.).},
}
@article {pmid41212235,
year = {2025},
author = {Yilmaz-Çolak, &#xc7;},
title = {The CRISPR-cas repertoire of Kluyvera ascorbata: insights from genomic data.},
journal = {Current genetics},
volume = {71},
number = {1},
pages = {28},
pmid = {41212235},
issn = {1432-0983},
mesh = {*CRISPR-Cas Systems/genetics ; Phylogeny ; *Genome, Bacterial ; *Kluyvera/genetics/classification/pathogenicity ; Genomics/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Plasmids/genetics ; },
abstract = {The genus Kluyvera has gained increasing attention due to their emerging role as opportunistic pathogens and their antibiotic resistance determinants. Various approaches have been employed to reveal genomic insights into the evolution and pathogenicity of Kluyvera species. However, detailed knowledge about Kluyvera-specific clustered regularly interspaced short palindromic repeats (CRISPR) is still missing. In this study, a genome-mining approach was employed for the characterization of CRISPR-Cas loci in a total of 13 complete Kluyvera genomes using CRISPRCasFinder and related tools. Out of 13 species, only K. ascorbata displayed multiple CRISPR-Cas arrays and a complete set of cas genes characteristics of a type I-E system. Spacer analysis revealed potential targets within phage and plasmid sequences, indicating historical exposure to mobile genetic elements. Furthermore, a phylogenetic tree constructed using the Cas3 protein sequence positioned K. ascorbata closely with other enteric bacteria, including Salmonella spp. and Citrobacter spp. This study provides the first detailed insight into the CRISPR-Cas architecture of K. ascorbata. Although there is no significant diversity of the CRISPR-Cas system identified in this species, it can emphasize a role as active immune defenses against invaders and offer a foundation for future functional and evolutionary investigations. Moreover, difficulties in identification of the genus Kluyvera can be overcome through the CRISPR-Cas system using next-generation diagnostics tools.},
}
@article {pmid41212595,
year = {2025},
author = {Wu, Y and Su, J and Sun, J and Li, Z and Yang, J and Wu, C and Liu, Z and Wang, J and Wang, Y and Liu, J and Duan, L and Wang, C and Zhu, G and Wen, X},
title = {Rapid detection of Seneca Valley virus based on reverse transcription recombinase polymerase amplification and CRISPR-Cas13a.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2585719},
pmid = {41212595},
issn = {2150-5608},
mesh = {Animals ; Swine ; *CRISPR-Cas Systems ; *Swine Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Picornaviridae/isolation &amp; purification/genetics ; *Picornaviridae Infections/diagnosis/veterinary/virology ; Recombinases/genetics ; *Nucleic Acid Amplification Techniques/methods ; Reverse Transcription ; },
abstract = {The Seneca Valley virus (SVV) is an emerging novel etiological agent that triggers vesicular stomatitis in pigs. SVV leads to considerable financial detriment to the global swine industry. Availability of expeditious, simple, and precise detection techniques would aid in the diagnosis and control of Seneca Valley disease. In this study, we developed a novel SVV detection assay based on reverse transcription recombinase polymerase amplification (RT-RPA)/CRISPR-Cas13a. This technique can specifically identify SVV with no cross-reactivity with other viruses, such as the porcine reproductive and respiratory syndrome virus, pseudorabies virus, classical swine fever virus, and foot-and-mouth disease virus. It boasts a detection threshold as sensitive as 2.43 copies/μL, matching the proficiency of reverse transcriptase quantitative PCR in clinical diagnostics. The streamlined RT-RPA/CRISPR-Cas13a assay for SVV minimizes procedural complexity and the likelihood of contamination. Integrating this method with nucleic acid acquisition via nucleic acid releaser enables a turnaround time of only 40 min from sample acquisition to result determination, underscoring its suitability for on-site diagnostics and affirming its role as a robust instrument for the monitoring and detection of SVV.},
}
@article {pmid41213129,
year = {2026},
author = {Ajdanian, L and Villot, S and Karikari, B and Torkamaneh, D},
title = {Technological advances in trait development: from conventional breeding and untargeted mutagenesis to precision genome editing.},
journal = {Genome},
volume = {69},
number = {},
pages = {1-13},
doi = {10.1139/gen-2025-0020},
pmid = {41213129},
issn = {1480-3321},
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *Mutagenesis ; Plants, Genetically Modified/genetics ; *Genome, Plant ; CRISPR-Cas Systems ; },
abstract = {Plant biotechnology has revolutionized modern agriculture by enabling precise and efficient crop improvement strategies. This review explores the evolution of selective breeding, mutation breeding, and precision breeding, highlighting their applications in Canada's agricultural sector. Conventional selective breeding has been instrumental in developing high-yielding and disease-resistant cultivars, while mutation breeding, through physical and chemical mutagenesis, has introduced valuable genetic diversity. The advent of transgenic breeding allowed for the direct insertion of foreign genes, leading to the development of crops with herbicide tolerance, pest resistance, and improved nutritional content. However, concerns over regulatory restrictions and public acceptance have driven the rapid adoption of genome editing tools, which enable precise modifications without introducing foreign DNA. Canada has played a key role in applying these biotechnological innovations, successfully developing genetically modified canola, CRISPR-edited wheat, stress-resistant soybean, and barley and oat cultivars improved for stress resistance and yield. While each breeding approach presents distinct advantages and limitations, integrating conventional and molecular techniques is essential for maximizing genetic potential, ensuring agriculture, and effectively food security challenges.},
}
@article {pmid41213207,
year = {2026},
author = {Chen, J and Wang, Z and Du, Y and Liu, B and Chen, Y and Chen, G and Cui, W},
title = {Injectable CRISPRa-microspheres for targeted A20 activation rescue age-related osteogenic impairment via senescence mitigation.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123830},
doi = {10.1016/j.biomaterials.2025.123830},
pmid = {41213207},
issn = {1878-5905},
mesh = {Animals ; *Osteogenesis/genetics ; *Cellular Senescence/genetics ; Mice ; *Microspheres ; *Tumor Necrosis Factor alpha-Induced Protein 3/metabolism/genetics ; Mesenchymal Stem Cells/cytology/metabolism ; *Aging ; *CRISPR-Cas Systems/genetics ; Mice, Inbred C57BL ; Gene Editing/methods ; Bone Regeneration ; Male ; Nanoparticles/chemistry ; Injections ; Humans ; },
abstract = {Age-related osteogenic failure in bone defect repair remains a significant clinical challenge, primarily due to persistent chronic inflammation-induced stem cell senescence. To address this, we engineered injectable CRISPRa-based gene-editing microspheres (GEMs), utilizing microfluidic-synthesized lipid nanoparticles (cLNPs) to co-deliver dCas9-VP64/sgRNA. This platform allows for precise spatiotemporal activation of tumor necrosis factor alpha-induced protein 3 (TNFAIP3/A20) within bone marrow stromal cells (BMSCs), effectively reprogramming the senescence-osteogenesis axis. Our study identifies A20 as a key regulator of the senescence-associated secretory phenotype (SASP) and osteogenic impairment in aged BMSCs. In vitro, GEMs reduced senescence markers (p16 and p21) by over 30 %, while increasing osteogenic gene expression (RUNX2 and ALP) by 4 ∼ 5-fold, and suppressed inflammatory cytokines IL-6 and TNF-α by more than 30 %. In vivo, in aged mice with critical-sized bone defects, GEMs achieved a significant bone regeneration and promoted vascularization 3.1 times faster (CD31[+] staining) compared to controls. This GEM system offers a promising, clinically viable strategy for recalibrating age-related skeletal disorders, demonstrated by the precise targeting of host stem cells in situ and achieving approximately 80 % defect healing in aged bone defect models.},
}
@article {pmid41213415,
year = {2026},
author = {Xu, YY and Zhou, SM and Wang, LY and Zhang, R and Li, K and Qian, ZY and Xiao, L},
title = {Methods for detecting off-target effects of CRISPR/Cas9.},
journal = {Biotechnology advances},
volume = {86},
number = {},
pages = {108750},
doi = {10.1016/j.biotechadv.2025.108750},
pmid = {41213415},
issn = {1873-1899},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; },
abstract = {The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.},
}
@article {pmid41213945,
year = {2025},
author = {Hoetzel, J and Wang, T and Suess, B},
title = {Beyond the niche - unlocking the full potential of synthetic riboswitches.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9897},
pmid = {41213945},
issn = {2041-1723},
support = {SU402/12-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; WA 5722/1-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Riboswitch/genetics ; *Synthetic Biology/methods ; Nucleic Acid Conformation ; CRISPR-Cas Systems ; RNA/genetics ; Aptamers, Nucleotide/genetics ; },
abstract = {Synthetic riboswitches have undergone great development in the past decade, evolving into valuable regulatory tools. Operating entirely at the RNA level and independently of auxiliary proteins, they offer a promising alternative to protein-based systems such as TetON/OFF or CRISPR-Cas. As compact, modular RNA elements they unite sensing and regulatory functions within a single molecule, giving them the advantages of high modularity, portability and low metabolic burden. Here, we explore the unique features of synthetic riboswitches, highlight key applications, assess current bottlenecks and limitations and put them in context with emerging solutions, to emphasise the potential of synthetic riboswitches.},
}
@article {pmid41213972,
year = {2025},
author = {Chisholm, CG and Bartlett, R and Brown, ML and Proctor, EJ and Farrawell, NE and Gorman, J and Delerue, F and Ittner, LM and Vine-Perrow, KL and Ecroyd, H and Cashman, NR and Saunders, DN and McAlary, L and Lum, JS and Yerbury, JJ},
title = {Development of a targeted BioPROTAC degrader selective for misfolded SOD1.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9713},
pmid = {41213972},
issn = {2041-1723},
mesh = {Animals ; *Superoxide Dismutase-1/metabolism/genetics/chemistry ; *Amyotrophic Lateral Sclerosis/genetics/metabolism/pathology/drug therapy ; Mice, Transgenic ; Mice ; Protein Folding ; Humans ; Disease Models, Animal ; Proteolysis ; Motor Neurons/metabolism/pathology ; Ubiquitin-Protein Ligases/metabolism/genetics ; CRISPR-Cas Systems ; Disease Progression ; HEK293 Cells ; },
abstract = {The accumulation of misfolded proteins underlies a broad range of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Due to their dynamic nature, these misfolded proteins have proven challenging to target therapeutically. Here, we specifically target misfolded disease variants of the ALS-associated protein superoxide dismutase 1 (SOD1), using a biological proteolysis targeting chimera (BioPROTAC) composed of a SOD1-specific intrabody and an E3 ubiquitin ligase. Screening of intrabodies and E3 ligases for optimal BioPROTAC construction reveals a candidate capable of degrading multiple disease variants of SOD1, preventing their aggregation in cells. Using CRISPR/Cas9 technology to develop a BioPROTAC transgenic mouse line, we demonstrate that the presence of the BioPROTAC delays disease progression in the SOD1[G93A] mouse model of ALS. Delayed disease progression is associated with protection of motor neurons, a reduction of insoluble SOD1 accumulation and preservation of innervated neuromuscular junctions. These findings provide proof-of-concept evidence and a platform for developing BioPROTACs as a therapeutic strategy for the targeted degradation of neurotoxic misfolded species in the context of neurodegenerative diseases.},
}
@article {pmid41214117,
year = {2025},
author = {Bakthavachalam, V and Sanborn, MA and Mathayan, M and Salunkhe, SS and Wood, JM and Maienschein-Cline, M and Setty, S and Kabeer, BSA and Rehman, J and Prabhakar, BS},
title = {CRISPR/Cas9-mediated deletion of MADD induces cell cycle arrest and apoptosis in anaplastic thyroid cancer cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {39264},
pmid = {41214117},
issn = {2045-2322},
support = {I01 BX004697/BX/BLRD VA/United States ; VA Merit Review, Award # 5 I01 BX004697//United States (U.S.) Department of Veteran's Affairs Biomedical Laboratory Research and Development Services (BLRD)/ ; },
mesh = {*Apoptosis/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Thyroid Carcinoma, Anaplastic/genetics/pathology/metabolism ; Animals ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; Mice ; *Thyroid Neoplasms/genetics/pathology ; Cell Proliferation/genetics ; *Death Domain Receptor Signaling Adaptor Proteins/genetics ; Gene Expression Regulation, Neoplastic ; Cell Movement/genetics ; Gene Deletion ; Guanine Nucleotide Exchange Factors ; },
abstract = {Anaplastic thyroid cancer (ATC) is an aggressive malignancy with a poor prognosis and limited treatment options. Previous studies have shown that selective downregulation of the MADD (MAP-kinase-activating death domain-containing protein) gene isoform increases ATC cell susceptibility to TRAIL-induced apoptosis. However, the existence of multiple MADD gene isoforms raised the possibility of functional compensation. This study aimed to definitively evaluate the role of MADD in ATC by employing CRISPR-Cas9 to Cas9 to target exon 3, a conserved exon expressed in all known MADD isoforms, resulting in functional knockout of MADD expression. CRISPR-Cas9-mediated MADD knockout, performed in three ATC cell lines (8505 C, C643, and HTH7) with distinct mutational backgrounds, significantly impaired ATC cell function in vitro, as indicated by reduced viability, increased apoptosis, decreased migration, and G0/G1 cell cycle arrest. RNA-seq analysis revealed alterations in genes related to cell survival, proliferation, and metastasis. In the orthotopic ATC mouse model, MADD deletion dramatically suppressed tumor growth, reduced lung metastases, and prolonged survival. Our findings demonstrate that MADD plays a crucial role in ATC cell survival, proliferation, and metastasis. The consistent effects observed across multiple cell lines and in vivo models suggest that MADD may represent a promising therapeutic target for this aggressive malignancy.},
}
@article {pmid41214358,
year = {2025},
author = {Liu, XY and Li, YF and Zhou, JZ and Huang, JQ and Wang, LJ and Yang, L and Li, CY and Fang, X and Chen, XY},
title = {Gamma-selinene synthase catalyzes the first step of dihydroagarofuran sesquiterpene alkaloid biosynthesis in Tripterygium.},
journal = {Science China. Life sciences},
volume = {68},
number = {12},
pages = {3696-3710},
pmid = {41214358},
issn = {1869-1889},
mesh = {*Tripterygium/genetics/enzymology/metabolism ; *Sesquiterpenes/metabolism/chemistry ; *Alkaloids/biosynthesis ; *Plant Proteins/metabolism/genetics ; Biosynthetic Pathways ; Phylogeny ; CRISPR-Cas Systems ; },
abstract = {Tripterygium wilfordii and T. hypoglaucum, which belong to family Celastraceae, are perennial vine shrubs with medicinal uses in treating rheumatoid arthritis, cancer, and male contraception. Among the bioactive ingredients, the macrocyclic dihydroagarofuran sesquiterpene alkaloids (DASAs), also serving as chemotaxonomic markers of Celastraceae, are well-known for cytotoxicity and insecticidal properties. Despite intensive phytochemical investigations over the last half-century, the DASAs biosynthetic pathway remains unsolved. Here, we mined multi-omics data of Tripterygium species and identified 14 sesquiterpene synthase genes. Detailed characterization revealed that a group of enzymes (TwTPS5, TwTPS16, ThTPS5) catalyzed the key step committed to the biosynthesis of DASAs in Tripterygium, as evidenced by CRISPR/Cas9 knockout of TwTPS5 in T. wilfordii. Our biochemical assay showed that these enzymes converted FPP to gamma-selinene. Homologues in other Celastraceae species (EaTPS3, CaTPS5, and CaTPS6) also produced gamma-selinene, suggesting functional conservation in this DASAs-producing lineage. Notably, knocking out TwTPS5 resulted in a reduction of DASAs and enhanced accumulation of pharmaceutically valuable compounds, generating a safer and innovative medicinal plant resource for future applications.},
}
@article {pmid41214365,
year = {2025},
author = {Tzou, FY and Hong, CL and Chen, KH and Vaughen, JP and Lin, WS and Hsu, CH and Rivas-Serna, IM and Hsu, KY and Ho, SM and Panganiban, MR and Hsieh, HT and Li, YJ and Hsiao, Y and Yeh, HC and Yu, CY and Tang, HW and Chou, YH and Wu, CL and Lo, CC and Mazurak, VC and Clandinin, MT and Huang, SY and Chan, CC},
title = {Functional profiling and visualization of the sphingolipid metabolic network in vivo.},
journal = {EMBO reports},
volume = {26},
number = {24},
pages = {6380-6417},
pmid = {41214365},
issn = {1469-3178},
support = {113-2320-B-002-022-MY3//National Science and Technology Council (NSTC)/ ; 114-2311-B-002-006-//National Science and Technology Council (NSTC)/ ; 112-2311-B-001-036-MY3//National Science and Technology Council (NSTC)/ ; EX114-11228NI//National Health Research Institutes (NHRI)/ ; NHRI-13A1-CG-CO-08-2325-2//National Health Research Institutes (NHRI)/ ; 114L8522//National Taiwan University ()/ ; 114L891303//National Taiwan University ()/ ; 114L910203//National Taiwan University ()/ ; 113-L3003//National Taiwan University Hospital (NTUH)/ ; },
mesh = {*Sphingolipids/metabolism ; Animals ; *Metabolic Networks and Pathways ; Brain/metabolism ; Mice ; Humans ; Sphingomyelin Phosphodiesterase/metabolism/genetics ; Membrane Microdomains/metabolism ; CRISPR-Cas Systems ; Ethanolamines/metabolism ; Sphingomyelins ; },
abstract = {Sphingolipids govern diverse cellular processes; their dysregulation underlies numerous diseases. Despite extensive characterizations, understanding the orchestration of the sphingolipid network within living organisms remains challenging. We established a versatile genetic platform of CRISPR-engineered reporters of 52 sphingolipid regulators, recapitulating endogenous gene activity and protein distribution. This platform further allows conditional protein degradation for functional characterization. In addition, we developed the biosensor OlyA[w] to detect ceramide phosphoethanolamine and visualize membrane raft dynamics in vivo. Using this platform, we established comprehensive profiles of the sphingolipid metabolic network in the brain at the transcriptional and translational levels. The highly heterogeneous patterns indicate extensive coordination between distinct cell types and regions, suggesting the brain functions as a coherent unit to execute specific steps of sphingolipid metabolism. As a proof-of-concept application, we showed cell type-specific requirements of sphingomyelinases, including CG6962/dSMPD4 and CG3376/aSMase, degrading distinct subcellular pools of ceramide phosphoethanolamine to maintain brain function. These findings establish a foundation for future studies on brain sphingolipid metabolism and showcase the utilization of this genetic platform in elucidating in vivo mechanisms of sphingolipid metabolism.},
}
@article {pmid41215480,
year = {2025},
author = {Ali, RM and Arshad, H and Zafar, AQ and Gull, S and Gul, A and Mansoor, F and Zahid, R},
title = {A Comprehensive Review on Engineering Lactic Acid Bacteria: Emerging Genetic Tools and Synthetic Biology Strategies.},
journal = {Biotechnology and applied biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1002/bab.70079},
pmid = {41215480},
issn = {1470-8744},
abstract = {Lactic acid bacteria (LAB) are pivotal in food, pharmaceutical, and environmental applications due to their metabolic versatility and probiotic potential. This review explores the advancements in genetic engineering and synthetic biology strategies to enhance LAB functionality. We examine the genomic architecture of key LAB species, such as Lactobacillus and Lactococcus, highlighting their natural genetic traits and metabolic constraints. Emerging genetic tools, including electroporation, conjugation, and CRISPR-Cas systems, have revolutionized LAB modification, enabling precise gene editing and expression control. Synthetic biology approaches, such as genetic circuits, riboswitches, and biosensor development, offer novel pathways for optimizing LAB for functional foods, mucosal therapeutics, and industrial biotechnology. We discuss applications in probiotic delivery, bioremediation, and agricultural enhancement, emphasizing LAB's role in producing bioactive metabolites and combating pathogens. Challenges, including plasmid instability, metabolic burden, and regulatory hurdles, are addressed alongside socio-ethical considerations for genetically modified LAB. The integration of genome-scale engineering and CRISPR-based technologies holds promise for overcoming these barriers, paving the way for next-generation LAB with enhanced stress tolerance and tailored functionalities. This review synthesizes current knowledge and future prospects, underscoring the transformative potential of engineered LAB in addressing global health, environmental, and industrial needs while navigating biosafety and public perception challenges.},
}
@article {pmid41215577,
year = {2025},
author = {Uombe, NPI and Velikkakam, T and Silveira, ACA and Rodrigues, CC and Borges, BC and Teixeira, TL and Pereira, CL and Servato, JPS and Melo, NS and Mortara, RA and Silveira, JFD and Silva, CVD},
title = {Trypanosoma cruzi P21 Is a Pleiotropic Protein That Is Involved in Parasite Host Cell Invasion and Intracellular Parasitism.},
journal = {MicrobiologyOpen},
volume = {14},
number = {6},
pages = {e70154},
pmid = {41215577},
issn = {2045-8827},
support = {//This study was supported by grants and fellowships from FAPESP, FAPEMIG, CAPES, and CNPq. FAPESP Grant number: 2016/15000-4 and 2019/05049-4 (Postdoctoral fellowship); FAPEMIG - RED-00198-23./ ; },
mesh = {*Trypanosoma cruzi/pathogenicity ; *Protozoan Proteins/metabolism ; *Intercellular Signaling Peptides and Proteins/metabolism ; Virulence ; Gene Knockout Techniques ; *Host-Parasite Interactions ; CRISPR-Cas Systems ; Animals ; Mice ; Heart/parasitology ; Vero Cells ; Chlorocebus aethiops ; Mice, Inbred C57BL ; Male ; Mice, Knockout ; Parasite Load ; *Chagas Disease/parasitology ; },
abstract = {We characterized the secreted Trypanosoma cruzi P21 protein and hypothesized its role in parasite invasion and multiplication. To investigate the role of T. cruzi P21 protein in host-parasite interactions, specifically focusing on the low-virulence G strain. P21 knockout parasites were generated using CRISPR/Cas9. Cell invasion, multiplication, egress, and tissue parasitism were assessed in vitro and in vivo, comparing knockout and control parasites. P21 knockout significantly reduced parasite invasion and multiplication in Vero cells. In vivo, knockout parasites also showed reduced heart tissue parasitism in infected mice, despite no observable systemic parasitemia. Accordingly, P21 knockout trypomastigote egress was reduced in Vero cells. P21 plays a pleiotropic role in T. cruzi infection, differentially impacting parasite biology in the low-virulent G strain. In the G strain, P21 promotes invasion and persistence, potentially through mechanisms distinct from its role in the Y strain previously described. This highlights its potential as a therapeutic target for Chagas disease, warranting further investigation into strain-specific functions.},
}
@article {pmid41215733,
year = {2026},
author = {Claisse, O and Mosterd, C and Marrec, CL and Samot, J},
title = {Defense Systems and Prophage Detection in Streptococcus mutans Strains.},
journal = {Molecular oral microbiology},
volume = {41},
number = {2},
pages = {57-68},
pmid = {41215733},
issn = {2041-1014},
support = {//Association France Parkinson/ ; },
mesh = {*Streptococcus mutans/virology/genetics/isolation &amp; purification ; *Prophages/genetics/isolation &amp; purification ; Humans ; CRISPR-Cas Systems/genetics ; Saliva/microbiology ; Toxin-Antitoxin Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Genome, Bacterial ; Computational Biology ; DNA Restriction-Modification Enzymes/genetics ; },
abstract = {Although the species is extensively studied, limited data are available on antiphage defense systems (APDSs) in Streptococcus mutans. The present study aimed to explore the diversity and the occurrence of APDSs and to search for prophages in the genomes of clinical isolates of S. mutans using bioinformatics tools. Forty-four clinical isolates of S. mutans were obtained from saliva samples of people with Parkinson's disease. Genomic DNA was extracted, sequenced using Illumina MiSeq technology, and analyzed for the presence of defense systems using DefenseFinder and PADLOC. CRISPR-Cas systems were characterized using CRISPRCasFinder, and prophages were detected by the PhiSpy pipeline from RAST. AcrFinder and AcrHub were used to identify anti-CRISPR proteins. Each strain harbored between 6 and 12 APDS, with restriction-modification systems being the most prevalent, followed by the MazEF toxin-antitoxin system and CRISPR-Cas systems. Type II-C CRISPR-Cas systems were not identified here in S. mutans. Novel variations in type II-A signature protein Cas9 were identified, allowing their classification into four distinct groups. Variability in direct repeat sequences within the same CRISPR array was also observed, and 80% of the spacers were classified as targeting "dark matter". A unique prophage, phi_37bPJ2, was detected, showing high similarity with previously described phages. The AcrIIA5 protein encoded by phi_37bPJ2 was conserved and suggested to remain functionally active. This study reveals the diversity of APDSs in S. mutans and the limited presence of prophages. The findings provide a foundation for future research on the evolutionary dynamics of these systems and their role in S. mutans adaptation to phage pressure.},
}
@article {pmid41216030,
year = {2025},
author = {Naki, D and Gophna, U},
title = {Evolutionary insights into provirus-encoded CRISPR-Cas systems in halophilic archaea.},
journal = {microLife},
volume = {6},
number = {},
pages = {uqaf033},
pmid = {41216030},
issn = {2633-6693},
abstract = {Prokaryotic microorganisms coexist with mobile genetic elements (MGEs), which can be both genetic threats and evolutionary catalysts. In Haloferax lucentense, a halophilic archaeon, we have recently identified an unusual genomic arrangement: a complete type I-B CRISPR-Cas system encoded on a megaplasmid and an incomplete type I-B system within an integrated provirus in the main chromosome. The provirus-encoded system lacks the adaptation genes (cas1, cas2, and cas4), suggesting its potential reliance on the megaplasmid-encoded CRISPR-Cas module for the acquisition of new spacers. This arrangement suggests a potential instance of "adaptive outsourcing," where a provirus might leverage a co-resident MGE for a key function. Through comparative genomics, we show that similar proviral CRISPR-Cas systems are found in distantly related haloarchaea (e.g. Natrinema and Halobacterium), indicating probable virus-mediated horizontal transfer and suggesting they may function as mobile defense modules. Phylogenetic analysis highlights distinct evolutionary origins of the two systems: the plasmid system clusters with other Haloferax CRISPR-Cas systems, while the proviral system clusters with those from other genera, consistent with horizontal acquisition. Interestingly, spacer analysis reveals that the proviral systems predominantly target viral sequences, while the plasmid system appears to target both plasmids and viral sequences, a distribution mirroring broader trends observed in other plasmid- and chromosome-encoded CRISPR systems. This observed targeting preference suggests a potential for complementarity that could support a model of cooperative immunity, where each system may protect its genetic "owner" from competition and, indirectly, the host.},
}
@article {pmid41217051,
year = {2025},
author = {Alam, SS and Mehdi, A and Zafar, A and Ali, S and Rehman, AU and Liaqat, I and Peng, L and Kanwal, F and Afzal, S and Haq, IU and Aftab, MN},
title = {Advances in microbial biofuel production by metabolic and enzyme engineering, synthetic biology, metagenomics, and genome editing applications.},
journal = {Emerging topics in life sciences},
volume = {8},
number = {3},
pages = {107-124},
pmid = {41217051},
issn = {2397-8554},
mesh = {*Biofuels/microbiology ; *Metagenomics/methods ; *Gene Editing/methods ; *Synthetic Biology/methods ; *Metabolic Engineering/methods ; CRISPR-Cas Systems ; },
abstract = {Microorganisms are the primary source of genetic diversity on earth due to their unparalleled metabolic and functional variability. With the depletion of fossil fuels, a sustainable alternative approach is the use of biofuels, where plant biomass as feedstock is essentially degraded to sugars with the aid of microbe-derived enzymes, followed by the conversion of those sugars to biofuels. Several cellulolytic and non-cellulolytic enzymes are involved in biofuel synthesis. Molecular cloning, along with the advancements in genetic and metabolic engineering in microbial cells, plays a significant contribution to biofuel overproduction. Advanced molecular technologies such as metagenomics and synthetic biology approaches are also being used to construct effective microorganisms for biofuel manufacturing. Obtaining novel enzymes from undiscovered microbial consortia and functional gene analysis is possible through a metagenomics approach. While synthetic biology provides engineered biological systems to generate required biofuel productivity, the CRISPR-Cas genome editing tool is another revolutionary tool being utilized for efficient biofuel production. This article provides a brief overview of different methods of biofuel production using microorganisms.},
}
@article {pmid41217062,
year = {2025},
author = {Vieyra, F and Pindi, C and Lisi, GP and Morzan, UN and Palermo, G},
title = {Design Rules for Expanding PAM Compatibility in CRISPR-Cas9 from the VQR, VRER and EQR variants.},
journal = {The journal of physical chemistry. B},
volume = {129},
number = {46},
pages = {11949-11958},
doi = {10.1021/acs.jpcb.5c06153},
pmid = {41217062},
issn = {1520-5207},
mesh = {Molecular Dynamics Simulation ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism/genetics ; *CRISPR-Associated Protein 9/chemistry/metabolism/genetics ; Gene Editing ; },
abstract = {Expanding the range of Protospacer Adjacent Motifs (PAMs) recognized by CRISPR-Cas9 is essential for broadening genome-editing applications. Here, we combine molecular dynamics simulations with graph-theory and centrality analyses to dissect the principles of PAM recognition in three Cas9 variants - VQR, VRER, and EQR - that target noncanonical PAMs. We show that efficient recognition is not dictated solely by direct contacts between PAM-interacting residues and DNA but also by a distal network that stabilizes the PAM-binding domain and preserves long-range communication with REC3, a hub that relays signals to the HNH nuclease. A key role emerges for the D1135 V/E substitution, which enables stable DNA binding by K1107 and preserves key DNA phosphate locking interactions via S1109, securing stable PAM engagement. In contrast, variants carrying only R-to-Q substitutions at PAM-contacting residues, though predicted to enhance adenine recognition, destabilize the PAM-binding cleft, perturb REC3 dynamics, and disrupt allosteric coupling to HNH. Together, these findings establish that PAM recognition requires local stabilization, distal coupling, and entropic tuning, rather than a simple consequence of base-specific contacts. This framework provides guiding principles for engineering Cas9 variants with expanded PAM compatibility and improved editing efficiency.},
}
@article {pmid41217936,
year = {2025},
author = {He, X and Deng, L and Zhou, S and Gu, T and Li, X and Zhu, S and Luo, X and Huo, D and Hou, C},
title = {Breaking the PAM Restriction: A Universal Double Stranded DNA Detection Method Based on the Sticky End-Mediated CRISPR/Cas12a Coupled RPA and Its Application to KRAS G12C Single Base Mutations.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26886-26896},
doi = {10.1021/acs.analchem.5c05908},
pmid = {41217936},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; *Proto-Oncogene Proteins p21(ras)/genetics ; *DNA/analysis/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Point Mutation ; *CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; *Recombinases/metabolism ; Bacterial Proteins ; },
abstract = {The CRISPR/Cas12a system facilitates efficient and specific nucleic acid detection, but its dependence on Protospacer Adjacent Motif (PAM) sequences and the complexity of existing sticky end-based methods pose challenges for stable and portable applications. To address these issues, this study developed a universal dsDNA detection method by integrating the sticky end-mediated CRISPR/Cas12a with recombinase polymerase amplification (RPA). By incorporating NlaIII recognition sites into RPA primers, precise cleavage of amplification products was achieved, generating uniform sticky ends and eliminating reliance on PAM sites. In comparison to flat end dsDNA containing PAM sites, the use of sticky end dsDNA significantly enhanced Cas12a activity. This strategy demonstrated sensitivity and specificity, achieving a detection limit of 40 aM and successfully identifying KRAS G12C mutations at a frequency of 0.1%, with genomic DNA results aligning with those obtained from FastNGS. Furthermore, we preliminarily explored a one-tube detection strategy, which effectively streamlined the operational process and reduced aerosol contamination. In summary, we established a simple, sensitive, and universal PAM-free CRISPR/Cas12a detection platform that integrates the advantages of isothermal amplification with a standardized sticky end design, thereby offering broad application prospects in molecular diagnostics and clinical translation.},
}
@article {pmid41218116,
year = {2025},
author = {Wang, X and Cramer, Z and Leu, NA and Monaghan, K and Durning, K and Adams-Tzivelekidis, S and Rhoades, JH and Heintz, J and Tian, Y and Rico, J and Mendez, D and Petroni, R and King, AC and Kim, MS and Matsuda, R and Hanselman, O and Shin, AE and Carrera Rodríguez, MF and Brodsky, IE and Rustgi, A and Li, N and Lengner, CJ and Andrés Blanco, M},
title = {Forward genetic screening in engineered colorectal cancer organoids identifies regulators of metastasis.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {46},
pages = {e2510910122},
pmid = {41218116},
issn = {1091-6490},
support = {P30 DK050306/DK/NIDDK NIH HHS/United States ; F31CA250267-02/GF/NIH HHS/United States ; F31AI160741-01/GF/NIH HHS/United States ; R01 CA168654/CA/NCI NIH HHS/United States ; T32 CA285274/CA/NCI NIH HHS/United States ; R01 CA279317-01/GF/NIH HHS/United States ; P30-DK050306/GF/NIH HHS/United States ; R01 CA266641/CA/NCI NIH HHS/United States ; R01 CA277795/CA/NCI NIH HHS/United States ; },
mesh = {*Colorectal Neoplasms/genetics/pathology ; Animals ; *Organoids/pathology/metabolism ; Mice ; Humans ; *Neoplasm Metastasis/genetics ; CRISPR-Cas Systems ; *Genetic Testing/methods ; Cell Movement/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness ; *Adenocarcinoma/genetics/pathology ; },
abstract = {Metastatic outgrowth requires that cancer cells delaminate from the primary tumor, intravasate, survive in circulation, extravasate, migrate to, and proliferate at a distal site. Recurrent genetic drivers of metastasis remain elusive, suggesting that unlike the early steps of oncogenesis, metastasis drivers may be variable. We develop a framework for identifying metastasis regulators using CRISPR/Cas9-based screening in a genetically defined organoid model of colorectal adenocarcinoma. We conduct in vitro screens for invasion and migration alongside orthotopic, in vivo screens for gain of metastasis in a syngeneic mouse model. We identify CTNNA1 and BCL2L13 as bona fide metastasis-specific suppressors which do not confer any selective advantage in primary tumors. CTNNA1 loss promotes cell invasion and migration, and BCL2L13 loss promotes anchorage-independent survival and non-cell-autonomous changes to macrophage polarization. This study demonstrates proof of principle that large-scale genetic screening can be performed in tumor-organoid models in vivo and identifies novel regulators of metastasis.},
}
@article {pmid41218271,
year = {2026},
author = {Fang, T and Peng, C and Ding, R and Fan, Y and Jia, J and Chen, J and Zhang, X and Wang, D and Li, J},
title = {Biomimetic nanoplatform-mediated CRISPR/Cas9 delivery for dual-pathway metabolic blockade in head and neck squamous cell carcinoma.},
journal = {Biomaterials},
volume = {328},
number = {},
pages = {123837},
doi = {10.1016/j.biomaterials.2025.123837},
pmid = {41218271},
issn = {1878-5905},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; Animals ; *Squamous Cell Carcinoma of Head and Neck/metabolism/drug therapy/pathology ; Cell Line, Tumor ; *Nanoparticles/chemistry ; *Head and Neck Neoplasms/metabolism/drug therapy/pathology ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics/metabolism ; *Biomimetic Materials/chemistry ; Glycolysis/drug effects ; Mice ; Mice, Nude ; },
abstract = {Head and neck squamous cell carcinoma (HNSCC) continues to exhibit a poor prognosis, largely due to late diagnosis and the development of cisplatin resistance. Tumor proliferation in HNSCC is closely associated with upregulation of key glycolytic enzymes. However, monotherapeutic targeting of glycolysis paradoxically enhances compensatory glutaminolysis via glutamate overproduction. To overcome this metabolic adaptation, we developed biomimetic nanoparticles P-T-p@CM, fabricated from the pH/reduction dual-responsive copolymer poly(lactic acid)-polyhistidine-polyethylenimine (PLA-pHis-ss-PEI). This nanoplatform enables coordinated co-delivery of telaglenastat (a glutaminase 1 (GLS1) inhibitor) and a CRISPR-Cas9 plasmid encoding sgRNA targeting HIF-1α. This system utilizes homologous cancer cell membrane coating for precise tumor homing, with stimuli-responsive release enabling simultaneous dual metabolic blockade: CRISPR-mediated HIF-1α knockout attenuates glycolysis while telaglenastat suppresses glutamine-to-glutamate conversion. Metabolic analyses confirmed significant reduction in Glycolysis proton efflux rate (GlycoPER), the oxygen consumption rate (OCR) and ATP generation, as well as the related metabolites including the lactate production and glutamate. This dual-starvation strategy depleted energy reserves and biosynthetic precursors, inducing severe metabolic disruption. Notably, in vivo studies showed a 90 % tumor inhibition rate (TIR) after 15 days of treatment, through enhanced apoptosis, reduced proliferation, and tumor glucose/glutamate depletion. Collectively, P-T-p@CM establishes a paradigm-shifting approach to disrupt metabolic compensation in the treatment of HNSCC.},
}
@article {pmid41218364,
year = {2025},
author = {Huang, X and Xiao, T and Zhao, X and Yang, Z and Sun, Z and Lu, K},
title = {Olfactory perception of trifluralin by GOBP2 decreases the susceptibility of Spodoptera litura to insecticides through modulation of 20E signaling pathway-mediated metabolic detoxification.},
journal = {Journal of hazardous materials},
volume = {500},
number = {},
pages = {140417},
doi = {10.1016/j.jhazmat.2025.140417},
pmid = {41218364},
issn = {1873-3336},
mesh = {Animals ; *Insecticides/toxicity/pharmacology ; *Spodoptera/drug effects/metabolism/genetics ; Signal Transduction/drug effects ; Inactivation, Metabolic ; *Trifluralin/pharmacology/toxicity ; Larva/drug effects/metabolism ; *Insect Proteins/genetics/metabolism ; Chlorpyrifos/toxicity ; CRISPR-Cas Systems ; *Herbicides/toxicity/pharmacology ; Oxazines ; },
abstract = {Herbicide contamination has emerged as a critical ecological concern due to its persistent environmental accumulation and unintended impacts on non-target species. This study reveals that olfactory exposure to the volatile herbicide trifluralin enhances metabolic detoxification in Spodoptera litura larvae, significantly reducing their susceptibility to indoxacarb and chlorpyrifos. Building on our previous discovery of GOBP2's trifluralin-binding capacity, CRISPR/Cas9-generated GOBP2 knockout (GOBP2[KO]) larvae exhibited compromised loss of cross-tolerance, accompanied by impaired induction of carboxylesterases (COEs) and glutathione S-transferases (GSTs). Mechanistically, trifluralin perception through GOBP2 activated a 20-hydroxyecdysone (20E)-dependent pathway, upregulating both 20E biosynthesis genes and ecdysone receptor (EcR)/ultraspiracle (USP) complexes in wild-type insects. Systemic disruption of this signaling axis via RNAi-mediated EcR/USP co-silencing abolished trifluralin-induced detoxification enzyme activation and restored insecticide vulnerability. Molecular validation through dual-luciferase assays and yeast-one hybrid systems confirmed EcR/USP-mediated transactivation of COE/GSTe genes via conserved response elements. Functional characterization identified GSTe1 and GSTe16 as key effectors, with GSTe16 exhibiting effective metabolic efficiency against indoxacarb (19.25 % degradation) and chlorpyrifos (13.04 % degradation), while GSTe1 showed significant activity toward indoxacarb metabolism (14.96 %) and chlorpyrifos degradation (10.17 %). Genetic evidence from CRISPR/Cas9-ablated S. litura and transgenic Drosophila models further established causal relationships between GSTe expression levels and insecticide tolerance. Our integrated analysis establishes that GOBP2-mediated endocrine signaling constitutes a central axis in trifluralin-induced insecticide tolerance, directly bridging herbicide perception to metabolic resistance through 20E-dependent regulatory cascades.},
}
@article {pmid41218598,
year = {2025},
author = {Nazarudeen, A and Aswathy, VA and Rauf, AA and Aswathy, UV and Mahesh Chandran, VR and Abhirami, N and Sudhina, S and Chandran, A and Ayyappan, JP},
title = {Targeting Circular RNAs (circRNAs) in Atherosclerosis Using CRISPR Technology.},
journal = {The journal of gene medicine},
volume = {27},
number = {11},
pages = {e70048},
doi = {10.1002/jgm.70048},
pmid = {41218598},
issn = {1521-2254},
support = {//University of Kerala/ ; //Start-up Research Grant from the Science and Engineering Research Board (SERB), India/ ; //Start-up grant for new faculty from the University Grants Commission (UGC), India/ ; //University of Kerala research support in the form of Plan and Nonplan funds/ ; },
mesh = {*RNA, Circular/genetics ; Humans ; *Atherosclerosis/genetics/therapy/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; Gene Editing/methods ; Genetic Therapy/methods ; Lipid Metabolism/genetics ; },
abstract = {Atherosclerosis is a chronic inflammatory condition that remains a major global cause of cardiovascular morbidity and death. Circular RNAs (circRNAs), emerging as key regulators of biological processes, have been linked to atherosclerosis because of their functions in inflammation, lipid metabolism, and plaque stability. This review explores the biogenesis and cellular functions of circRNAs, highlighting specific circRNAs, such as circANRIL, circHIPK, and circRSF1, which influence atherosclerosis progressions and development. CRISPR-Cas technology, specifically Cas9 and Cas13, has transformed the way atherosclerosis is studied and potentially treated. Targeting PCSK9, LDLR, and APOB to modify lipid metabolism, including lowering LDL cholesterol and repairing mutations in familial hypercholesterolemia, has been made possible using CRISPR-Cas9 in atherosclerosis models. In parallel, CRISPR-Cas13 offers a novel approach for RNA-level intervention by selectively editing circRNAs, providing a dynamic approach to regulate atherosclerosis-related pathways. In order to convert these findings into therapeutic treatments, future research should focus on elucidating the mechanics of circRNA, which in turn determines CRISPR-Cas13, and designing specific delivery systems. This review paper demonstrates the revolutionary promise of circRNA research and CRISPR innovation in the treatment of atherosclerosis and underscores the need for extensive preclinical validation to bridge the gap towards clinical use.},
}
@article {pmid41218606,
year = {2025},
author = {Shevade, K and Yang, YA and Feng, K and Mader, K and Sevim, V and Parsons, J and Arora, G and Elfawy, H and Mace, R and Federman, S and Esanov, R and Shafer, S and Chow, ED and Przybyla, L},
title = {Simultaneous capture of single cell RNA-seq, ATAC-seq, and CRISPR perturbation enables multiomic screens to identify gene regulatory relationships.},
journal = {Cell reports methods},
volume = {5},
number = {12},
pages = {101222},
pmid = {41218606},
issn = {2667-2375},
mesh = {Humans ; *Single-Cell Analysis/methods ; *Gene Regulatory Networks/genetics ; *RNA-Seq/methods ; Induced Pluripotent Stem Cells/metabolism ; *Chromatin Immunoprecipitation Sequencing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Dasatinib/pharmacology ; *CRISPR-Cas Systems/genetics ; Cell Line, Tumor ; Chromatin/metabolism/genetics ; Transposases/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Here, we introduce CRISPR and transcriptomics-assay for transposase-accessible chromatin (CAT-ATAC), a technique that adds CRISPR guide RNA (gRNA) capture to the existing 10× Genomics Multiome assay, generating linked transcriptome, chromatin accessibility, and perturbation identity data from the same individual cells. We demonstrate up to 77% capture rate for both arrayed and pooled delivery of lentiviral gRNAs in induced pluripotent stem cells (iPSCs) and cancer cell lines. This capability allows us to construct gene regulatory networks (GRNs) in cells under drug and genetic perturbations. By applying CAT-ATAC, we identified a GRN associated with dasatinib resistance, indirectly activated by the HIC2 gene. Using loss-of-function experiments, we further validated that ZFPM2, a component of the predicted GRN, also contributes to dasatinib resistance. CAT-ATAC can thus be used to generate high-content multidimensional genotype-phenotype maps to reveal gene and cellular interactions and functions.},
}
@article {pmid41218722,
year = {2026},
author = {Madugula, SS and Jayasinghe-Arachchige, VM and Norgan Radler, CR and Wang, S and Liu, J},
title = {Structure-Based Classification of CRISPR/Cas9 Proteins: A Machine Learning Approach to Elucidating Cas9 Allostery.},
journal = {Journal of molecular biology},
volume = {438},
number = {2},
pages = {169538},
doi = {10.1016/j.jmb.2025.169538},
pmid = {41218722},
issn = {1089-8638},
support = {R21 GM144860/GM/NIGMS NIH HHS/United States ; },
mesh = {*Machine Learning ; Allosteric Regulation ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/chemistry/genetics/metabolism ; Molecular Dynamics Simulation ; Streptococcus pyogenes/genetics/enzymology ; Gene Editing/methods ; Protein Conformation ; },
abstract = {The CRISPR/Cas9 system is a powerful gene-editing tool. Its specificity and stability rely on complex allosteric regulation. Understanding these allosteric regulations is essential for developing high-fidelity Cas9 variants with reduced off-target effects. Here, we used a novel structure-based machine learning (ML) approach to systematically identify long-range allosteric networks in Cas9. Our ML model was trained using all available Cas9 structures, ensuring a comprehensive representation of Cas9's structural landscape. We then applied this model to Streptococcus pyogenes Cas9 (SpCas9) to demonstrate the feature selection process. Using Cα-Cα inter-residue distances, we mapped key allosteric networks and refined them through a two-stage SHAP feature selection (FS) strategy, reducing a vast feature space to 28 critical Lysine-Arginine (Lys-Arg) residue pairs that mediate SpCas9 interdomain communication, stability, and specificity. These Lys-Arg pairs initially shared a 46.5 Å inter-residue distance, but molecular dynamics simulations revealed distinct stabilization behaviors, indicating a hierarchical allosteric network. Further mutational analysis of R78A-K855A (M1) and R765A-K1246A (M2) identified an "electrostatic valley," a stabilizing network where positively charged residues interact with negatively charged DNA to maintain SpCas9's structural integrity. Disrupting this valley through direct (M2) or allosteric (M1) mutations destabilized SpCas9's DNA-bound conformation, leading to distinct pathways for improving SpCas9 specificity. This study provides a new framework for understanding allostery in Cas9, integrating ML-driven structural analysis with MD simulations. By identifying key allosteric residues and introducing the electrostatic valley as a central concept, we offer a rational strategy for engineering high-fidelity Cas9 variants. Beyond Cas9, our approach can be applied to uncover allosteric hotspots in other enzyme regulations and rational protein design.},
}
@article {pmid41219189,
year = {2025},
author = {Uchigashima, M and Iguchi, R and Fujii, K and Shiku, K and Kumar, P and Liu, X and Isogai, M and Hoshino, C and Abe, M and Nozumi, M and Okamura, Y and Igarashi, M and Sakimura, K and Bise, R and Lavis, LD and Mikuni, T},
title = {Single-cell synaptome mapping of endogenous protein subpopulations in mammalian brain.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9705},
pmid = {41219189},
issn = {2041-1723},
support = {JP19dm0207080, JP21wm0525014, and JP24wm0625117//Japan Agency for Medical Research and Development (AMED)/ ; JPMJPR16F9//MEXT | JST | Precursory Research for Embryonic Science and Technology (PRESTO)/ ; CDA00043/2019-C//Human Frontier Science Program (HFSP)/ ; N/A//Uehara Memorial Foundation/ ; JP20K21461, JP20H03349, JP20H05918, JP23K18160, and JP24K02130//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JPMJFR231M//MEXT | Japan Science and Technology Agency (JST)/ ; N/A//Kowa Life Science Foundation/ ; },
mesh = {Animals ; *Synapses/metabolism ; *Brain/metabolism/cytology ; *Single-Cell Analysis/methods ; Mice ; *Neurons/metabolism ; CRISPR-Cas Systems ; Mice, Inbred C57BL ; },
abstract = {Different spatial or temporal protein populations, such as cell-surface/intracellular or pre-existing/nascent subpopulations, determine the basal and activity-induced functions of individual synapses within a neuron in vivo. Here, we developed a simple and generalizable platform to image different spatial and temporal subpopulations of endogenous proteins at thousands of synapses in single neurons in the mammalian brain. The platform is based on the development, improvement and integration of CRISPR-Cas9-mediated protein labeling methods, chemical tag labeling techniques, and a semi-automatic analytical pipeline. The combined platform enables whole-cell mapping of total, cell-surface, intracellular, pre-existing, nascent or nascent-and-surface populations of endogenous proteins, such as receptor, scaffold and signaling proteins, at thousands of synapses in individual neurons in living or fixed mouse brain. Our single-cell "synaptome" mapping of endogenous protein subpopulations comprehensively visualizes the spatial representation of synapse diversity in protein localization, trafficking and turnover, providing valuable insights into single-cell organization and computations in the brain.},
}
@article {pmid41219509,
year = {2025},
author = {Johnson, KA and Goswami, HN and Catchpole, RJ and Ahmadizadeh, F and Zhao, P and Wells, L and Li, H and Terns, MP},
title = {A phage-encoded anti-CRISPR protein co-opts host enolase to prevent type III CRISPR immunity.},
journal = {Nature microbiology},
volume = {10},
number = {12},
pages = {3162-3175},
pmid = {41219509},
issn = {2058-5276},
support = {2400220//National Science Foundation (NSF)/ ; R35GM118160//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R35 GM118160/GM/NIGMS NIH HHS/United States ; GR000347//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; R35 GM152081/GM/NIGMS NIH HHS/United States ; },
mesh = {*Phosphopyruvate Hydratase/metabolism/genetics ; *Streptococcus thermophilus/virology/immunology/genetics ; *Viral Proteins/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; *Streptococcus Phages/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems provide powerful adaptive immunity against phage infection. In response, phages use anti-CRISPR (Acr) proteins to evade CRISPR immunity. The few type III Acrs identified so far show conditional effectiveness in countering type III immunity or rely on unknown or poorly understood inhibitory mechanisms. Here we report the discovery of AcrIIIA2, a type III-A Acr encoded by Streptococcus thermophilus phages. Biochemical and structural analyses reveal that phage AcrIIIA2 co-opts host enolase, a highly abundant glycolysis enzyme, to form a ternary complex with the S. thermophilus type III-A (Csm) CRISPR ribonucleoprotein complex, obstructing its immune responses. The enolase-chaperoned AcrIIIA2 blocks the initial step of phage RNA binding, thereby preventing downstream type III anti-phage immune responses. Enolase participates in the anti-immune response by serving as an essential structural scaffold, stabilizing Acr-CRISPR interactions. These findings uncover a new anti-defence strategy that exploits a well-conserved host factor to block CRISPR immunity.},
}
@article {pmid41219611,
year = {2025},
author = {Javaid, MM and Ahmed, J and Ahmed, M and Awan, MJA and Waqas, MAB and Ali, Z and Saeed, NA},
title = {CRISPR-Cas9 based editing of the susceptibility allele TaLr34 enhances leaf rust resistance in bread wheat without yield penalty.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {236},
pmid = {41219611},
issn = {1438-7948},
mesh = {*Triticum/genetics/microbiology/growth &amp; development ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Plant Diseases/genetics/microbiology ; *Gene Editing ; Alleles ; *Plant Proteins/genetics/metabolism ; Basidiomycota/pathogenicity ; Plant Leaves/microbiology/genetics ; Plant Breeding ; },
abstract = {Wheat (Triticum aestivum L.) is widely grown and consumed cereal crop around the world, but most wheat-producing regions suffer from rust diseases, especially stripe and leaf rust, which has caused a devastating global pandemic and severely reduced grain yields. The most effective way to control rust problem in wheat is to sow and breed durable, rust resistant wheat varieties. Conventional breeding for disease-resistant crops primarily relies on resistance (R) genes; however, the effectiveness of R gene-mediated resistance is often compromised by mutations in the pathogen. In this study, we employed CRISPR-Cas9-based genome editing as an advanced breeding tool to enhance rust resistance in the bread wheat cultivar Galaxy-13 by knocking out the homologs of the susceptibility allele TaLr34, specifically targeting the conserved regions within exon 11. Out of 21 transformed plants, five carried successful editing and exhibited resistance to moderate resistance against leaf rust. The TaLr34 mutants were evaluated for leaf rust resistance under both glasshouse and field conditions over three consecutive growing seasons at multiple geographical locations. Our results demonstrate that CRISPR-Cas9-mediated knockout of TaLr34 provides a robust strategy for achieving durable leaf rust resistance in the high-yielding elite wheat cultivar Galaxy-13 without compromising grain yield and agronomic performance.},
}
@article {pmid41219913,
year = {2025},
author = {Muttappagol, M and Sharma, M and Adhikari, DN and Suma, R and Borgohain, T and Saikia, B and Keithellakpam, A and Dutt, A and Laskar, A and Hiremath, S and Chikkaputtaiah, C},
title = {Towards Climate-Resilient Crops: Integrative Strategies for Reproductive Stage Abiotic Stress Tolerance.},
journal = {Physiologia plantarum},
volume = {177},
number = {6},
pages = {e70635},
doi = {10.1111/ppl.70635},
pmid = {41219913},
issn = {1399-3054},
support = {MMP025301//Council of Scientific and Industrial Research, India/ ; CRG/2022/007073 TSA//Anusandhan National Research Foundation (ANRF), Government of India/ ; BT/PR38410/GET/119/310/2020//Department of Biotechnology, Government of India (DBT)/ ; },
mesh = {*Crops, Agricultural/physiology/genetics ; *Stress, Physiological ; Plant Breeding ; Reproduction/physiology ; Droughts ; },
abstract = {Abiotic stresses such as drought, heat, cold, and salinity, threaten global crop productivity, particularly during the highly sensitive reproductive phase. These stresses disrupt female gametophyte formation, ovule development, fertilization, and seed maturation, resulting in reduced fertility and yield losses. While the mechanisms of vegetative stress tolerance are relatively well studied, reproductive-stage resilience, especially the resilience of the female gametophyte, remains insufficiently understood, despite its decisive role in determining yield stability. Recent advances provide an opportunity to close this gap through an integrative perspective. Physiological assessments and high-throughput phenotyping offer reliable tools to evaluate gametophyte and embryo performance under stress. Multi-omics approaches, including genomics, transcriptomics, proteomics, and metabolomics, are covering regulatory pathways underlying reproductive success. Emerging technologies such as CRISPR/Cas genome editing, biomarker discovery and machine learning further accelerate trait identification and their application in breeding pipelines. The novelty of this review lies in its focus on the female gametophyte as an overlooked determinant of stress resilience and in presenting a systems-level framework that integrates physiology, omics, breeding, and AI. This integration does more than catalogue stress effects; it connects mechanistic insights with breeding strategies and scales them with computational tools. The ultimate outcome is climate-resilient varieties that sustain productivity under stress, which support farmer livelihoods and contribute to global food security.},
}
@article {pmid41219964,
year = {2025},
author = {George, NA and Zhou, Z and Anantharaman, K and Hug, LA},
title = {Discarded diversity: novel megaphages, auxiliary metabolic genes, and virally encoded CRISPR-Cas systems in landfills.},
journal = {Virology journal},
volume = {22},
number = {1},
pages = {370},
pmid = {41219964},
issn = {1743-422X},
support = {R35 GM143024/GM/NIGMS NIH HHS/United States ; 2016-03686//Natural Sciences and Engineering Research Council of Canada/ ; Tier II chair//Canada Research Chairs/ ; R35GM143024/GM/NIGMS NIH HHS/United States ; },
mesh = {*Bacteriophages/genetics/classification/isolation &amp; purification ; *CRISPR-Cas Systems ; *Waste Disposal Facilities ; Metagenomics ; Archaea/virology/genetics ; *Bacteria/virology/genetics ; Genome, Viral ; Host Microbial Interactions ; Computational Biology ; North America ; },
abstract = {BACKGROUND: Viruses are the most abundant microbial entities on the planet, impacting microbial community structure and ecosystem services. Despite outnumbering bacteria and archaea by an order of magnitude, viruses have been comparatively underrepresented in reference databases. Metagenomic examinations have illustrated that viruses of bacteria and archaea have been specifically understudied in engineered environments. Here we employed metagenomic and computational biology methods to examine the diversity, host interactions, and genetic systems of viruses predicted from 27 samples taken from three municipal landfills across North America.<br><br>RESULTS: We identified numerous viruses that are not represented in reference databases, including the third largest bacteriophage genome identified to date (~&#x2009;678 kbp), and noted a large diversity of viruses in landfills that has limited overlap across landfills and is distinct from viromes in other systems. Host-virus interactions were examined via host CRISPR spacer to viral protospacer mapping which captured hyper-targeted viral populations and six viral populations predicted to infect hosts across multiple phyla. Auxiliary metabolic genes (AMGs) were identified with the potential to augment hosts' methane, sulfur, and contaminant degradation metabolisms, including AMGs not previously reported in the literature. CRISPR arrays and CRISPR-Cas systems were identified from predicted viral genomes, including the two largest bacteriophage genomes to contain these genetic features. Some virally encoded Cas effector-like proteins appear distinct relative to previously reported Cas effectors and are interesting targets for potential genome editing tools.<br><br>CONCLUSIONS: Our observations indicate landfills, as heterogeneous contaminated sites with unique selective pressures, are key locations for diverse viruses and atypical virus-host dynamics.},
}
@article {pmid41220317,
year = {2025},
author = {Rayad, N and Chowdhury, EA and Meno-Tetang, GML},
title = {The Impact of QSP Modeling on the Design and Optimization of Gene Therapy Approaches.},
journal = {CPT: pharmacometrics &amp; systems pharmacology},
volume = {14},
number = {11},
pages = {1760-1764},
pmid = {41220317},
issn = {2163-8306},
mesh = {*Genetic Therapy/methods ; Humans ; Gene Editing/methods ; Dependovirus/genetics ; Genetic Vectors ; Animals ; RNA, Messenger/administration &amp; dosage/genetics ; Nanoparticles/administration &amp; dosage ; CRISPR-Cas Systems ; *Models, Biological ; Lipids/chemistry ; Tissue Distribution ; Liposomes ; },
abstract = {Quantitative Systems Pharmacology (QSP) is increasingly utilized to support the design and translation of gene therapies. This perspective outlines the application of QSP modeling across three domains of gene therapy: mRNA-based therapeutics, adeno-associated virus (AAV) vectors, and genome editing systems. We highlight opportunities for dose optimization, biomarker interpretation, and mechanistic understanding, while addressing current limitations in model generalizability, data sparsity, and translational relevance. Examples include QSP platforms for lipid nanoparticle (LNP)-delivered mRNA, physiologically based pharmacokinetics (PBPK)-informed AAV biodistribution models, and CRISPR-Cas9-based editing systems. These case studies demonstrate QSP's value in de-risking development and personalizing therapies for rare and complex diseases.},
}
@article {pmid41220980,
year = {2025},
author = {Yuan, X and Yu, X and Zhao, W and Sun, J},
title = {A Practical CRISPR-Based Method for Rapid Genome Editing in Caulobacter crescentus.},
journal = {Bio-protocol},
volume = {15},
number = {21},
pages = {e5490},
pmid = {41220980},
issn = {2331-8325},
abstract = {The RNA-guided Cas enzyme specifically cuts chromosomes and introduces a targeted double-strand break, facilitating multiple kinds of genome editing, including gene deletion, insertion, and replacement. Caulobacter crescentus and its relatives, such as Agrobacterium fabrum and Sinorhizobium meliloti, have been widely studied for industrial, agricultural, and biomedical applications; however, their genetic manipulations are usually characterized as time-consuming and labor-intensive. C. crescentus and its relatives are known to be CRISPR/Cas-recalcitrant organisms due to intrinsic limitations of SpCas9 expression and possible CRISPR escapes. By fusing a reporting gene to the C terminus of SpCas9M and precisely manipulating the expression of SpCas9M, we developed a CRISPR/SpCas9M-reporting system and achieved efficient genome editing in C. crescentus and relatives. Here, we describe a protocol for rapid, marker-less, and convenient gene deletion by using the CRISPR/SpCas9M-reporting system in C. crescentus, as an example. Key features • CRISPR-SpCas9M-reporting system overcomes the limitation of CRISPR escape and achieves a high apparent editing efficiency. • The method enables multiple kinds of genome editing, generating in-frame and marker-less chromosomal modifications. • The method completes a cycle of genome editing within one week. • The method could be readily applied for genome editing in C. crescentus, A. fabrum, and S. meliloti.},
}
@article {pmid41221123,
year = {2025},
author = {van Karnebeek, CDM and Gailus-Durner, V and Engelke, UF and Seisenberger, C and Marschall, S and Dragano, NRV and da Silva-Buttkus, P and Leuchtenberger, S and Fuchs, H and Hrabě de Angelis, M and Wevers, RA and Coughlin, CR and Lefeber, DJ},
title = {New treatment for pyridoxine-dependent epilepsy due to ALDH7A1 deficiency: first proof-of-principle of upstream enzyme inhibition in the mouse.},
journal = {Brain communications},
volume = {7},
number = {6},
pages = {fcaf397},
pmid = {41221123},
issn = {2632-1297},
abstract = {Pyridoxine-dependent epilepsy (PDE) due to recessive ALDH7A1 mutations is characterized by intractable epilepsy that is often unresponsive to antiseizure medications. Irrespective of pyridoxine (vitamin B6) supplementation and lysine reduction therapy, patients present severe residual neurocognitive deficits. We evaluated upstream inhibition of 2-aminoadipic semialdehyde synthase (AASS) as a novel therapeutic strategy to reduce the accumulating metabolites (α-aminoadipic semialdehyde, Δ[1]-piperideine-6-carboxylate, pipecolic acid, 6-oxo-pipecolic acid and 2S,6S-/2s,6R-oxopropylpiperidine-2-carboxylic acid) considered neurotoxic. We utilized an existing mouse knockout model of hyperlysinaemia (Aass-knockout) and generated a PDE model, a Aldh7a1 single knockout model via CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated protein) and generated the double-knockout Aass/Aldh7a1 mice. Next-generation metabolomics screening was performed to measure all known biomarkers in brain, liver and plasma of wild-type and mutant mice. Metabolomics confirmed the known metabolite markers for Aldh7a1-knockout and Aass knockout mice in all samples. The potentially neurotoxic metabolites (Δ[1]-piperideine-6-carboxylate, pipecolic acid, 6-oxo-pipecolic acid and 2S,6S-/2s,6R-oxopropylpiperidine-2-carboxylic acid) significantly decreased in double-knockout Aass/Aldh7a1 mice brain and liver tissues compared to Aldh7a1-knockout mice. Plasma analysis revealed a significant reduction of known biomarkers, suggesting a reliable monitoring option in human patients. We demonstrate the first mammalian evidence that AASS inhibition is a viable strategy to rescue abnormal brain metabolism associated with PDE. This may target the intellectual disability and neurologic deficits caused by persistent lysine catabolic-related neurotoxicity despite adequate vitamin B6 supplementation.},
}
@article {pmid41221676,
year = {2025},
author = {Petersen, F and Westermann, S and Smialkovska, V and Mathony, J and Feldmann, A and Niopek, D},
title = {Engineering the Link: From Genome Interaction Maps to Functional Insight.},
journal = {Advanced biology},
volume = {9},
number = {12},
pages = {e00525},
pmid = {41221676},
issn = {2701-0198},
support = {//Baden-W&#xfc;rttemberg Stiftung/ ; VH-NG-1604//Helmholtz Association/ ; 453202693//Deutsche Forschungsgemeinschaft/ ; //Carl-Zeiss-Stiftung/ ; //Aventis Foundation/ ; //Health + Life Science Alliance Heidelberg Mannheim/ ; //Hans und Ria Messer Stiftung/ ; ERC-STG 101115812/ERC_/European Research Council/International ; },
mesh = {Humans ; *Chromatin/genetics/metabolism ; *Genetic Engineering/methods ; Animals ; *Genome ; Promoter Regions, Genetic ; CRISPR-Cas Systems ; Gene Editing/methods ; },
abstract = {The 3D organization of the genome constitutes a spatial layer of information processing that helps govern gene expression and thus cell function. Advances in chromosome conformation capture sequencing have enabled detailed assessment of chromatin architecture, from enhancer-promoter loops to topological domains and higher-order contacts, across cell types and developmental states. While the ability to investigate genome conformation is maturing, the field faces a central challenge: The link between chromatin interactions and cellular function remains largely correlative, leaving their causality unresolved. This review explores how recent developments in genome engineering enable the targeted manipulation of 3D chromatin architecture - specifically DNA loops - to illuminate causal links between genome structure and function. Synthetic strategies are introduced that rewire enhancer-promoter communication through engineered chromatin loops, leveraging programmable DNA-binding platforms such as zinc fingers, transcription activator-like effectors (TALEs), and CRISPR-Cas9. The current limitations of these approaches related to efficiency, scalability, and specificity are also highlighted, and the strategies to address them are outlined. As these systems mature, programmable 3D genome engineering is emerging as a transformative pillar of synthetic biology, complementing sequence-based editing as a core modality for both understanding and ultimately reprogramming genome function.},
}
@article {pmid41221894,
year = {2025},
author = {Wei, R and Wang, S and Pan, Y and Pan, W and Li, N and Tang, B},
title = {CRISPR-coupled triple cascade amplification for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {97},
pages = {19241-19244},
doi = {10.1039/d5cc04993h},
pmid = {41221894},
issn = {1364-548X},
mesh = {*Mycoplasma pneumoniae/isolation &amp; purification/genetics ; *Influenza A Virus, H1N1 Subtype/isolation &amp; purification/genetics ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Saliva/microbiology/virology ; Limit of Detection ; },
abstract = {We developed a CRISPR-coupled triple cascade system integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a, and catalytic hairpin assembly (CHA) for simultaneous lateral flow detection of Mycoplasma pneumoniae and H1N1 in saliva samples, achieving a LOD of 10 aM for H1N1 RNA and 25 aM for MP DNA on a single LFA.},
}
@article {pmid41222266,
year = {2025},
author = {Monahan, CC and Held, K and Yang, H and Sotelo, G and Grieshaber, N and Grieshaber, S and Omsland, A},
title = {Ectopic overexpression and CRISPRi-based knockdown of Chlamydia trachomatis ObgE inhibits RB replication and EB reformation.},
journal = {Journal of bacteriology},
volume = {207},
number = {12},
pages = {e0028225},
pmid = {41222266},
issn = {1098-5530},
support = {R01 AI130072/AI/NIAID NIH HHS/United States ; T32 GM008336/GM/NIGMS NIH HHS/United States ; T32 GM152310/GM/NIGMS NIH HHS/United States ; R01AI130072/NH/NIH HHS/United States ; },
mesh = {*Chlamydia trachomatis/genetics/metabolism/growth &amp; development/physiology ; *Bacterial Proteins/genetics/metabolism ; Humans ; Gene Expression Regulation, Bacterial ; Gene Knockdown Techniques ; HeLa Cells ; Chlamydia Infections/microbiology ; CRISPR-Cas Systems ; },
abstract = {Chlamydia trachomatis is an obligate intracellular bacterium of major clinical significance. While untreated sexually transmitted infections can result in pelvic inflammatory disease and infertility, ocular infections can cause the blinding disease trachoma. During infection of host cells, C. trachomatis transitions between the non-replicative, infectious elementary body (EB) and the replicative, non-infectious reticulate body (RB). ObgE is a GTPase that can promote morphological differentiation in some bacteria. In C. trachomatis, obgE is maximally expressed from 16 to 24hpi, a timeframe that is associated with logarithmic growth and the onset of production of infectious progeny; therefore, ObgE is predicted to have significance during Chlamydia replication and/or morphological transitions. To determine the role of ObgE during the C. trachomatis developmental cycle, we assessed the effects of ObgE ectopic overexpression and CRISPRi-based knockdown of obgE on RB replication and EB formation. When ectopic overexpression of ObgE was induced, we observed a significant decrease in infectious progeny but no changes in bacterial ultrastructure. These data suggest that during ectopic overexpression of ObgE, RBs can transition into EBs; however, EBs are diminished in their ability to establish new infections. CRISPRi-based knockdown of obgE resulted in a 2-log decrease in bacterial yield and infectious progeny. Ultrastructural analysis revealed that knockdown of obgE resulted in small, underdeveloped inclusions with few cells inside. In total, while ectopic overexpression of ObgE negatively affects production of infectious EBs, CRISPRi-based knockdown of obgE severely affects RB replication, inclusion development, and generation of EBs.IMPORTANCEThe pathogenesis of C. trachomatis is reliant on the transition between the non-replicative, infectious elementary body (EB) and the replicative, non-infectious reticulate body (RB). Therefore, understanding the molecular determinants of Chlamydia developmental transitions is of the utmost importance. ObgE has been shown to regulate morphological transitions in other bacteria and is thus predicted to have relevance during regulation of the Chlamydia developmental cycle. Using both ectopic overexpression and CRISPRi-based knockdown of ObgE/obgE, we identify the significance of balanced ObgE expression for RB replication and the formation of infectious EBs. Our findings further expand our knowledge of how developmental transitions in Chlamydia are regulated.},
}
@article {pmid41222440,
year = {2025},
author = {Ming, W and Zhu, Y and Li, L and Wang, T and Pan, A and Xu, Q and Ji, H and Qin, Y and Wu, L},
title = {Allele-Specific CRISPR-Cas9-Based Ratiometric Fluorescence Platform for Portable EGFR L858R Mutation Detection.},
journal = {Analytical chemistry},
volume = {97},
number = {46},
pages = {25832-25839},
doi = {10.1021/acs.analchem.5c05548},
pmid = {41222440},
issn = {1520-6882},
mesh = {Humans ; ErbB Receptors/genetics ; *CRISPR-Cas Systems/genetics ; Alleles ; Mutation ; *Lung Neoplasms/genetics/diagnosis ; *Carcinoma, Non-Small-Cell Lung/genetics/diagnosis ; Fluorescence ; Point-of-Care Testing ; },
abstract = {Effective detection of low-abundance EGFR L858R mutation from circulating tumor DNA (ctDNA) is critical for early stage NSCLC diagnosis. Here, a portable CRISPR-Cas9-based ratiometric fluorescence sensor was proposed. Typically, allele-specific activation of Cas9 and the trans-cleavage of Cy5/BHQ2-labeled blocker DNAs were achieved by engineering sgRNA to position the L858R mutation within the PAM region of Cas9, resulting in increased Cy5 fluorescence. Simultaneously, the attenuated fluorescence of HBC-530 was observed because the released input RNA bound to the Pepper aptamer, which resulted from the decreased melting temperature (Tm) of the blocker DNA-RNA hybrid. Thus, a dual-channel ratiometric readout can be readily attained. Ultimately, visual point-of-care testing (POCT) of L858R at 0.01% allele frequency with single-nucleotide specificity was realized using a compact 3D-printed device. The validation result of 22 collected plasma samples demonstrated 100% concordance with the clinical diagnoses. This platform provides a cost-effective and accessible solution for NSCLC screening, making it particularly suitable for resource-limited healthcare settings.},
}
@article {pmid41222482,
year = {2025},
author = {Chen, W and He, Y and Yuan, J and Yang, F},
title = {Catalytic hairpin assembly cascade-initiated proximity with self-priming amplification for CRISPR-enhanced ultrasensitive detection of coronary heart disease-associated microRNAs.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {45},
pages = {9254-9261},
doi = {10.1039/d5ay01456e},
pmid = {41222482},
issn = {1759-9679},
mesh = {*MicroRNAs/genetics/analysis/blood ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Coronary Disease/genetics/diagnosis ; *Biosensing Techniques/methods ; },
abstract = {Accurate detection of specific microRNAs (miRNAs) is essential for the early diagnosis of coronary heart disease. Emerging technologies, including functional nuclease-mediated target amplification and DNA nanotechnology, offer substantial potential for precise miRNA identification in clinical diagnostics. This study presents a highly sensitive and specific biosensing platform that integrates catalytic hairpin assembly (CHA) cascade-initiated proximity based self-priming amplification and CRISPR/Cas12a-mediated signal generation for miRNA quantification. Target miRNA initiates the CHA cascade, yielding a toehold-bearing CHA product. This toehold subsequently enables "Variable primer" extension, transcribing double-stranded DNA (dsDNA). The resultant dsDNA activates CRISPR/Cas12a, triggering collateral cleavage and signal amplification. Leveraging this dual-amplification strategy (CHA and CRISPR/Cas12a), the assay achieves a sub-femtomolar detection limit (0.36 fM). Dual-sequence verification, including CHA and CRISPR/Cas12a recognition, ensures exceptional specificity. Validation using spiked serum samples confirmed precise miRNA quantification. Collectively, this biosensor demonstrates significant promise for clinical molecular diagnostics.},
}
@article {pmid41222688,
year = {2026},
author = {Yuan, A and Sun, T and Deng, Z and Ye, L and Shang, Y and Xie, W and Peng, H},
title = {A multifunctional approach: merging CRISPR/Cas technology with DNA nanomachines for advanced biosensing.},
journal = {Analytical and bioanalytical chemistry},
volume = {418},
number = {2},
pages = {513-531},
pmid = {41222688},
issn = {1618-2650},
support = {2023YFA0915102//National Key Research and Development Program of China/ ; 22276199//National Natural Science Foundation of China/ ; 22306195//National Natural Science Foundation of China/ ; XDB0750100//Strategic Priority Research Program of the Chinese Academy of Sciences/ ; },
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *DNA/chemistry/genetics ; Humans ; *Nanostructures/chemistry ; Animals ; },
abstract = {CRISPR/Cas systems have revolutionized nucleic acid recognition, offering unparalleled programmability for biosensing. Simultaneously, DNA nanomachines have emerged as powerful bioanalytical tools due to their structural precision, biocompatibility, and multifunctional capabilities. The strategic integration of these platforms synergistically enhances sensitivity, specificity, and multiplexing potential, creating next-generation bioanalytical systems. While promising, this convergence presents unique engineering challenges and necessitates critical evaluation. This review systematically analyzes the target recognition mechanisms of CRISPR/Cas systems and critically evaluates prevalent signal readout modalities. A core focus is the critical assessment of innovative biosensing strategies that leverage DNA nanomachines-particularly dynamic walker systems-coupled with CRISPR/Cas activation. We highlight representative integrated platforms, detailing their operational principles and objectively examining their demonstrated advantages against inherent limitations such as signal leakage, complex assembly, and in vivo applicability constraints. By providing a critical analysis of the synergistic potential and current constraints of CRISPR/Cas-DNA nanomachine integration, this review aims to guide rational design towards robust, clinically translatable precision diagnostics and therapeutics.},
}
@article {pmid41222695,
year = {2026},
author = {Yusuf, M},
title = {CRISPR screens reveal YAP/TEAD axis as a mediator of drug-tolerant cells in EGFR-mutant NSCLC.},
journal = {Naunyn-Schmiedeberg's archives of pharmacology},
volume = {399},
number = {4},
pages = {5761-5776},
pmid = {41222695},
issn = {1432-1912},
mesh = {Humans ; ErbB Receptors/genetics/antagonists &amp; inhibitors ; *Carcinoma, Non-Small-Cell Lung/genetics/drug therapy/metabolism/pathology ; *Lung Neoplasms/genetics/drug therapy/metabolism/pathology ; Drug Resistance, Neoplasm/genetics ; Mutation ; Cell Line, Tumor ; YAP-Signaling Proteins ; *Transcription Factors/genetics/metabolism ; Hippo Signaling Pathway ; *Antineoplastic Agents/pharmacology ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Protein Kinase Inhibitors/pharmacology ; CRISPR-Cas Systems ; Acrylamides/pharmacology ; Signal Transduction/drug effects ; Protein Serine-Threonine Kinases/metabolism ; Aniline Compounds/pharmacology ; Gefitinib/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; Indoles ; Pyrimidines ; },
abstract = {Despite initial responses, most patients with metastatic lung cancer-including those with EGFR mutations-ultimately develop resistance to targeted therapies. To systematically uncover mechanisms underlying this resistance, genome-wide CRISPR knockout and activation screens were conducted in EGFR-mutant lung cancer cell lines treated with EGFR inhibitors such as osimertinib and gefitinib. These screens highlighted a recurrent involvement of genes associated with the Hippo signaling pathway. Notably, a subset of tumor cells, termed 'persister' cells, survive initial osimertinib exposure by engaging non-genetic, transcriptional adaptation mechanisms that promote drug tolerance. Our studies, integrating both genetic and pharmacological approaches, identified Hippo pathway activation as a key driver of this drug-tolerant state. Importantly, co-inhibition of EGFR and the Hippo signaling axis led to a pronounced reduction in cell viability in both established cell lines and patient-derived organoids. These findings propose that dual targeting of EGFR and Hippo signaling may offer a promising therapeutic approach to overcome resistance in EGFR-mutant lung cancer.},
}
@article {pmid41223195,
year = {2025},
author = {Kimata, K and Satou, K},
title = {Improved CRISPR/Cas9 off-target prediction with DNABERT and epigenetic features.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335863},
pmid = {41223195},
issn = {1932-6203},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Epigenesis, Genetic ; *Gene Editing/methods ; Genome, Human ; Deep Learning ; },
abstract = {CRISPR/Cas9 is a powerful genome editing tool, but its clinical application is hindered by off-target effects. Accurate computational prediction of these unintended edits is crucial for ensuring the safety and efficacy of therapeutic applications. While various deep learning models have been developed, most are trained only on task-specific data, failing to leverage the vast knowledge embedded in entire genomes. To address this limitation, we introduce a novel approach that integrates DNABERT, a deep learning model pre-trained on the human genome, with epigenetic features (H3K4me3, H3K27ac, and ATAC-seq). We conducted a comprehensive benchmark of our model, DNABERT-Epi, against five state-of-the-art methods across seven distinct off-target datasets. Our results demonstrate that the pre-trained DNABERT-based models achieve competitive or even superior performance. Rigorous ablation studies quantitatively confirmed that both genomic pre-training and the integration of epigenetic features are critical factors that significantly enhance predictive accuracy. Furthermore, by applying advanced interpretability techniques (SHAP and Integrated Gradients), we identified the specific epigenetic marks and sequence-level patterns that influence the model's predictions, offering insights into its decision-making process. This study is the first to establish the significant potential of a pre-trained DNA foundation model for CRISPR/Cas9 off-target prediction. Our findings underscore that leveraging both large-scale genomic knowledge and multi-modal data is a key strategy for advancing the development of safer genome editing tools.},
}
@article {pmid41223249,
year = {2025},
author = {Pan, X and Huang, P and Ali, SS and Renslo, B and Greenberg, Z and Erwin, N and Li, Y and Ding, Z and Hutchinson, TE and Warnecke, A and Fernandez, NE and Staecker, H and He, M},
title = {Extracellular vesicle-mediated gene editing for the treatment of nonsyndromic progressive hearing loss in adult mice.},
journal = {Science translational medicine},
volume = {17},
number = {824},
pages = {eadn3993},
pmid = {41223249},
issn = {1946-6242},
support = {R35 GM133794/GM/NIGMS NIH HHS/United States ; U01 AI191208/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Extracellular Vesicles/metabolism ; *Gene Editing/methods ; *Hearing Loss/therapy/genetics ; Mice ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Genetic Therapy/methods ; Ribonucleoproteins/metabolism ; Hair Cells, Auditory/metabolism ; Electroporation ; Humans ; RNA, Guide, CRISPR-Cas Systems/metabolism ; },
abstract = {The clinical translation of gene therapy has been challenging in part because of the limitations of current delivery approaches. Herein, we report an efficient nonviral genome editor delivery approach using extracellular vesicles (EVs) carrying single-guide RNA (sgRNA): CRISPR-Cas9 ribonucleoprotein (RNP) complexes for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet-based electroporation system (μDES), we achieved a 10-fold enhancement in loading efficiency and more than 1000-fold increase in processing throughput for loading RNP complexes into EVs compared with conventional high-voltage pulsed electroporation. μDES generated uniform microdroplets containing EVs and RNPs by applying direct current-controlled low voltage (up to 60 V) to transiently permeabilize membranes and enable efficient cargo encapsulation while maintaining EV integrity at both the protein and morphological levels. In the Myo7a[WT/Sh1] mouse model of autosomal dominant progressive hearing loss, which may model MYO7A-associated DFNA11 hearing loss in humans, we demonstrated the effective delivery of RNPs by EVs into cochlear hair cells by cross-sectional and whole-mount confocal imaging. The injection of RNP-EVs via the posterior semicircular canal in 4-week-old Myo7a[WT/Sh1] mice resulted in a reduction in Myo7a[Sh1] messenger RNA expression and evidence of hearing preservation, as measured by auditory brainstem responses, compared with untreated ears and EV only-injected mice. This study highlights the potential of μDES-produced RNP-EVs for gene editing as a treatment for progressive nonsyndromic hearing loss in patients.},
}
@article {pmid41223903,
year = {2025},
author = {Alsultan, A and Karim, SM and Al-Saadi, M and Alsallami, D and Ben Said, M and Belkahia, H},
title = {Rapid and sensitive detection of Theileria equi using a novel integrated RPACRISPR/Cas13a lateral flow assay.},
journal = {Journal of equine veterinary science},
volume = {155},
number = {},
pages = {105732},
doi = {10.1016/j.jevs.2025.105732},
pmid = {41223903},
issn = {0737-0806},
mesh = {Horses ; Animals ; *Theileria/isolation &amp; purification ; *Theileriasis/diagnosis/parasitology ; *Horse Diseases/diagnosis/parasitology ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/veterinary/methods ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: Equine piroplasmosis (EP), caused by the intracellular protozoa Theileria equi, Babesia caballi, and Theileria haneyi, represents a major health and economic threat to the equine industry worldwide. Existing diagnostic methods, including PCR, serology, and microscopy, are constrained by their dependence on specialized equipment, lengthy protocols, and the requirement for skilled personnel.<br><br>AIM: This study aimed to develop a rapid, accurate, and field-deployable molecular diagnostic assay for T. equi.<br><br>METHODS: A nucleic acid-based diagnostic platform combining recombinase polymerase amplification (RPA) with CRISPR/Cas13-mediated detection and lateral flow device (LFD) readout was developed. The assay targets a conserved region of the erythrocyte merozoite antigen 1 (EMA-1) gene of T. equi. Validation was performed using 22 blood samples collected from horses, as well as specificity controls including B. caballi- and Anaplasma phagocytophilum-infected samples, synthetic EMA-1 DNA, and non-template controls. All assay steps were conducted at room temperature.<br><br>RESULTS: The integrated RPA-CRISPR/Cas13-LFD assay generated clear visual results within 50 minutes. It demonstrated complete specificity with no false positives across all tested samples. The method effectively differentiated horses infected with T. equi, including both clinically affected and asymptomatic individuals, from healthy, uninfected animals, confirming its high accuracy and reliability.<br><br>CONCLUSION: The developed assay provides a rapid, precise, and equipment-free diagnostic platform suitable for both field and clinical environments. Although the current protocol relies on DNA extraction, future optimization will aim to enable direct detection from unprocessed blood samples, thereby further simplifying point-of-care diagnostics for equine piroplasmosis.},
}
@article {pmid41224955,
year = {2026},
author = {Gil, JS and Lee, S and Koo, T},
title = {Therapeutic in vivo genome editing: innovations and challenges in rAAV vector-based CRISPR delivery.},
journal = {Gene therapy},
volume = {33},
number = {1},
pages = {97-106},
pmid = {41224955},
issn = {1476-5462},
mesh = {*Dependovirus/genetics ; *Gene Editing/methods ; *Genetic Vectors/genetics ; *Genetic Therapy/methods ; Humans ; *CRISPR-Cas Systems ; Animals ; Gene Transfer Techniques ; },
abstract = {The integration of CRISPR systems with recombinant adeno-associated virus (rAAV) vectors has opened new possibilities for therapeutic genome editing, offering potential treatments for both genetic and non-genetic disorders. rAAV vectors have emerged as promising vehicles for in vivo gene therapy due to their favorable safety profile, high tissue specificity, and ability to induce sustained transgene expression. However, their limited packaging capacity has been a significant challenge for delivering large CRISPR molecules. To overcome this limitation, innovative strategies have been developed, including the use of compact Cas orthologs, dual rAAV vector systems, and trans-splicing rAAV vectors. These approaches have significantly improved the efficiency of genome editing for therapeutic applications. This review presents recent advancements in rAAV-CRISPR-mediated in vivo gene therapy, highlighting key technological innovations, current challenges, and the therapeutic potential of these strategies in the development of next-generation gene therapies.},
}
@article {pmid41225007,
year = {2026},
author = {Noh, H and Hashem, Z and Boms, E and Najafov, A},
title = {SIGLEC12 mediates plasma membrane rupture during necroptotic cell death.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {460-466},
pmid = {41225007},
issn = {1476-4687},
support = {S10 OD020103/OD/NIH HHS/United States ; R35 GM146861/GM/NIGMS NIH HHS/United States ; S10 OD021685/OD/NIH HHS/United States ; S10 OD021684/OD/NIH HHS/United States ; S10 OD028630/OD/NIH HHS/United States ; P30 CA142543/CA/NCI NIH HHS/United States ; },
mesh = {Animals ; Humans ; Mice ; *Cell Membrane/metabolism/pathology ; CRISPR-Cas Systems ; HEK293 Cells ; *Necroptosis/genetics ; Phosphorylation ; Protein Kinases/metabolism ; Membrane Proteins ; Lectins ; },
abstract = {Necroptosis is a form of lytic cell death that is overactivated during infections and in inflammatory pathologies[1]. NINJ1 was recently found to be a mediator of plasma membrane rupture (PMR) during pyroptosis, toxin-induced necrosis, apoptosis, and ferroptosis[2,3], but the mediator of PMR during necroptotic cell death remained unknown. Here, using a CRISPR-Cas9-based genome-wide knockout approach, we identify SIGLEC12 as a key mediator of necroptosis downstream of MLKL at the PMR step. Cells with knockdown or knockout of SIGLEC12 are defective in necroptosis-induced PMR and demonstrate ballooning morphology. During necroptosis, SIGLEC12 undergoes dephosphorylation, interacts with MLKL, forms cytosolic puncta and assembles into fibrils. Notably, SIGLEC12 is cleaved by TMPRSS4 during necroptosis to produce a 20-kDa fragment highly homologous to NINJ1, and this cleavage event is required and sufficient to induce PMR during necroptosis. A SIGLEC12 variant associated with cancer (Ser458Phe) and a variant found in the general human population (Arg528Trp) attenuate SIGLEC12 cleavage by TMPRSS4. Knockout of Siglec12 in mouse cells does not affect PMR, suggesting a species-specific role. Our identification of SIGLEC12 as a mediator of PMR expands our understanding of how programmed necrosis is executed and offers new approaches for targeting this proinflammatory form of cell death in human diseases.},
}
@article {pmid41225502,
year = {2025},
author = {Huang, S and Zhou, F and Jiang, Z and Jiang, S and Yang, Q and Yang, L and Huang, J and Shi, J and Ding, Y and Li, E and Li, Y},
title = {Development and validation of PmMAD7 for efficient gene editing in Penaeus monodon.},
journal = {BMC biotechnology},
volume = {25},
number = {1},
pages = {125},
pmid = {41225502},
issn = {1472-6750},
support = {2022YFD2401900//National Key R &amp; D Program of China/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Penaeidae/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based gene editing has become a promising approach for enhancing traits in aquaculture species. Nevertheless, traditional CRISPR-Cas systems encounter challenges, including significant off-target effects and strict protospacer adjacent motif (PAM) requirements, which constrain their use in crustaceans such as Penaeus monodon. To address these limitations, this research has developed PmMAD7, a codon-optimized CRISPR system specifically designed for P. monodon, which incorporates nuclear localization signals to improve editing efficiency and precision.<br><br>RESULTS: This research successfully synthesized and delivered PmMAD7 mRNA and crRNAs targeting the ECH1 and AQP4 genes into the hemocytes of P. monodon. Quantitative PCR analysis demonstrated that PmMAD7 achieved significant gene silencing, reducing the expression levels of ECH1 and AQP4 by 81.5% and 78.33%, respectively. Next-generation sequencing confirmed targeted insertions and deletions at the gene loci, with knockout efficiencies of 14.81% for ECH1 and 20.57% for AQP4, which were significantly higher than those obtained with LbCas12a (7.14% and 12.43%, respectively). Furthermore, functional analysis indicated that ECH1 knockout resulted in increased cell volume and mortality, while AQP4 knockout led to decreased cell volume and reduced viability. These specific results highlight the first successful demonstration of MAD7-based genome editing in shrimp. The broader PAM compatibility and enhanced editing efficiency of PmMAD7 provide a versatile platform for gene editing in shrimp.<br><br>CONCLUSION: PmMAD7 constitutes an enhanced CRISPR editing tool specifically designed for P. monodon, exhibiting superior precision, expanded PAM compatibility, and enhanced editing efficacy relative to conventional Cas12a systems. These results lay the groundwork for the advancement of gene editing applications in crustaceans and contribute to sustainable genetic improvements in aquaculture.},
}
@article {pmid41226505,
year = {2025},
author = {Qian, Y and Liu, X and Wang, B and Li, D and Wu, Z and Tong, J},
title = {Genome-Wide Identification of the LsaPHR1 Gene Family and Preliminary Functional Validation of LsaPHR1.1 in Phosphorus Tolerance in Lactuca sativa.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
pmid = {41226505},
issn = {1422-0067},
support = {KJCX20240508//Special Innovation Ability Construction Fund of Beijing Academy of Agricultural and Forestry Sciences/ ; QNJJ202248//Beijing Academy of Agriculture and Forestry Science Youth Research Fund/ ; KYCX202506//Innovation and Development Program of Beijing Vegetable Research Center/ ; CARS-24-B-02//China Agriculture Research System of the MOF and MARA/ ; KJCX20200205//Project for Scientific and Technological Innovation Capability Building of the Beijing Academy of Agriculture and Forestry Sciences/ ; },
mesh = {*Lactuca/genetics/metabolism ; *Phosphorus/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Stress, Physiological/genetics ; *Multigene Family ; CRISPR-Cas Systems ; Chlorophyll/metabolism ; Gene Editing ; },
abstract = {Phosphorus (P) is a limiting nutrient for plant growth and productivity. Improving P use efficiency is important for crop production. In Lactuca sativa (lettuce), five phosphate starvation response 1 (PHR1) genes were identified and characterized through a bioinformatics approach. The expression patterns of LsaPHR1s were examined using qRT-PCR under various treatments, including devoid phosphorus (DP), low phosphorus (LP), high phosphorus (HP), darkness, ABA, IAA, and MeJA. The results indicate that LsaPHR1s in lettuce responded to phosphorus stress, hormones, and darkness. Furthermore, we engineered LsaPHR1.1 knock-out mutants via CRISPR/Cas9-mediated genome editing. Then, the mutants were subjected to phosphorus stress (DP, LP, and HP). In contrast to WT, the mutants improved nitrate and ammonium contents, increased antioxidant enzyme activity, and elevated antioxidant and chlorophyll contents. Our results offer a potential strategy for improving phosphorus stress tolerance in lettuce, which holds great significance for maintaining yield and quality.},
}
@article {pmid41226716,
year = {2025},
author = {Zhao, R and Zhu, J and Wang, J and Wang, D and Liu, X and Han, L and Li, S},
title = {Functional Characterization of Fp2Cas9, a Cold-Adapted Type II-C CRISPR Nuclease from Flavobacterium psychrophilum.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
pmid = {41226716},
issn = {1422-0067},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; 2024XT01, 2023TD45//the Central Public Interest Scientific Institution Basal Research Fund, CAFS/ ; Identification, Evaluation, and Breeding of Characteristic Aquatic Animal Germplasm Resources in Heilongjiang Province.//Heilongjiang Province Seed Industry Innovation and Development Fund Project/ ; },
mesh = {*Flavobacterium/enzymology/genetics ; Zebrafish/genetics ; Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; Cold Temperature ; *CRISPR-Associated Protein 9/metabolism/genetics ; *Bacterial Proteins/genetics/metabolism ; },
abstract = {Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5'-SNAAAG-3', and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems.},
}
@article {pmid41226719,
year = {2025},
author = {Amiri, S and Adibzadeh, S and Khazaei Monfared, Y and Kaboli, S and Arashkia, A and Barkhordari, F and Mahmoudian, M and Kheirandish, MH and Trotta, F and Davami, F},
title = {Efficient Delivery of CRISPR-Cas9 RNP Complexes with Cyclodextrin-Based Nanosponges for Enhanced Genome Editing: TILD-CRISPR Integration.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
pmid = {41226719},
issn = {1422-0067},
support = {BD-66002183//Pasteur Institute of Iran/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Cyclodextrins/chemistry ; CHO Cells ; Cricetulus ; *Ribonucleoproteins/genetics ; Green Fluorescent Proteins/genetics ; *Nanostructures/chemistry ; },
abstract = {The CRISPR-Cas9 system has transformed biomedical research by enabling precise genetic modifications. However, efficient delivery of CRISPR components remains a major hurdle for therapeutic applications. To address this, we employed a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly) system to deliver an integrating GFP gene using the TILD-CRISPR method, which couples donor DNA linearization with RNP complexes. The physicochemical properties, loading efficiency, and cellular uptake of RNP with Ppoly were studied. After transfection, antibiotic selection and single-cell cloning were performed. Junction PCR was then performed on the isolated clones, and we compared the knock-in efficiency of Ppoly with that of the commercial CRISPRMAX™ reagent (Thermo Fisher, Invitrogen™, Waltham, MA, USA). The results demonstrate the encapsulation efficiency of over 90% for RNP and Ppoly, and cell viability remaining above 80%, reflecting the minimal toxicity of this approach. These attributes facilitated successful GFP gene integration using the TILD-CRISPR with RNP delivered via cyclodextrin-based nanosponges. The present method achieved a remarkable 50% integration efficiency in CHO-K1 cells, significantly outperforming the 14% observed with CRISPRMAX™ while maintaining lower cytotoxicity. This study highlights a promising platform for precise and efficient genome editing, with strong potential for therapeutic and regenerative medicine applications.},
}
@article {pmid41226739,
year = {2025},
author = {Arana, &#xc1;J and Veiga-Rua, S and Cora, D and Gónzalez-Gómez, MA and Seijas, A and Carballeda, M and Polo, D and Cuesta, A and Piñeiro, Y and Rivas, J and Novo, M and Al-Soufi, W and Martínez, P and Sánchez, L and Robledo, D},
title = {Comparative Analysis of CRISPR/Cas9 Delivery Methods in Marine Teleost Cell Lines.},
journal = {International journal of molecular sciences},
volume = {26},
number = {21},
pages = {},
pmid = {41226739},
issn = {1422-0067},
support = {CRISPR-NANOXEN//Universidade de Santiago de Compostela/ ; Optimizing CRISPR/Cas9 genome editing to improve disease resistance in aquaculture//Fundaci&#xf3;n Caixa Rural Galega Tom&#xe1;s Notario Vacas/ ; investment line n&#xba; 1 and component number 17, which includes the Complementary RTDI Plan for Marine Science//Spain's Recovery and Resilience Plan/ ; No 101076432&#x202f;(FishTRIM)//European Union ERC Starting Grant programme 2022/ ; (BBS/E/20002172, BBS/E/D/30002275, BBS/E/D/10002070 and BBS/E/RL/230002A)//Oportunius programme of the Axencia Galega the Innovaci&#xf3;n (GAIN, Xunta de Galicia)/ ; RYC2023-044793-I//Ministerio de Ciencia, Innovaci&#xf3;n y Universidades/ ; 10.13039/501100011033//Agencia Estatal de Investigaci&#xf3;n/ ; },
mesh = {Animals ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Cell Line ; Electroporation/methods ; *Fishes/genetics ; Transfection/methods ; *Gene Transfer Techniques ; },
abstract = {Gene editing technologies such as CRISPR/Cas9 have revolutionized functional genomics, yet their application in marine fish cell lines remains limited by inefficient delivery. This study compares three delivery strategies-electroporation, lipid nanoparticles (LNPs), and magnetofection using gelatin-coated superparamagnetic iron oxide nanoparticles (SPIONs)-for CRISPR/Cas9-mediated editing of the ifi27l2a gene in DLB-1 and SaB-1 cell lines. We evaluated transfection and editing efficiency, intracellular Cas9 localization, and genomic stability of the target locus. Electroporation achieved up to 95% editing in SaB-1 under optimized conditions, but only 30% in DLB-1, which exhibited locus-specific genomic rearrangements. Diversa LNPs enabled intracellular delivery and moderate editing (~25%) in DLB-1 but yielded only minimal editing in SaB-1, while SPION-based magnetofection resulted in efficient uptake but no detectable editing, highlighting post-entry barriers. Confocal imaging and fluorescence correlation spectroscopy suggested that nuclear localization and Cas9 aggregation may influence editing success, highlighting the importance of intracellular trafficking in CRISPR/Cas9 delivery. Our findings demonstrate that CRISPR/Cas9 delivery efficiency is cell line-dependent and governed by intracellular trafficking and genomic integrity. These insights provide a practical framework for optimizing gene editing in marine teleosts, advancing both basic research and selective breeding in aquaculture.},
}
@article {pmid41229123,
year = {2026},
author = {Ma, Y and Wei, W and Yang, Z and Zhou, Y and Dong, T and Wang, T and Xia, X and Ma, Y and Zhou, M and Gao, Y and Yu, B and Wang, C and Ruan, L and Hong, K and Gu, C},
title = {Exosomes as nonviral carrier for targeted delivery of CRISPR-Cas12a for therapeutic HIV-1 proviral DNA editing.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {2},
pages = {1152-1171},
pmid = {41229123},
issn = {1525-0024},
mesh = {*Exosomes/metabolism/genetics ; Humans ; *HIV-1/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Animals ; *HIV Infections/therapy/virology/genetics ; Mice ; *Proviruses/genetics ; CD4-Positive T-Lymphocytes/metabolism ; Disease Models, Animal ; Genetic Therapy/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; Gene Transfer Techniques ; DNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Current strategies to treat HIV infection including traditional cART and immunotherapy can effectively suppress viral replication but are unable to eliminate the latent viral reservoir, particularly within circulating immune cells. Although genome editing by CRISPR-Cas provides a promising cure for HIV-1, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we developed an exosome-mediated targeted CRISPR-Cas12a delivery system (EMT-Cas12a), an engineered exosome system enabling targeted delivery of mRNA of Cas12a and crRNAs to CD4+ T cells. The EMT-Cas12a system uniquely optimizes cell-specific targeting, CRISPR-Cas12a expression, crRNAs maturation, nuclear entry efficiency, accuracy cleavage with major delins and achieving dramatically HIV suppression in both cellular and humanized mouse models. Compared with single-crRNA approaches, the multiple crRNA arrays strategy demonstrates enhanced antiviral efficacy in HIV-infected mouse model, ex-vivo-expanded PBMCs from HIV+ subjects and especially in vitro cell lines without detectable HIV DNA. Critically, the system exhibits no detectable off-target effects and restores CD4+ T cell counts in vivo and ex vivo PBMCs, indicating its dual therapeutic potential for viral clearance and immune reconstitution. Altogether, in vitro and in vivo excision of HIV-1 proviral DNA can be achieved via EMT-Cas12a delivery, which could advance efforts toward human clinical trials.},
}
@article {pmid41229344,
year = {2025},
author = {Sures, K and Esser, SP and Bornemann, TLV and Moore, CJ and Soares, AR and Plewka, J and Figueroa-Gonzalez, PA and Ruff, SE and Moraru, C and Probst, AJ},
title = {Acquisition of Spacers from Foreign Prokaryotic Genomes by CRISPR-Cas Systems in Natural Environments.},
journal = {Genome biology and evolution},
volume = {17},
number = {11},
pages = {},
pmid = {41229344},
issn = {1759-6653},
mesh = {*CRISPR-Cas Systems ; *Genome, Archaeal ; *Archaea/genetics ; Genome, Bacterial ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics ; Metagenome ; Evolution, Molecular ; *DNA, Intergenic/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems of bacteria and archaea provide immunities against mobile genetic elements, like viruses. In addition, protospacer analyses revealed a very specific acquisition of CRISPR spacers derived from genomes of related species or from closely interacting episymbiont genomes as recently shown for subsurface archaea. However, the origin of most of the spacers that can be found in CRISPR-Cas systems from natural environments has not been deciphered. Here, by analyzing CRISPR-Cas systems of metagenome-assembled genomes (MAGs) from two subsurface environments spanning more than 1 Tb of sequencing data, we show that a substantial proportion of CRISPR spacers are acquired from DNA of other prokaryotes inhabiting the same environment. As such, we found that the number of respective spacers can be up to three times higher than the number of self-targeting spacers. Statistical analyses demonstrated that the acquisition of CRISPR spacers from other prokaryotic genomes is partly explained by the relative abundance of the MAG containing the protospacer, as well as by other factors, such as the total number of CRISPR arrays present in a MAG with the respective spacers. Further, we found that spacer acquisition from foreign prokaryotic DNA occurs in almost all types of CRISPR-Cas systems, but shows preferences for subtypes of CRISPR-Cas systems that differ across the investigated ecosystems. Taken together, our results shed new light on the diversity of CRISPR spacers in natural microbial communities and provide an explanation for some of the many unmatched spacers in public databases.},
}
@article {pmid41230228,
year = {2025},
author = {Park, S and Mani, V and Ha, K and Kim, JA and Lee, S},
title = {Plant synthetic biology: from knowledge to biomolecules.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1562216},
pmid = {41230228},
issn = {1664-462X},
abstract = {Plant synthetic biology is rapidly emerging as an innovative approach to solving complex problems in human health and agriculture. Although conventional metabolic engineering primarily focuses on microbial systems for large-scale biomolecules production, these platforms often face limitations in expressing plant-derived enzymes and synthesizing structurally complex molecules. In contrast, recent advances in plant synthetic biology have integrated multidisciplinary tools, from molecular biology and biochemistry to synthetic circuit design and computational modeling, to engineer plant systems with enhanced traits. These include improved yield, nutritional quality, environmental resilience, and synthesis of pharmaceutically relevant functional biomolecules. This review focuses on the fundamental technologies that have enabled such advances, which include DNA synthesis, programmable gene circuits, and CRISPR/Cas-based genome editing. We discussed recent applications of reprogramming plant metabolic pathways and existing obstacles, such as transformation efficiency, regulatory bottlenecks, and pathway instability. This review provides key case studies and a forward-looking perspective on the evolution of plant synthetic biology as a robust foundation for sustainable biomanufacturing and production of functional biomolecules.},
}
@article {pmid41230595,
year = {2026},
author = {Kim, K and Kachiprath, B and Yeo, IC and Shim, KY and Kwak, IS and Jeong, CB},
title = {Environmental DNA-based RPA-CRISPR/Cas12a assay for on-site detection of chironomid larvae in aquatic environments.},
journal = {Pest management science},
volume = {82},
number = {3},
pages = {2458-2467},
pmid = {41230595},
issn = {1526-4998},
support = {//Incheon National University Research Grant in 2021 (No. 2021-0447)/ ; },
mesh = {*Chironomidae/growth &amp; development/genetics ; Animals ; *DNA, Environmental/analysis ; Larva/growth &amp; development/genetics ; *CRISPR-Cas Systems ; *Environmental Monitoring/methods ; *Nucleic Acid Amplification Techniques/methods ; },
abstract = {BACKGROUND: Freshwater chironomid midges pose a significant threat to drinking water quality and freshwater ecosystems in South Korea by disrupting ecological balance and causing biofouling in drinking water treatment systems. To enable rapid detection and provide early warnings of chironomid presence, we developed a highly sensitive and accurate diagnostic assay based on environmental DNA (eDNA) analysis. This method integrates recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated cleavage, and visualization through either fluorescence-based detection or a lateral flow assay (LFA).<br><br>RESULTS: The RPA-CRISPR/Cas12a assays demonstrated remarkable specificity for chironomid eDNA, as confirmed through field surveys and microscopic observations. Although both fluorescence-based and LFA-based RPA-CRISPR/Cas12a assays exhibited comparable detection limits of 10 copies/&#x3bc;L, the LFA method proved more convenient for on-site applications due to its ease of deployment in the field.<br><br>CONCLUSION: Our eDNA-based RPA-CRISPR/Cas12a assay addresses the limitations of traditional biomonitoring by requiring only a small sampling volume, thereby minimizing habitat disturbance, while offering much higher sensitivity. This assay represents a valuable tool for on-site detection of invasive species and for monitoring environmental and public health threats (e.g., chironomids). &#xa9; 2025 The Author(s). Pest Management Science published by John Wiley &amp; Sons Ltd on behalf of Society of Chemical Industry.},
}
@article {pmid41230612,
year = {2025},
author = {Hao, J and Gong, X and Duan, X and Qin, W and Ren, H and ShenTu, X and Ye, Z and Yu, X},
title = {Ultrasensitive electrochemical detection of parvovirus B19 DNA by combining CRISPR-Cas12a and multivalent framework nucleic acids.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {46},
pages = {9357-9364},
doi = {10.1039/d5ay01285f},
pmid = {41230612},
issn = {1759-9679},
mesh = {*Electrochemical Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Parvovirus B19, Human/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Biosensing Techniques/methods ; Humans ; Limit of Detection ; CRISPR-Associated Proteins ; *Endodeoxyribonucleases/genetics ; Bacterial Proteins ; },
abstract = {The rapid and ultrasensitive detection of parvovirus B19 (B19V) DNA is critical for preventing severe complications in high-risk populations, such as fetal hydrops in pregnant women and aplastic crisis in immunocompromised patients. The absence of clinically approved vaccines or antivirals against B19V thus mandates the urgent development of accessible in vitro diagnostics to enable time-critical interventions and contain community transmission. Herein, we developed an electrochemical biosensor as a proof-of-concept for B19V by integrating CRISPR-Cas12a with multivalent framework nucleic acids (FNAs), namely, 12 nm tetrahedral DNA nanostructures (TDNs). Target B19V DNA activates Cas12a to indiscriminately cleave the biotin-modified ssDNA protruding from the four vertices of the TDNs, while the TDNs precisely orient ssDNA probes on electrodes, minimizing nonspecific adsorption. This method takes advantage of the target-specific cleavage ability of CRISPR-Cas12a (Cas12a-crRNA complex, 10-12 nm) and the unique structural and functional features of 12 nm TDNs. The comparable dimensions of the Cas12a-crRNA complex and the TDN suggest a potential synergistic effect, which contributes to the observed signal amplification and high detection sensitivity. The developed platform is user-friendly, has a low detection limit (2.19 fM), and shows high selectivity. This work establishes a foundational biosensing platform, demonstrating potential for ultrasensitive nucleic acid detection. By combining the accuracy of CRISPR-Cas12a and the benefits of FNAs, this method provides a more efficient, amplification-free, and reliable approach that holds promise for future development in point-of-care diagnostics and other applications.},
}
@article {pmid41231321,
year = {2025},
author = {Kumari, S and Keshari, AK and Singh, SK and Pandey, S and Singh, A},
title = {CRISPR-Based genome editing in pulses: current approaches, challenges, and future prospects.},
journal = {Plant molecular biology},
volume = {115},
number = {6},
pages = {126},
pmid = {41231321},
issn = {1573-5028},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *Fabaceae/genetics ; Plant Breeding/methods ; Plants, Genetically Modified/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Genetic Engineering/methods ; },
abstract = {Legumes are the second most important food crop after cereals for the world population. It is a significant protein source for developing countries and integral to global food security. However, various agroecological constraints and biotic and abiotic factors often compromise the production of pulses. Legumes are long-term neglected crops worldwide and follow traditional breeding, leading to a time-consuming, labor-intensive, less economically feasible program associated with linkage drag. Recent sequencing attempts in the twenty-first century, with the development of an enormous repertoire of genetic and genomic resources, allowed scientists to accelerate the improvement of legumes with modern genome editing tools. One such promising tool is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), which has revolutionized and transformed the landscape of genetic engineering. The emergence of CRISPR/Cas systems has redefined precision breeding, offering unprecedented control over genome manipulation in legume crops. It has tremendous potential for crop improvement and can precisely make changes at genomic locations with incredible accuracy. Therefore, identifying the desired genes and their precise manipulation has enormous implications for legume crop improvement. This review will give an overview of the genome editing tools available for crop improvement and the efficiency of different transformation methods in legume crops. It will also discuss the current status of genome editing in legume crops, including challenges and future perspectives.},
}
@article {pmid41231539,
year = {2025},
author = {Jia, N and Zhou, YJ and Gao, J},
title = {Engineering recombination machinery facilitates the construction of yeast cell factories.},
journal = {FEMS yeast research},
volume = {25},
number = {},
pages = {},
pmid = {41231539},
issn = {1567-1364},
support = {22478382//National Natural Science Foundation of China/ ; E411040705//Energy Revolution S&amp;T Program of Yulin Innovation Institute of Clean Energy/ ; },
mesh = {*Metabolic Engineering/methods ; *Gene Editing/methods ; *DNA End-Joining Repair ; *Saccharomyces cerevisiae/genetics/metabolism ; *Homologous Recombination ; *Recombination, Genetic ; CRISPR-Cas Systems ; DNA Breaks, Double-Stranded ; },
abstract = {Advances in genome editing have been promoted by programmable nucleases like CRISPR-Cas9, which triggers endogenous DNA repair mechanisms by inducing double-strand break (DSB). Cellular responses to DSBs are governed by competing repair pathways: error-prone non-homologous end joining (NHEJ) and high-fidelity homologous recombination (HR). This review systematically compares the molecular mechanisms and key regulators of NHEJ and HR, with a focus on recent breakthroughs in recombination engineering in non-conventional yeasts. These advances address challenges in precise genome editing, enabling robust metabolic engineering of yeast cell factories for sustainable bioproduction.},
}
@article {pmid41231855,
year = {2025},
author = {Liu, S and Bai, J and Zhan, B and Yao, J and Zhang, J and Yi, J and Dong, M and Li, Q and Shen, Y and Chen, Y and Zhao, Y},
title = {Development of an RNA aptamer-assisted CRISPR/Cas9 system for efficiently generating and isolating Cas9-free mutants in plant.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011931},
pmid = {41231855},
issn = {1553-7404},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Aptamers, Nucleotide/genetics ; Plants, Genetically Modified/genetics ; Mutation ; *Arabidopsis/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 gene-editing system is a powerful tool in plant genetic engineering; however, screening for Cas9-free edited plants remains complex and time-consuming. To address this limitation, we developed an RNA aptamer-assisted CRISPR/Cas9 system, termed 3WJ-4 × Bro/Cas9. In this system, the engineered RNA aptamer 3WJ-4 × Bro functions as a transcriptional reporter, serving as an alternative to traditional fluorescent proteins and thus avoiding their potential interference with Cas9 activity. Compared to the conventional GFP/Cas9 system, 3WJ-4 × Bro/Cas9 showed more efficient transformation and higher accuracy in fluorescence-based selection of positive T1 transformants, without significantly affecting plant growth. Furthermore, 3WJ-4 × Bro/Cas9 achieved a 78.6% increase in the T1 mutation rate compared to GFP/Cas9, with the homozygous mutation rate reaching 1.78%. In addition, 3WJ-4 × Bro/Cas9 enabled fluorescence-based visual screening in the T2 generation for rapid identification of Cas9-free mutants, improving sorting efficiency by 30.2% over the GFP-based method. Moreover, 3WJ-4 × Bro/Cas9 enabled more efficient generation of homozygous double-target mutants compared to GFP/Cas9. These results demonstrate that the 3WJ-4 × Bro/Cas9 system provides a non-transgenic, efficient, and broadly applicable strategy for plant genome editing and selection.},
}
@article {pmid41231980,
year = {2025},
author = {Gelsinger, DR and Ronda, C and Ma, J and Kar, OB and Edwards, M and Huang, Y and Mavros, CF and Sun, Y and Perdue, T and Vo, PL and Ivanov, II and Sternberg, SH and Wang, HH},
title = {Metagenomic editing of commensal bacteria in vivo using CRISPR-associated transposases.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6774},
pages = {eadx7604},
pmid = {41231980},
issn = {1095-9203},
support = {U01 AI163069/AI/NIAID NIH HHS/United States ; R01 CA272898/CA/NCI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R21 AI126305/AI/NIAID NIH HHS/United States ; R21 AI146817/AI/NIAID NIH HHS/United States ; R01 EB027793/EB/NIBIB NIH HHS/United States ; R01 DK118044/DK/NIDDK NIH HHS/United States ; R01 AI132403/AI/NIAID NIH HHS/United States ; R01 EB031935/EB/NIBIB NIH HHS/United States ; R01 AI144808/AI/NIAID NIH HHS/United States ; R01 DK098378/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Gastrointestinal Microbiome/genetics ; *Gene Editing/methods ; *Bacteroides/genetics/growth &amp; development ; Humans ; Metagenomics/methods ; *CRISPR-Cas Systems ; Symbiosis ; Mice, Inbred C57BL ; Metagenome ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Although metagenomic sequencing has revealed a rich microbial biodiversity in the mammalian gut, methods to genetically alter specific species in the microbiome are highly limited. Here, we introduce Metagenomic Editing (MetaEdit) as a platform technology for microbiome engineering that uses optimized CRISPR-associated transposases delivered by a broadly conjugative vector to directly modify diverse native commensal bacteria from mice and humans with new pathways at single-nucleotide genomic resolution. Using MetaEdit, we achieved in vivo genetic capture of native murine Bacteroides by integrating a metabolic payload that enables tunable growth control in the mammalian gut with dietary inulin. We further show in vivo editing of segmented filamentous bacteria, an immunomodulatory small-intestinal microbial species recalcitrant to cultivation. Collectively, this work provides a paradigm to precisely manipulate individual bacteria in native communities across gigabases of their metagenomic repertoire.},
}
@article {pmid41232012,
year = {2025},
author = {Maire, A and Bikard, D},
title = {Programmable DNA insertion in native gut bacteria.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6774},
pages = {676-677},
doi = {10.1126/science.aec3823},
pmid = {41232012},
issn = {1095-9203},
mesh = {Animals ; *Gastrointestinal Microbiome/genetics ; Mice ; *Gene Editing/methods ; CRISPR-Cas Systems ; *Bacteria/genetics ; *Mutagenesis, Insertional ; DNA, Bacterial/genetics ; },
abstract = {A gene-editing approach enables modification of bacteria within the mouse gut.},
}
@article {pmid41232532,
year = {2025},
author = {Yang, Y},
title = {What we can learn from the first personalized CRISPR-treated baby to tackle genetic brain disorders.},
journal = {Neuron},
volume = {113},
number = {22},
pages = {3697-3702},
doi = {10.1016/j.neuron.2025.10.023},
pmid = {41232532},
issn = {1097-4199},
mesh = {Humans ; *Gene Editing/methods ; *Genetic Therapy/methods ; *CRISPR-Cas Systems/genetics ; *Brain Diseases/genetics/therapy ; *Precision Medicine/methods ; Infant ; },
abstract = {The landmark report on personalized CRISPR genome editing to treat an infant (baby KJ) with a life-threatening liver disease sparked widespread attention,[1] ushering in a new era of precision genetic intervention. This piece discusses the key challenges and opportunities in translating this milestone into treatments for genetic brain disorders.},
}
@article {pmid41232706,
year = {2026},
author = {Puri, B and Majumder, S and Gaikwad, AB},
title = {Targeting LncRNA MEG3 to modulate ER stress and autophagy: A CRISPR/Cas9-based strategy in AKI-to-CKD transition.},
journal = {Experimental cell research},
volume = {454},
number = {1},
pages = {114826},
doi = {10.1016/j.yexcr.2025.114826},
pmid = {41232706},
issn = {1090-2422},
mesh = {*Endoplasmic Reticulum Stress/genetics ; *Autophagy/genetics ; *RNA, Long Noncoding/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; *Acute Kidney Injury/genetics/pathology/metabolism ; Rats ; *Renal Insufficiency, Chronic/genetics/pathology/metabolism ; Fibrosis ; Cell Line ; Apoptosis/genetics ; },
abstract = {Acute kidney injury (AKI) to chronic kidney disease (CKD) transition is a progressive, long-term kidney dysfunction driven by complex pathophysiological processes, including persistent endoplasmic reticulum (ER) stress and impaired autophagy, contributing to fibrosis. Long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3) has been implicated in endoplasmic reticulum (ER) stress and autophagy in several diseases, but its role in kidney injury and fibrosis during AKI-to-CKD transition remains unclear. Our previous transcriptomic analysis revealed that MEG3 is dysregulated during this transition, prompting us to explore its functional role. In this study, we investigated the function of MEG3 in ER stress-autophagy crosstalk during the AKI-to-CKD transition. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR/Cas9)-mediated MEG3 knockout in NRK-52E cells was confirmed by T7 endonuclease assay, quantitative real-time polymerase chain reaction (qRT-PCR), and fluorescence in-situ hybridization (FISH) assay. Functionally, MEG3 knockout markedly attenuated ER stress and apoptosis, as shown by reduced expression of BiP, CHOP, ATF6, ATF4, p-PERK, p-eIF2α, along with restoration of the Bax/Bcl-2 balance. Autophagy activity was restored, with increased Beclin-1 and LC3B expression and decreased p62 accumulation. Furthermore, fibrotic progression was reduced, as indicated by lower levels of fibronectin and collagen I. Notably, tauroursodeoxycholic acid (TUDCA, 400 μM) acted synergistically with MEG3 knockout, further suppressing ER stress and fibrosis markers compared to either treatment alone. These findings demonstrate that MEG3 promotes maladaptive ER stress and impaired autophagy in tubular epithelial cells, driving AKI-to-CKD transition. Targeting MEG3 through CRISPR-based strategies or in combination with TUDCA may represent a promising therapeutic strategy to mitigate fibrosis and slow disease progression.},
}
@article {pmid41232934,
year = {2025},
author = {Li, Z and Li, F and Hua, L and Chai, F and Xie, L and Wang, D and Zhang, S and Zheng, C and Wang, Z and Jiang, X},
title = {Unlocking Zeptomolar Single-Molecule Detection by Synergizing Digital Microfluidics and Digital CRISPR.},
journal = {Journal of the American Chemical Society},
volume = {147},
number = {47},
pages = {43870-43883},
doi = {10.1021/jacs.5c15767},
pmid = {41232934},
issn = {1520-5126},
mesh = {Humans ; Immunoassay/methods ; Limit of Detection ; *CRISPR-Cas Systems ; Natriuretic Peptide, Brain/blood ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Tumor Necrosis Factor-alpha/blood ; *Single Molecule Imaging ; Interleukin-6/blood ; *Microfluidics ; Biomarkers/blood ; Peptide Fragments/blood ; },
abstract = {Accurate diagnosis relies on the highly sensitive and quantitative detection of multiple immune-related biomarkers. However, current detection methods still face significant limitations in sensitivity, specificity, and background signal control. Here, we introduce DDA (Dual-Digital immunoAssay), a fully automated, universal immunoassay platform that synergizes digital microfluidics with digital Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based amplification. This "dual-digital" strategy pushes the detection limit into the zeptomolar (zM) regime, enabling unprecedented sensitivity for single-molecule analysis. The DDA platform is built upon a digital microfluidic microwell array chip, integrating magnetic bead-based immunocapture with RNA-guided CRISPR/Cas13a signal amplification. This system enables a fully automated, "sample-in, answer-out" workflow. By systematically optimizing the entire process, DDA significantly reduces background noise and enhances detection sensitivity, achieving a limit of detection (LOD) down to 100 zM for key protein biomarkers. This represents a >100-fold improvement over leading commercial ultrasensitive assays. With single-molecule resolution and full automation, DDA provides a robust solution for the precise quantification of low-abundance immune biomarkers. As a proof-of-concept, we demonstrate its ability to accurately quantify key heart-failure-associated biomarkers, including NT-proBNP (LOD: 1 aM), IL-6 (LOD: 1.5 aM), and TNF-α (LOD: 2.5 aM), directly in complex serum samples. This platform holds great promise for automated multibiomarker screening and risk assessment, showcasing its powerful potential for the early diagnosis of major diseases such as cardiovascular diseases, cancers, neurodegenerative disorders, and infectious diseases.},
}
@article {pmid41233591,
year = {2025},
author = {Li, L and Gao, J and Yi, D and Sheft, AP and Schimenti, JC and Ding, X},
title = {A primordial germ cell-like-cell platform enables CRISPRi screen for epigenetic fertility modifiers.},
journal = {EMBO reports},
volume = {26},
number = {23},
pages = {6044-6078},
pmid = {41233591},
issn = {1469-3178},
support = {CO29155//New York State Stem Cell Science (NYSTEM)/ ; 1P50HD096723//HHS | NIH | Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD)/ ; C30293GG//New York State Stem Cell Science (NYSTEM)/ ; P50 HD096723/HD/NICHD NIH HHS/United States ; R01 HD082568/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Germ Cells/metabolism/cytology ; *Epigenesis, Genetic ; Male ; Positive Regulatory Domain I-Binding Factor 1/genetics/metabolism ; Cell Differentiation/genetics ; Nanog Homeobox Protein/genetics/metabolism ; *Fertility/genetics ; Transcription Factor AP-2/genetics/metabolism ; Transcription Factors/genetics/metabolism ; *CRISPR-Cas Systems ; Female ; Repressor Proteins/genetics/metabolism ; RNA-Binding Proteins/genetics ; DNA-Binding Proteins/genetics/metabolism ; },
abstract = {Primordial germ cells (PGCs) are the precursors of gametes, and the ability to derive PGC-like cells (PGCLCs) from pluripotent stem cells has transformed germline research. A key limitation remains producing PGCLCs in sufficient numbers for large-scale applications. Here, we show that overexpression of Nanog plus three PGC master regulators - Prdm1, Prdm14, and Tfap2c - in mouse epiblast-like cells and formative embryonic stem cells yields abundant and highly enriched PGCLCs without costly recombinant cytokines. Nanog enhances the PGC regulatory network, suppresses somatic differentiation, and stabilizes PGCLC fate. Transcriptomically, these PGCLCs are developmentally more advanced than cytokine-induced counterparts and can be sustained long-term or differentiated into spermatogonia-like cells. Using this platform, we conduct a CRISPRi screen of 701 epigenetic genes to identify those needed for PGCLC formation. Downregulation of Ncor2, a histone deacetylase (HDAC) recruiter, has the greatest impact. Additionally, the HDAC inhibitors valproic acid and sodium butyrate suppress PGCLC formation and sperm counts of in utero-exposed animals. This work establishes a scalable system for functional screening of genes that influence germline development.},
}
@article {pmid41233602,
year = {2025},
author = {Strefeler, A and Baker, ZN and Chollet, S and Foged, MM and Guerra, RM and Ivanisevic, J and Gallart-Ayala, H and Pagliarini, DJ and Jourdain, AA},
title = {Uridine-sensitized screening identifies demethoxy-coenzyme Q and NUDT5 as regulators of nucleotide synthesis.},
journal = {Nature metabolism},
volume = {7},
number = {11},
pages = {2221-2235},
pmid = {41233602},
issn = {2522-5812},
support = {R35 GM131795/GM/NIGMS NIH HHS/United States ; 310030_200796//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; },
mesh = {*Pyrophosphatases/metabolism/genetics ; Humans ; *Nucleotides/biosynthesis ; *Uridine/metabolism/pharmacology ; Phosphoribosyl Pyrophosphate/metabolism ; CRISPR-Cas Systems ; Pyrimidines/biosynthesis ; },
abstract = {Rapidly proliferating cells require large amounts of nucleotides, making nucleotide metabolism a widely exploited therapeutic target against cancer, autoinflammatory disorders and viral infections. However, regulation of nucleotide metabolism remains incompletely understood. Here, we reveal regulators of de novo pyrimidine synthesis. Using uridine-sensitized CRISPR-Cas9 screening, we show that coenzyme Q (CoQ) is dispensable for pyrimidine synthesis, in the presence of the demethoxy-CoQ intermediate as alternative electron acceptor. We further report that the ADP-ribose pyrophosphatase NUDT5 directly binds PPAT, the rate-limiting enzyme in purine synthesis, which inhibits its activity and preserves the phosphoribosyl pyrophosphate (PRPP) pool. In the absence of NUDT5, hyperactive purine synthesis exhausts the PRPP pool at the expense of pyrimidine synthesis, which promotes resistance to purine and pyrimidine nucleobase analogues. Of note, the interaction between NUDT5 and PPAT is disrupted by PRPP, highlighting an intricate allosteric regulation. Overall, our findings reveal a fundamental mechanism of nucleotide balance and position NUDT5 as a regulator of nucleobase analogue metabolism.},
}
@article {pmid41233858,
year = {2025},
author = {Mayer, J and Pack, M and Montenarh, M and Götz, C},
title = {Gene expression changes in pancreatic α-cell lines following knock-out Of either CK2α or CK2α'.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {69},
pmid = {41233858},
issn = {0717-6287},
mesh = {*Casein Kinase II/genetics/metabolism ; *Glucagon-Secreting Cells/metabolism ; Animals ; Mice ; Cell Line ; Gene Knockout Techniques ; *Gene Expression/genetics ; },
abstract = {BACKGROUND: Protein kinase CK2 is known to exist as a tetramer of two catalytic α- or α'- subunits and two non-catalytic β-subunits, or as multimers of this tetramer. Moreover, CK2α (CSNK2A1) and CK2α' (CSNK2A2) are also active in the absence of CK2β (CSNK2B). Very little is known about specific functions of the individual subunits of protein kinase CK2.<br><br>RESULTS: In order to study the effects of CK2&#x3b1; and CK2&#x3b1;' on gene expression, we used the Mus musculus pancreatic &#x3b1;-cell line &#x3b1;TC1 and two derivatives with either CK2&#x3b1; (KO1 cells) or CK2&#x3b1;' (KO2 cells) expression knocked-out by CRISPR/Cas technology. We found numerous genes deregulated in both KO1 and KO2 cells compared to the parental cells. Applying stringent thresholds, 266 genes were found down-regulated and 153 genes up-regulated in KO1 cells, 233 genes were found down-regulated and 84 genes up-regulated in KO2 cells. Dozens of genes were found deregulated in a similar fashion in both KO1 and KO2 cells. We found altered expression of genes involved in the differentiation of pancreatic cells, including Hox genes, and in the regulation of glucagon synthesis or secretion. Moreover, many of the deregulated genes play an important role in developmental processes and in neuronal cell biology.<br><br>CONCLUSION: Our findings reveal individual and shared functions of the CK2&#x3b1; and CK2&#x3b1;' catalytic subunits, in particular regarding their involvement in regulating gene expression.},
}
@article {pmid41234307,
year = {2025},
author = {Tuncel, A and Kim, HU and Kim, MC},
title = {Editorial: Engineering future crops through genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1720325},
pmid = {41234307},
issn = {1664-462X},
}
@article {pmid41235827,
year = {2026},
author = {Chen, W and Zhang, X and Fan, R and Li, X and Guan, F and Wan, G and Kong, W and Qi, X and Pan, S and Shi, S and Su, Y and Gao, S and Huang, W and Xian, X and Liu, J and Wang, Y and Ma, Y},
title = {Generating golden Syrian hamsters with conditional alleles via zygote microinjection of CRISPR/Cas9.},
journal = {Animal models and experimental medicine},
volume = {9},
number = {2},
pages = {308-318},
pmid = {41235827},
issn = {2576-2095},
support = {2060204//State Key Laboratory Special Fund/ ; Peking University, 202411//Open Research Project in State Key Laboratory of Vascular Homeostasis and Remodeling/ ; 2023-PT180-01//The Non-profit Central Research Institute Fund of the Chinese Academy of Medical Sciences/ ; HH24KYZX0007//Haihe Laboratory of Cell Ecosystem Innovation Fund/ ; 2021-I2M-1-024//CAMS Innovation Fund for Medical Sciences/ ; 2022-I2M-1-020//CAMS Innovation Fund for Medical Sciences/ ; 2023-I2M-2-001//CAMS Innovation Fund for Medical Sciences/ ; 2021YFF0702802//the National Key Research and Development Program of China from the Ministry of Science and Technology/ ; },
mesh = {Animals ; *CRISPR-Cas Systems/genetics ; *Mesocricetus/genetics ; *Microinjections ; Cricetinae ; *Zygote ; Alleles ; Gene Knockout Techniques ; Female ; Male ; Animals, Genetically Modified ; },
abstract = {BACKGROUND: The golden Syrian hamster is a valuable animal model for studying carcinogenesis, metabolic disorders, cardiovascular diseases, and viral infections due to its biological and pathological similarities to humans. However, the development of genetically engineered hamsters has lagged behind that of mice and rats, largely because of an embryonic development block at the two-cell stage in vitro. Although CRISPR/Cas9-mediated gene knockout has been achieved in hamsters, precise DNA fragment insertion or conditional knockout (cKO) models have not previously been reported, likely due to technical limitations in embryo manipulation and insufficient efficiency of homology-directed repair (HDR).<br><br>METHODS: In this study, we generated conditional alleles of the ApoF gene in golden Syrian hamsters. A two-cut strategy was applied using Cas9 protein, two sgRNAs, and a single donor plasmid containing exon 2 flanked by loxP sites and two ~0.8&#x2009;kb homology arms. A mixture of Cas9 protein, sgRNAs, and the donor plasmid was microinjected into the pronuclei of one-cell stage hamster embryos.<br><br>RESULTS: The efficiency of CRISPR/Cas9-mediated loxP knock-in reached up to 27%, and the genetically modified floxed alleles were successfully transmitted through the germline. The functionality of the inserted loxP sites was validated by in&#xa0;vivo Cre-mediated recombination following local administration of AAV vectors, including AAV-cTnT-Cre in the heart and AAV-CMV-Cre in the brain.<br><br>CONCLUSIONS: To our knowledge, this work represents the first successful establishment of a conditional knockout model in the golden Syrian hamster, providing a valuable tool for mechanistic studies of gene function and disease modeling.},
}
@article {pmid41235931,
year = {2026},
author = {Ioannidou, C and Gregoriou, ME and Schetelig, MF and Drosopoulou, E and Mathiopoulos, KD and Bourtzis, K},
title = {CRISPR/Cas9-based white pupae mutant lines in Bactrocera spp. for sterile insect technique applications.},
journal = {Insect science},
volume = {33},
number = {2},
pages = {463-475},
pmid = {41235931},
issn = {1744-7917},
support = {//Insect Pest Control Subprogramme of the Joint FAO/IAEA Centre of Nuclear Techniques in Food and Agriculture/ ; 101059523//European Union&#xb4;s Horizon Europe Research and Innovation Program REACT/ ; //Two postgraduate programs of the Department of Biochemistry and Biotechnology of the University of Thessaly ("Biotechnology-Quality Assessment in Nutrition and the Environment" and "Applications of Molecular Biology-Genetics-Diagnostic Biomarkers")/ ; //"Synthetic Biology: from omics technologies to genomic engineering (OMIC-ENGINE)" (MIS 5002636) which is implemented under the Action "Reinforcement of the Research and Innovation Infrastructure", funded by the Operational Programme "Competitiveness, Entrepreneurship and Innovation" (NSRF 2014-2020) and co-financed by Greece and the European Union (European Regional Development Fund)/ ; },
mesh = {Animals ; *Tephritidae/genetics/growth &amp; development ; *CRISPR-Cas Systems ; Male ; *Pest Control, Biological/methods ; Pupa/genetics/growth &amp; development ; Female ; Gene Editing ; },
abstract = {The Bactrocera genus includes highly invasive fruit and vegetable pest species such as Bactrocera dorsalis, Bactrocera correcta, and Bactrocera oleae. The sterile insect technique (SIT) is a biological control method used to suppress populations of the invasive Bactrocera fruit flies by releasing sterilized male insects that compete with wild males for mates, reducing reproduction and eventually pest numbers. The effectiveness of the SIT against insect pests can be enhanced through male-only releases, achieved via genetic sexing strains (GSS) that enable early-stage sex separation. To overcome limitations faced when developing a new GSS through the classical genetic approach, a novel "neoclassical approach" has been proposed, focusing on the identification of genetic markers, the induction of desired phenotypes through genome editing, and the linkage of selectable markers to male sex. In this study, we evaluated the white pupae gene as a selectable marker for GSS development in 3 Bactrocera species. The white pupae orthologous genes have been identified, and, through CRISPR/Cas9 mutagenesis, the 3rd exon of the white pupae gene was knocked out resulting in white pupae lines in Bactrocera dorsalis, Bactrocera correcta, and Bactrocera oleae species. These results demonstrate the applicability of CRISPR/Cas9-mediated disruption of the conserved white pupae gene as a selectable marker in multiple Bactrocera species, supporting the development of genetic sexing systems for SIT-based pest management.},
}
@article {pmid41236017,
year = {2026},
author = {Zhang, C and Goldsmith, SR and Htut, M and Rhee, JW and Vyas, V and Clark, MC and Armenian, SH and Forman, SJ and Wang, X},
title = {CRISPR Knockdown of CHIP Genes in Macrophages Drives Increased Production of Inflammatory Cytokines in CAR-T Cell Therapy.},
journal = {Transplantation and cellular therapy},
volume = {32},
number = {1},
pages = {10.e1-10.e5},
doi = {10.1016/j.jtct.2025.08.022},
pmid = {41236017},
issn = {2666-6367},
mesh = {Humans ; *Macrophages/metabolism/immunology ; *Cytokines/metabolism ; DNA Methyltransferase 3A ; *Immunotherapy, Adoptive/methods/adverse effects ; *CRISPR-Cas Systems ; Dioxygenases/genetics ; Receptors, Chimeric Antigen ; Gene Knockdown Techniques ; Proto-Oncogene Proteins/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; DNA-Binding Proteins/genetics ; Repressor Proteins/genetics ; DNA (Cytosine-5-)-Methyltransferases/genetics ; Gene Editing ; },
abstract = {Clonal hematopoiesis of indeterminate potential (CHIP) is characterized by somatic mutations in leukemogenic genes (e.g., DNMT3A, TET2, and ASXL1) in hematopoietic stem cells and is linked to age-related clonal expansion and inflammation. We previously showed that patients with CHIP receiving chimeric antigen receptor (CAR)-T cell therapy have a higher risk of developing clinically significant cytokine release syndrome (CRS) compared to those without CHIP. Here, we investigated the mechanisms through which CHIP mutations can contribute to CAR-T-related toxicities. To model the interaction of CAR-T cell-mediated inflammatory response and macrophages harboring CHIP gene mutations, we employed CRISPR-based gene editing to knock down three key CHIP-associated genes (DNMT3A, TET2, and ASXL1) in human macrophages, using multiple guide RNAs for each. We co-cultured gene-edited macrophages with autologous CS1 and BCMA CAR-T cells and multiple myeloma (MM) tumor cells. Compared to nonedited macrophages, DNMT3A-, TET2-, and ASXL1-edited macrophages secreted significantly higher levels of pro-inflammatory cytokines characteristic of CRS, including IL-6, MCP-1, and IL-1β (P < .05 to .01). These results suggest that the presence of CHIP mutations in human macrophages may exacerbate the inflammatory response during CAR-T therapy. This study highlights CHIP mutations as potential biomarkers for identifying patients at high risk of developing CRS and for guiding personalized prophylactic interventions in MM CAR-T therapy.},
}
@article {pmid41236144,
year = {2025},
author = {Apsley, EJ and Riepsaame, J and Cheng, YC and Cowley, SA and Becker, EBE},
title = {Cerebellar organoids model cell type-specific FOXP2 expression during human cerebellar development.},
journal = {Disease models &amp; mechanisms},
volume = {18},
number = {11},
pages = {},
pmid = {41236144},
issn = {1754-8411},
support = {BB/M011224/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //UK Research and Innovation/ ; },
mesh = {Humans ; *Forkhead Transcription Factors/metabolism/genetics ; *Organoids/metabolism ; *Cerebellum/metabolism/growth &amp; development/embryology/cytology ; Induced Pluripotent Stem Cells/metabolism/cytology ; Purkinje Cells/metabolism ; Gene Expression Regulation, Developmental ; Cell Differentiation/genetics ; *Models, Biological ; CRISPR-Cas Systems/genetics ; },
abstract = {Human cerebellar development is unique and cannot be fully replicated in animal models. Although human stem cell-derived cerebellar organoid models are increasingly being applied to model cerebellar diseases, their potential to provide insight into normal human cerebellar development remains underexplored. Here, we used CRISPR-based gene editing in cerebellar organoids as an approach for modelling specific features of early human cerebellar development. Forkhead box protein P2 (FOXP2) is a transcription factor associated with speech and language development that is highly expressed in the developing brain. However, little attention has been directed to the study of FOXP2 in the early developing cerebellum. We generated a fluorescent FOXP2 reporter line in human induced pluripotent stem cells to enable the characterisation of FOXP2-expressing cells during cerebellar organoid differentiation. Through transcriptomic profiling of FOXP2 reporter cerebellar organoids and cross-referencing with existing cerebellar datasets, we describe the expression and identify potential downstream targets of FOXP2 in the early developing human cerebellum. Our results highlight expression of FOXP2 in early human Purkinje cells and cerebellar nuclei neurons, and the vulnerability of these cell populations to neurodevelopmental disorders.},
}
@article {pmid41236435,
year = {2025},
author = {Chen, Y and Wang, Y and Liu, L and Yu, X and Zhang, Y and Xi, M and Xu, J and Yang, H and Xie, C and Wang, D},
title = {Targeting BnNAC038 improves drought tolerance with low yield penalty in Brassica napus.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {3},
pages = {e70571},
doi = {10.1111/tpj.70571},
pmid = {41236435},
issn = {1365-313X},
support = {241111112400//Key Research and Development Project of Henan Province/ ; 2023-YBNY-023//Key Research and Development Project of Shaanxi Province/ ; 32070324//National Natural Science Foundation of China/ ; 32301811//National Natural Science Foundation of China/ ; 2024YFD1200400//the National Key Research and Development Project from the Ministry of Agriculture and Rural Affairs of China/ ; },
mesh = {*Brassica napus/genetics/physiology/growth &amp; development ; Droughts ; *Plant Proteins/genetics/metabolism/physiology ; Gene Expression Regulation, Plant ; Photosynthesis/genetics ; CRISPR-Cas Systems ; Drought Resistance ; },
abstract = {Drought stress severely limits crop productivity in Brassica napus, yet strategies to enhance drought tolerance without compromising yield remain elusive. Here, we identify BnNAC038 as a negative regulator of drought responses in Brassica napus. CRISPR/Cas9-generated bnnac038 mutants exhibited improved drought survival, reduced water loss, and enhanced stomatal closure under drought conditions compared to wild-type (WT) plants. RNA-sequencing (RNA-seq) and DNA affinity purification sequencing (DAP-seq) analyses revealed that BnNAC038 directly represses drought-responsive genes, including BnSnRK2.6 (a key ABA signaling kinase), and genes involved in photosynthesis (BnPPC2) and gluconeogenesis (BnPGK). Field trials demonstrated that bnnac038 plants exhibit enhanced photosynthesis, accumulate more sucrose and glucose under drought, and exhibit increased biomass and seed yield compared to WT. Genetic interaction studies further showed that BnSnRK2.6 acts downstream of BnNAC038 to mediate drought tolerance. Our results indicate that targeted editing of BnNAC038 enhances drought tolerance while minimizing yield loss, providing a new strategy for developing drought-resilient Brassica napus varieties with minimal yield penalty.},
}
@article {pmid41236489,
year = {2025},
author = {Wang, Y and Kong, Y and Zhang, Y and He, Y and Wang, W and Lu, Y and Meng, J and Yuan, H},
title = {Long-Term Attenuation of Vascular Hyperpermeability in a Hereditary Angioedema Mouse Model by Adenine Base Editing.},
journal = {Allergy},
volume = {},
number = {},
pages = {},
doi = {10.1111/all.70152},
pmid = {41236489},
issn = {1398-9995},
support = {//the National Natural Science Foundation of China/ ; //West China Hospital, Sichuan University/ ; },
abstract = {BACKGROUND: Hereditary angioedema (HAE) is a rare and potentially life-threatening disorder caused by dysregulated kallikrein-kinin signaling and bradykinin-induced vascular hyperpermeability. Current therapies targeting this pathway are effective but require lifelong administration, underscoring the need for durable and potentially curative interventions. Adenine base editors (ABEs), engineered from CRISPR/Cas systems, enable precise single-nucleotide modifications with minimal genomic disruption, offering a promising strategy for long-term gene silencing.<br><br>METHODS: NG-ABE8e was delivered via AAV8 or lipid nanoparticles (LNP) to disrupt the exon 2 splice donor site of Klkb1 in Serping1[+/-] mice. Editing outcomes were quantified by high-throughput sequencing; serum kallikrein levels were measured by ELISA; and vascular permeability was evaluated using Evans blue dye extravasation, fluorescent tracer leakage, and VE-cadherin immunostaining. Safety evaluations included off-target analysis, histopathology, serum biochemistry, activated partial thromboplastin time (aPTT), and systemic hemodynamic stability.<br><br>RESULTS: AAV8-NG-ABE8e induced >&#x2009;60% A&#x2022;T-to-G&#x2022;C conversion at the target site, promoting exon 2 skipping and reducing Klkb1 mRNA and serum kallikrein levels by >&#x2009;85%, an effect sustained for at least 1&#x2009;year. AAV8-NG-ABE8e also reversed bradykinin-driven vascular hyperpermeability and attenuated inflammatory gene signatures. Prolongation of aPTT was observed only when circulating kallikrein levels declined below 2&#x2009;&#x3bc;g/mL. Similarly, LNP-mediated delivery of NG-ABE8e mRNA resulted in >&#x2009;90% reductions in Klkb1 expression and serum kallikrein levels. No evidence of significant long-term toxicity was detected.<br><br>CONCLUSION: These findings demonstrate that, in a murine model, NG-ABE8e-mediated disruption of Klkb1 enables durable suppression of serum kallikrein and vascular stabilization, suggesting its potential as a promising single-intervention strategy for the treatment of HAE.},
}
@article {pmid41236708,
year = {2026},
author = {Zahedi, S},
title = {CRISPR-Based Functional Genomics in Pluripotent Stem Cells.},
journal = {Stem cell reviews and reports},
volume = {22},
number = {1},
pages = {403-414},
pmid = {41236708},
issn = {2629-3277},
mesh = {Humans ; *Genomics/methods ; *CRISPR-Cas Systems/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Gene Editing/methods ; Animals ; Induced Pluripotent Stem Cells/metabolism ; },
abstract = {The integration of CRISPR-based functional genomics with pluripotent stem cell (PSC) technologies has been recognized as a transformative approach for investigating gene function, modeling human disease, and advancing regenerative medicine. The aim of this review is to provide a comprehensive evaluation of recent developments in CRISPR-Cas platforms, including gene knockouts, base and prime editing, and CRISPR activation or interference (CRISPRa/i), as applied to PSC systems. Studies employing human PSCs, including embryonic stem cells and induced pluripotent stem cells, have been examined to summarize methodologies for genome-wide screening, lineage tracing, and therapeutic engineering. Advances in editing efficiency, delivery strategies, and genomic safety have been reported, while limitations persist in the form of off-target modifications, epigenetic variability, and cell-type-specific responses. Notable applications include the generation of immune-evasive PSC lines, the development of organoid models for physiological and pathological studies, and the implementation of phenotypic screening for disease-relevant traits. Collectively, these technological and methodological advancements have established functional genomics of PSC-CRISPRSPR as a versatile and powerful framework for elucidating fundamental aspects of human biology, dissecting complex traits, and accelerating the translation of discoveries from experimental research to clinical implementation.},
}
@article {pmid41237780,
year = {2025},
author = {Sivakumar, S and Wang, Y and Goetsch, SC and Pandit, V and Wang, L and Zhao, H and Sundarrajan, A and Armendariz, D and Takeuchi, C and Deng, M and Nzima, M and Chen, WC and Dederich, AE and El Hayek, L and Gao, T and Gogate, A and Kaur, K and Kim, HB and McCoy, MK and Niederstrasser, H and Oura, S and Pinzon-Arteaga, CA and Sanghvi, M and Schmitz, DA and Yu, L and Zhang, Y and Zhou, Q and Kraus, WL and Xu, L and Wu, J and Posner, BA and Chahrour, MH and Hon, GC and Munshi, NV},
title = {Benchmarking and optimizing Perturb-seq in differentiating human pluripotent stem cells.},
journal = {Stem cell reports},
volume = {20},
number = {12},
pages = {102713},
pmid = {41237780},
issn = {2213-6711},
support = {R01 HL172898/HL/NHLBI NIH HHS/United States ; UM1 HG011996/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Cell Differentiation/genetics ; *Pluripotent Stem Cells/cytology/metabolism ; Benchmarking ; Gene Regulatory Networks ; Myocytes, Cardiac/cytology/metabolism ; CRISPR-Cas Systems/genetics ; High-Throughput Nucleotide Sequencing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Perturb-seq is a powerful approach to systematically assess how genes and enhancers impact the molecular and cellular pathways of development and disease. However, technical challenges have limited its application in stem-cell-based systems. Here, we benchmarked Perturb-seq across multiple CRISPRi modalities, on diverse genomic targets, in multiple human pluripotent stem cells, during directed differentiation to multiple lineages, and across multiple single guide RNA (sgRNA) delivery systems. To ensure cost-effective production of large-scale Perturb-seq datasets as part of the Impact of Genomic Variants on Function (IGVF) consortium, our optimized protocol dynamically assesses experiment quality across the weeks-long procedure. Our analysis of 1,996,260 sequenced cells across benchmarking datasets reveals shared regulatory networks linking disease-associated enhancers and genes with downstream targets during cardiomyocyte differentiation. This study establishes open tools and resources for interrogating genome function during stem cell differentiation.},
}
@article {pmid41237878,
year = {2025},
author = {Ashok, K and Ellango, R and Venkatesh, R and Bhargava, CN and Pavithran, S and Dhawane, Y and Manamohan, M and Jha, GK and Asokan, R},
title = {Differential expression of genes in pre-blastoderm embryos of oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) microinjected with white locus CRISPR/Cas9 ribo nucleo protein (RNP) complex.},
journal = {International journal of biological macromolecules},
volume = {333},
number = {Pt 2},
pages = {148735},
doi = {10.1016/j.ijbiomac.2025.148735},
pmid = {41237878},
issn = {1879-0003},
mesh = {Animals ; *Tephritidae/genetics/embryology ; *CRISPR-Cas Systems/genetics ; *Ribonucleoproteins/genetics ; *Gene Expression Regulation, Developmental ; Microinjections ; Gene Editing ; Gene Expression Profiling ; Insect Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Associated Protein 9/genetics ; Transcriptome ; },
abstract = {CRISPR/Cas9-based genome editing is currently revolutionizing applications in agriculture, human health, and other domains. Targeted gene editing employing CRISPR/Cas9 is achieved by delivering the various components viz. guide RNA, traCrRNA, and the Cas9 protein in DNA or RNA or RNP format. RNP enables fast, DNA-free editing without genomic integration. While innate immunity to guide RNA is studied in humans and plants, similar research in insects remains unexplored. In the present study, the RNP complex (sgRNA+Cas9) was administered via microinjection of embryos (eggs) in the pre-blastoderm (G0) stage. Transcriptome sequencing was performed using the Illumina Novoseq 6000 platform to understand the prevalence of innate immunity against the delivered guide RNA for the white locus of Bactrocera dorsalis. 33 up- and 67 down-regulated genes were found in the differential gene expression analysis from the RNA-Seq data. The correlation between RT-qPCR and RNA-Seq gene expression levels showed a strong coefficient of determination (R2 = 0.984). The genes related to stress, intron removal, and effector recognition were overexpressed. However, the genes responsible for growth and development were significantly downregulated. Thus, the present study identified the possible reasons that could play a vital role in influencing the editing outcomes employed through the RNP complex.},
}
@article {pmid41238027,
year = {2026},
author = {Zhang, Z and Tang, D and Dong, L and Hu, Y and Li, S and Wang, D and Wang, M and Wang, Q},
title = {Functional divergence of Msp porins in substrate uptake and stress resistance in Mycobacterium smegmatis.},
journal = {Microbial pathogenesis},
volume = {210},
number = {},
pages = {108185},
doi = {10.1016/j.micpath.2025.108185},
pmid = {41238027},
issn = {1096-1208},
mesh = {*Mycobacterium smegmatis/genetics/metabolism/growth &amp; development/drug effects ; *Porins/genetics/metabolism ; *Stress, Physiological ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Anti-Bacterial Agents/pharmacology ; Mutation ; CRISPR-Cas Systems ; Amino Acids/metabolism ; Drug Resistance, Bacterial ; Muramidase/pharmacology ; Hydrogen-Ion Concentration ; *Bacterial Outer Membrane Proteins/genetics/metabolism ; Carbon/metabolism ; },
abstract = {Mycobacterium smegmatis acquires external nutrients primarily through its outer membrane porins, notably the Msp proteins. Although the bacterium encodes four Msp porins (MspA, MspB, MspC, and MspD), previous work has identified MspA as the most abundant and functionally dominant porin, while regarding others as backup porins sharing considerable functional redundancy with MspA; however, their specific physiological roles and transcriptional dynamics remain poorly characterized. To dissect the individual function of each porin, we constructed a precise M. smegmatis Msp quadruple mutant using CRISPR-Cas9, and subsequently generated a panel of isogenic strains expressing individual porins at controlled levels. These strains were assessed in growth assays for their capacity to utilize various nutrients, including carbon sources, amino acids, and ions, as well as for their resistance to antibiotics, low pH, and lysozyme. We also employed Quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR) to examine the transcriptional responses of each msp gene in the wild-type strain under the tested conditions. Our results demonstrated distinct substrate preferences among the four Msp porins and revealed their differential contributions to resistance against environmental stressors; the transcriptional changes of each Msp porin in the wild-type strain correlated well with their expected roles. Furthermore, the engineered M. smegmatis quadruple mutant provides a robust platform for investigating the functions of other channel proteins in mycobacteria.},
}
@article {pmid41239088,
year = {2025},
author = {Xu, H and Chen, H and Li, Y and Wang, Y and Zeng, H and Xu, Z and Chen, T and Kong, D and Huang, W and Cheng, H and Zhou, H and Jiang, X and Feng, J},
title = {CRISPR/Cas12a-SERS biosensor based on sea urchin-like AuNPs for the detection of β-thalassemia mutant gene CD31.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {810},
pmid = {41239088},
issn = {1436-5073},
mesh = {*Biosensing Techniques/methods ; *beta-Thalassemia/genetics/diagnosis ; *CRISPR-Cas Systems ; Animals ; *Spectrum Analysis, Raman/methods ; *Gold/chemistry ; *Metal Nanoparticles/chemistry ; Mutation ; Sea Urchins/chemistry ; Limit of Detection ; Humans ; *Endodeoxyribonucleases/genetics/metabolism ; DNA, Single-Stranded/chemistry/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Sulfhydryl Compounds/chemistry ; Bacterial Proteins ; Benzoates ; },
abstract = {Beta-thalassemia is a single-gene recessive disorder caused by mutations in the HBB gene, and approximately 1.5% of the global population are carriers of β-thalassemia. It is therefore vital to establish a rapid and sensitive method to detect the mutant genes of β-thalassemia. In this study, a CRISPR/Cas12a-mediated amplification-free surface-enhanced Raman spectroscopy (SERS) biosensor was developed. This biosensor uses sea urchin-shaped gold nanoparticles (SUGNPs) as the SERS enhancement substrate and 4-mercaptobenzoic acid (4-MBA) as the Raman reporter. It couples the SUGNPs/4-MBA with magnetic beads through single-strand DNA (ssDNA) to form an SERS probe with magnetic responsiveness. The presence of the β-thalassemia target mutation gene CD31 activates the cleavage activity of Cas12a, leading to non-specific cleavage of single-stranded DNA (ssDNA) on the probe. This results in a significant reduction in SERS intensity. This signal change enables quantitative detection of the target gene, thereby significantly enhancing the sensitivity of nucleic acid testing. We employed magnetic separation technology to enrich target nucleic acids in serum while eliminating matrix interference, enabling specific recognition and quantitative detection of the mutated CD31 gene. This method exhibits excellent linearity over a concentration range 0.1 fM to 10 pM, with a detection limit of 0.1 fM and a detection time of only 40 min. Compared to traditional qPCR and other CRISPR methods, this approach is simple, rapid, and offers advantages such as high sensitivity, high specificity, and cost-effectiveness. By simply replacing the crRNA, it can detect multiple β-thalassemia and other disease genes, demonstrating broad clinical application potential.},
}
@article {pmid41239469,
year = {2025},
author = {Ma, J and Zhang, J and Guo, X},
title = {Harnessing CRISPR-Cas9 for Lactobacillus improvement in silage production: current knowledge and future perspectives.},
journal = {Journal of animal science and biotechnology},
volume = {16},
number = {1},
pages = {150},
pmid = {41239469},
issn = {1674-9782},
support = {U20A2002//National Natural Science Foundation of China/ ; },
abstract = {High-quality silage is the cornerstone to sustainable livestock development and animal food production. As the core fermentation bacteria of silage, Lactobacillus directly regulates silage fermentation by producing lactic acid, enzymes, and other bioactive molecules. However, traditional screening methods for functional strains are labor-intensive and time-consuming. Recent advances in synthetic biology, particularly the development of CRISPR-Cas genome editing technology, offer a revolutionary approach to designing Lactobacillus strains with customized traits. This review systematically reviewed the importance of silage in sustainable agricultural development and the limitations of current silage preparation and promotion. It also discussed the application of strain engineering approaches in optimizing the phenotypic performance of Lactobacillus for better silage. Building on this, we reviewed the research progress of CRISPR-Cas9 gene editing in Lactobacillus and discussed how to leverage its high efficiency and precision to optimize the strain's traits for improved silage quality and functionality. CRISPR-Cas9 toolkits are expected to achieve directed evolution of strain performance, ultimately yielding next-generation silage microbial inoculants with multiple functions, adaptability to multiple substrates, and eco-friendly characteristics. The use of such innovative biotechnologies would facilitate resource-efficient utilization, promote animal performance and health for sustainable development in livestock production.},
}
@article {pmid41240131,
year = {2025},
author = {Behera, L and Samal, KC and Mishra, A and Sahoo, JP and Dash, M and Mishra, A},
title = {CRISPR-driven innovations in rice (Oryza sativa L.) breeding: precision development of male sterile lines.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {89},
pmid = {41240131},
issn = {1573-4978},
mesh = {*Oryza/genetics ; *Plant Infertility/genetics ; *Plant Breeding/methods ; Gene Editing/methods ; Pollen/genetics ; *CRISPR-Cas Systems/genetics ; },
abstract = {Male sterility is a vital trait exploited in hybrid seed production to boost crop yield and improve quality. In rice, different male sterility systems have been developed, significantly advancing the production of high-yielding hybrids. The three main types of male sterility in rice are cytoplasmic male sterility (CMS), photoperiod-sensitive genic male sterility (PGMS), and genic male sterility (GMS). Among these, CMS is the most widely used, arising from interactions between mitochondrial (cytoplasmic) and nuclear genes, resulting in pollen dysfunction. PGMS, on the other hand, is influenced by environmental cues such as day length and temperature, while GMS is attributed to mutations in specific nuclear genes affecting anther or pollen development. A thorough understanding of the genetic and molecular mechanisms underlying these systems is essential for efficient hybrid rice breeding. CMS lines are typically crossed with maintainer and restorer lines carrying fertility-restoring genes to produce fertile F1 hybrids. Recent advancements in molecular biology, genomics, and genome editing technologies have accelerated the development of novel male-sterile and fertility-restoring lines, thereby enhancing the precision and scalability of hybrid breeding programs. These innovations are not only expanding the genetic base of hybrid rice but also making the production process more sustainable. As global food demand rises alongside climate uncertainties, the strategic use of male sterility in rice breeding holds immense potential for improving agricultural productivity.},
}
@article {pmid41240134,
year = {2025},
author = {Gong, J and Soleimani Samarkhazan, H and Siavashi, M and Servatian, N and Pirsavabi, F},
title = {CRISPR-Cas9 in leukemia immunotherapy: precision engineering of CAR-T cells and tumor-microenvironment modulation.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {90},
pmid = {41240134},
issn = {1573-4978},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Leukemia/therapy/immunology/genetics ; Gene Editing/methods ; Receptors, Chimeric Antigen/genetics/immunology ; *Tumor Microenvironment/immunology/genetics ; *Immunotherapy, Adoptive/methods ; Precision Medicine/methods ; *Immunotherapy/methods ; T-Lymphocytes/immunology ; Animals ; },
abstract = {Together with base editors, prime editors, and alternative nuclease platforms, CRISPR-Cas9 technology has transformed the field of genetic engineering by providing unprecedented precision in genome editing and creating new opportunities for therapeutic interventions. This technology comprises a versatile genome-editing toolkit for achieving a variety of therapeutic objectives. In the context of leukemia, a group of life-threatening hematologic malignancies, CRISPR-Cas9 has emerged as a transformative tool for immunotherapy. By enabling targeted modifications of immune cells, such as T cells, this technology enhances their ability to recognize and eradicate leukemic cells. CRISPR-Cas9 facilitates the disruption of immune checkpoint inhibitors, the insertion of chimeric antigen receptors (CARs), and the correction of genetic mutations that drive leukemia progression. These advancements have led to the development of more potent and personalized immunotherapies, such as CAR-T cell therapies, with improved efficacy and reduced off-target effects. Moreover, CRISPR-Cas9 allows researchers to model leukemia in vitro, providing deeper insights into disease mechanisms and accelerating the discovery of novel therapeutic targets. Despite challenges such as delivery efficiency and potential immunogenicity, the integration of CRISPR-Cas9 into leukemia immunotherapy represents a paradigm shift, offering hope for durable remissions and potentially curative outcomes. As clinical trials progress, this technology promises to redefine the standard of care for leukemia patients, ushering in a new era of precision medicine. This narrative review explores the revolutionary applications of CRISPR-Cas9 in redefining therapeutic strategies for leukemia.},
}
@article {pmid41240306,
year = {2026},
author = {Harada, A},
title = {Golgi Units as Modules in the Ribbon.},
journal = {Sub-cellular biochemistry},
volume = {110},
number = {},
pages = {35-42},
pmid = {41240306},
issn = {0306-0225},
mesh = {*Golgi Apparatus/metabolism/ultrastructure/genetics ; Glycosylation ; Humans ; Glycosaminoglycans/metabolism/biosynthesis ; CRISPR-Cas Systems ; HeLa Cells ; Animals ; },
abstract = {Using super-resolution microscopies, SCLIM and STORM, and CRISPR/Cas9 knockin technology, we show that the Golgi complex is assembled by a number of small "Golgi units" that are 1-3 μm in diameter. Each Golgi unit can be considered a functional and morphological module because it contains all sets of glycosylation enzymes that occupy small domains named "zones." Golgi units change shape dynamically. They attach, detach, fuse, and separate from each other. The zones of glycosylation enzymes rapidly move near the rim of the unit. However, the behaviors of N- and O-linked glycosylation enzyme zones differ from those of glycosaminoglycan (GAG)-synthesizing enzyme zones in localization and dynamics. Since depletion of giantin dissociates the Golgi units, it is responsible for their attachment. Giantin depletion also prevents the movement of GAG-synthesizing enzymes between units, leading to incomplete GAG synthesis. Here, we present the model of the Golgi and its glycosylation enzymes that can explain a number of characteristics of the glycosylation processes.},
}
@article {pmid41240344,
year = {2025},
author = {Banerjee, N and Walsh, B and Patel, R and Castelletto, ML and Hallem, EA},
title = {Protocol to generate stable knockout lines in the human-parasitic nematode Strongyloides stercoralis.},
journal = {STAR protocols},
volume = {6},
number = {4},
pages = {104201},
pmid = {41240344},
issn = {2666-1667},
mesh = {Animals ; *Strongyloides stercoralis/genetics ; *Gene Knockout Techniques/methods ; Humans ; CRISPR-Cas Systems/genetics ; Gerbillinae ; },
abstract = {A major limitation to the study of gene function in parasitic nematodes was the inability to make stable mutant lines. Here, we present a protocol for generating stable knockout lines in the human-parasitic nematode Strongyloides stercoralis. We describe steps for generating CRISPR components and microinjecting them into worms. We also detail procedures for identifying potential gene disruptions and propagating mutants by host passage in gerbils to generate stable homozygous knockout lines. This protocol enables studies of gene function in S. stercoralis. For complete details on the use and execution of this protocol, please refer to Banerjee et al.[1].},
}
@article {pmid41240616,
year = {2026},
author = {Zong, W and Xie, S and Chu, H and Han, S and Zhang, X},
title = {Beyond traditional antibacterial agents: Novel approaches to combat resistant pathogens.},
journal = {European journal of medicinal chemistry},
volume = {302},
number = {Pt 2},
pages = {118362},
doi = {10.1016/j.ejmech.2025.118362},
pmid = {41240616},
issn = {1768-3254},
mesh = {*Anti-Bacterial Agents/pharmacology/chemistry ; Humans ; *Bacterial Infections/drug therapy ; *Bacteria/drug effects ; *Drug Resistance, Bacterial/drug effects ; Liposomes/chemistry ; Animals ; Drug Delivery Systems ; Microbial Sensitivity Tests ; },
abstract = {As bacterial infections caused by antibiotic-resistant strains become increasingly prevalent, traditional antibacterial therapies face mounting challenges. These resilient pathogens not only complicate the treatment of common infections but also undermine the efficacy of therapies for major diseases. This growing threat underscores the urgent need for innovative therapeutic strategies. In recent years, the combinatorial use of antibacterial agents has emerged as a promising approach to enhance efficacy and combat resistant bacteria. This review first provides an overview of antibacterial classifications and their mechanisms of action against bacterial infections. It then explores two combined treatment strategies: antibacterial-antibacterial combinations and antibacterial-non-antibacterial pairings, alongside a drug delivery technology: antibacterial-loaded liposomes. Furthermore, we highlight emerging frontiers in antimicrobial strategies, including CRISPR-Cas technologies, AI-driven discovery platforms, nanomaterials beyond liposomes, microbiota-based therapies, and immunotherapeutic approaches. Finally, we offer a forward-looking perspective on the challenges and opportunities shaping the future of antibacterial development in the biomedical field.},
}
@article {pmid41240694,
year = {2026},
author = {Gao, Y and Dong, P and Lin, H and Tian, J},
title = {A circular crRNA-triggered CRISPR/Cas12a fluorescent platform for detection of myeloperoxidase activity.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 1},
pages = {127149},
doi = {10.1016/j.saa.2025.127149},
pmid = {41240694},
issn = {1873-3557},
mesh = {*Peroxidase/metabolism/analysis ; *CRISPR-Cas Systems/genetics ; Humans ; *RNA, Circular/chemistry ; Limit of Detection ; Spectrometry, Fluorescence/methods ; *CRISPR-Associated Proteins/metabolism ; *Biosensing Techniques/methods ; Animals ; Hypochlorous Acid/metabolism ; *Enzyme Assays/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Myeloperoxidase (MPO) is a key enzymatic biomarker for the diagnosis and therapeutic monitoring of acute leukemia. Here, we developed a CRISPR/Cas12a-based fluorescent sensing platform using a circular phosphorothioate-modified crRNA (crRNA-PS) for selective MPO activity detection. In this system, MPO catalyzes hypochlorous acid (HClO) generation, which oxidizes and linearizes the circular crRNA-PS, activating Cas12a-mediated trans-cleavage of a fluorescent reporter. The assay exhibited high sensitivity with detection limits of 0.79 ng/mL for MPO and 0.21 μM for HClO, along with excellent selectivity against reactive oxygen/nitrogen species and biomolecular interferents. Reliable performance was achieved in 1 % fetal bovine serum with recoveries of 92.87-112.54 %. The sensor also maintained stable responses over 8 weeks, indicating strong structural integrity of the circular crRNA-PS. Furthermore, the system was applied for inhibitor screening, yielding IC50 values of 127.2 μM for SHA and 0.81 μM for 4-ABAH. This strategy provides a rapid, sensitive, and robust platform for MPO detection with promising clinical potential.},
}
@article {pmid41240805,
year = {2026},
author = {Ghoreshi, ZA and Ali-Hassanzadeh, M and Mashayekhi-Sardoo, H and Askarpour, H and Arefinia, N},
title = {The identification of tuberculosis using CRISPR technique: A systematic review and meta-analysis.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {2},
pages = {117183},
doi = {10.1016/j.diagmicrobio.2025.117183},
pmid = {41240805},
issn = {1879-0070},
mesh = {Humans ; *Mycobacterium tuberculosis/genetics/isolation &amp; purification ; *Tuberculosis/diagnosis/microbiology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Sensitivity and Specificity ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Rapid and precise detection of Mycobacterium tuberculosis (MTB) is essential for effective management and control of tuberculosis. The diagnostic challenge is particularly acute for extrapulmonary TB, which accounts for approximately 15-20% of cases and often presents with paucibacillary samples. Clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for pathogenic diagnosis across diverse sample types owing to its specificity and adaptability. This systematic review and meta-analysis aimed to appraise the diagnostic accuracy of CRISPR-based techniques in identifying MTB.<br><br>METHODS: A comprehensive search was conducted in Medline, Scopus, Embase, and ISI Web of Science to retrieve relevant studies, adhering to PRISMA guidelines. Quality was assessed using the Joanna Briggs comprehensive checklist. Data synthesis and analyses, including subgroup analyses, were performed with Meta-Disc 1.4, examining variables like CRISPR variants, gene targets, pre-amplification techniques, and signal readout methods.<br><br>RESULTS: From 341 identified studies, 13 met the inclusion criteria, encompassing 1,572 MTB strains. The pooled sensitivity and specificity of CRISPR-based techniques were 0.91 (95%CI: 0.89-0.92) and 0.97 (95%CI: 0.95-0.98), respectively. The pooled diagnostic odds ratio was 498.67 (95%CI: 255.1-974.7), with an AUC of 0.99 in the SROC curve, denoting excellent diagnostic accuracy. Subgroup analysis by sample type revealed that sputum samples achieved sensitivity and specificity of 0.92 (95%CI: 0.89-0.94) and 0.97 (95%CI: 0.94-0.99), while extrapulmonary samples (BALF, pus, CSF) showed comparable performance with sensitivity of 0.89 (95%CI: 0.84-0.93) and specificity of 0.98 (95%CI: 0.94-0.99).<br><br>CONCLUSION: CRISPR-based methods exhibit substantial diagnostic sensitivity and specificity for detecting MTB across both pulmonary and extrapulmonary samples, with notable variances across different CRISPR variants and methodological approaches.},
}
@article {pmid41241097,
year = {2025},
author = {Hołubowicz, R and Gao, F and Du, SW and Rodrigues Menezes, C and Zhang, J and Hołubowicz, MW and Chen, PZ and Armbrust, N and Geilenkeuser, J and Liu, DR and Truong, DJ and Westmeyer, GG and Palczewska, G and Palczewski, K},
title = {Scalable purification enables high-quality virus-like particles for therapeutic translation.},
journal = {The Journal of biological chemistry},
volume = {301},
number = {12},
pages = {110946},
pmid = {41241097},
issn = {1083-351X},
support = {UG3 AI150551/AI/NIAID NIH HHS/United States ; U01 AI142756/AI/NIAID NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; P30 EY034070/EY/NEI NIH HHS/United States ; T32 GM008620/GM/NIGMS NIH HHS/United States ; R01 EY036994/EY/NEI NIH HHS/United States ; F30 EY033642/EY/NEI NIH HHS/United States ; R01 EY034501/EY/NEI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; Animals ; Mice ; *Gene Editing/methods ; *Leukemia Virus, Murine/genetics ; *Virion/genetics ; HEK293 Cells ; *Vaccines, Virus-Like Particle/isolation &amp; purification ; Ultracentrifugation ; },
abstract = {Emerging molecular therapies introduce enzymatic activity into cells by delivering genes, transcripts, or proteins. Owing to their robust cell-entry capacity, virus-like particles (VLPs) represent a technology of choice in genome editing, where low doses of heterologous proteins and nucleic acids are essential. However, clinical translation of VLP vectors is hindered by inadequate purification methods. Current approaches, relying primarily on ultracentrifugation, suffer from inconsistent product quality and poor scalability. Here, we report the development of a broadly applicable purification strategy that improves the purity and therapeutic efficacy of genome-editing VLPs. Considering the characteristic properties of murine leukemia virus-derived engineered VLPs and HIV-derived engineered nucleocytosolic vehicles for loading of programmable editors, we developed a workflow that involves single-modal and multimodal chromatographic steps, effectively removing host cell proteins and cell-culture contaminants while improving VLP integrity and biological activity. Our purified VLPs displayed superior protein composition, consistency, and enhanced functional delivery compared to VLPs partially purified by conventional ultracentrifugation methods. Mass spectrometric analysis revealed a substantial decrease in contaminants, with VLP-specific proteins comprising >90% of the final product. In vivo studies confirmed improved therapeutic outcomes when chromatographically purified VLPs were used. Our scalable purification platform addresses critical manufacturing bottlenecks and constitutes a starting point for further development of VLP therapeutics, enabling robust production of pure VLPs for diverse applications such as genome editing, vaccine development, and other uses that require intracellular protein delivery.},
}
@article {pmid41241162,
year = {2026},
author = {Huang, G and Tang, Y and Zhang, S and Ning, H and Xu, J and Li, F and Zhang, X},
title = {Multifunctional nano-polymer-based targeted delivery system for CRISPR/Cas9-Mediated hepatocellular carcinoma therapy.},
journal = {International journal of pharmaceutics},
volume = {687},
number = {},
pages = {126392},
doi = {10.1016/j.ijpharm.2025.126392},
pmid = {41241162},
issn = {1873-3476},
mesh = {*Carcinoma, Hepatocellular/therapy/genetics ; *Liver Neoplasms/therapy/genetics ; Humans ; *CRISPR-Cas Systems ; Gene Editing/methods ; Animals ; *Methyltransferases/genetics ; Cell Line, Tumor ; Plasmids/administration &amp; dosage ; Polyethylene Glycols/chemistry ; *Polymers/chemistry ; Mice, Inbred BALB C ; Deoxycholic Acid/chemistry/administration &amp; dosage ; *Nanoparticles/chemistry/administration &amp; dosage ; Mice, Nude ; Hep G2 Cells ; Apoptosis ; Gene Transfer Techniques ; Mice ; Genetic Therapy/methods ; Polyethyleneimine/chemistry ; Disaccharides ; },
abstract = {CRISPR/Cas9 gene-editing technology exhibits substantial therapeutic potential for hepatocellular carcinoma (HCC); however, the targeted delivery of the CRISPR/Cas9 system into tumor cells remains a critical challenge requiring urgent exploration. Methyltransferase-Like 3 (METTL3), a key methyltransferase, drives HCC proliferation via multiple mechanisms. To address this challenge, a multifunctional delivery system was developed to efficiently deliver CRISPR/Cas9 plasmids targeting METTL3 (pMETTL3) into HCC cells. The cationic PEI, which facilitated the adsorption of pMETTL3 and protected it from lysosomal degradation, served as the polymeric backbone and was modified with deoxycholic acid (DOCA) to enhance its hydrophobicity. Meanwhile, lactobionic acid (LA) was grafted onto the structure to actively target HCC cells. The resulting pMETTL3/LPD was further functionalized with pH-sensitive and cleavable polyethylene glycol (PEG), aiming to reduce toxicity and enhance prolonged circulation. Results demonstrated that the delivery system maintains stability in physiological pH environments while achieving significantly enhanced accumulation in tumor tissues. Furthermore, the efficient cellular uptake of CRISPR/Cas9 plasmids enables precise gene editing, thereby effectively disrupting METTL3 expression, inducing apoptosis, and ultimately inhibiting HCC growth. This study presents a promising therapeutic strategy targeting METTL3 for HCC treatment and further expands the application of CRISPR/Cas9 gene-editing technology in cancer therapy.},
}
@article {pmid41241617,
year = {2026},
author = {Kalogeropoulos, K and van Beljouw, SPB and Feldmann, D and van den Berg, DF and Brouns, SJJ},
title = {Proteases in bacteriophage defense systems and their potential in bioengineering.},
journal = {Trends in biochemical sciences},
volume = {51},
number = {1},
pages = {64-79},
doi = {10.1016/j.tibs.2025.10.006},
pmid = {41241617},
issn = {0968-0004},
mesh = {*Bacteriophages/enzymology/metabolism ; *Bioengineering/methods ; *Peptide Hydrolases/metabolism ; },
abstract = {Novel phage defense systems featuring diverse enzymatic activities are continually being discovered. Among these, defense systems employing proteolytic enzymes have been identified, revealing a previously unrecognized enzymatic activity in phage defense. These protease-associated defense systems represent an untapped reservoir for new biotechnological tools and may serve as a springboard for the development of proteome editors. This review outlines recent advancements in the discovery and characterization of protease-containing defense systems, proposes methods for further exploration and investigation of protease activity, and considers the prospect of protease defense systems for modulating protein processing and cell fate.},
}
@article {pmid41241768,
year = {2025},
author = {Lotfi, M and Farshchian Yazdi, Z and Hashemi, MR and Abbaszadegan, MR and Sharif, S},
title = {Guarding the genome: strategies for off-target detection and minimization in CRISPR/Cas9 system.},
journal = {Expert review of molecular diagnostics},
volume = {25},
number = {12},
pages = {883-902},
doi = {10.1080/14737159.2025.2581677},
pmid = {41241768},
issn = {1744-8352},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {INTRODUCTION: CRISPR-based genome editing has revolutionized molecular diagnostics and therapeutic applications, offering precise genetic modifications. However, off-target effects remain a significant challenge, potentially leading to unintended mutations and genomic instability. Addressing these concerns is crucial for ensuring the clinical safety and efficacy of CRISPR technologies.<br><br>AREAS COVERED: This review provides a comprehensive analysis of strategies for detecting and mitigating off-target effects in CRISPR genome editing. We discuss in silico, in vitro, and in vivo detection methods, highlighting their strengths and limitations. Additionally, we examine advancements in guide RNA (gRNA) engineering, improved Cas variants, optimized delivery systems, base and prime editing technologies, and anti-CRISPR proteins as key approaches to enhance genome-editing precision.<br><br>EXPERT OPINION: While significant progress has been made in off-target detection and mitigation, challenges remain in accurately assessing unintended genomic modifications in vivo and ensuring long-term safety in clinical applications. Future research should focus on developing high-fidelity CRISPR variants, refining computational prediction models, and integrating multi-modal detection systems. Advancing delivery mechanisms and regulatory strategies will be essential for translating CRISPR into safe and effective therapeutic interventions.},
}
@article {pmid41241835,
year = {2025},
author = {Zhang, J and Li, L and Zhu, Y and Qian, K and Xu, Q and Qin, Y and Wu, L},
title = {Amplification-Free CRISPR-Cas System Integrated Centrifugal Digital Microfluidic Platform Developed for Multiplexed Respiratory Pathogen Nucleic Acid Analysis.},
journal = {Analytical chemistry},
volume = {97},
number = {46},
pages = {25583-25591},
doi = {10.1021/acs.analchem.5c04524},
pmid = {41241835},
issn = {1520-6882},
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification ; *Influenza A Virus, H1N1 Subtype/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Humans ; Nucleic Acid Amplification Techniques ; *DNA, Bacterial/analysis/genetics ; *Lab-On-A-Chip Devices ; *Microfluidic Analytical Techniques/instrumentation ; *RNA, Viral/analysis/genetics ; Bacterial Proteins ; Penicillin-Binding Proteins ; },
abstract = {In response to the urgent demand for highly sensitive and rapid multiplex detection technologies in the prevention and control of respiratory infectious diseases, this study presents the development of an integrated CRISPR-Cas9/Cas13a detection platform based on a centrifugal digital microfluidic chip. It aims to overcome the reliance of traditional real-time fluorescence quantitative PCR on specialized equipment and trained personnel. Additionally, it addresses the issue of false positives commonly associated with existing isothermal amplification technologies, while also meeting the requirement for preamplification in sensitive CRISPR-based detection methods. In this study, Methicillin-resistant Staphylococcus aureus (MRSA) and influenza A virus subtype H1N1 were selected as model pathogens. The off-chip CRISPR-Cas9/Cas13a dual nucleic acid detection system was initially developed and optimized to enable highly specific detection of MRSA-mecA DNA at a concentration of 173 pM and H1N1-HA RNA at a concentration of 117 pM. Subsequently, the optimal centrifugal digital chip structure was designed and screened to achieve a droplet filling rate of 99.6%. The optimized CRISPR system was finally integrated into the digital chip, resulting in significantly improved sensitivity, reaching 0.7 copies/μL for MRSA DNA and 1.2 copies/μL for H1N1 RNA within a 20 min reaction time at 37 °C. Furthermore, both the negative and positive detection rates achieved 100% accuracy across all 20 simulated clinical samples. The platform integrates centrifugal digital droplet segmentation technology with the CRISPR-Cas system in an innovative manner, enabling subcopy sensitivity detection without the need for nucleic acid preamplification. Therefore, this convenient, cost-effective, and contamination-resistant method provides a reliable solution for the rapid detection of respiratory pathogens in resource-constrained scenarios.},
}
@article {pmid41241907,
year = {2026},
author = {Treichel, AJ and Bazzini, AA and Tornini, VA},
title = {Functional Testing of Microproteins in a Vertebrate Model of Development.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2992},
number = {},
pages = {183-201},
pmid = {41241907},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; CRISPR-Cas Systems ; *Zebrafish Proteins/genetics/metabolism ; Ribosomes/metabolism/genetics ; Gene Expression Regulation, Developmental ; Embryonic Development/genetics ; Micropeptides ; },
abstract = {While thousands of putative microproteins have been identified through ribosome profiling, reporter assays, and mass spectrometry-based methods, their functional testing has remained challenging. Advances in genome sequencing and CRISPR/Cas technologies enable the prioritization and testing of candidate microprotein functions for roles in development, for example, in the maternal-to-zygotic transition or in neurodevelopment. Here, we describe the functional testing of microproteins in vivo using a vertebrate model of early development, Danio rerio (zebrafish).},
}
@article {pmid41242144,
year = {2026},
author = {Yu, ES and Jang, H and Kwon, JM and Jeong, H and Park, J and Kang, BH and Rho, D and Son, S and Kang, T and Jeong, KH},
title = {Nanoplasmonic real-time RT-RPA and CRISPR/Cas12a detection for rapid point-of-care molecular diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {294},
number = {},
pages = {118216},
doi = {10.1016/j.bios.2025.118216},
pmid = {41242144},
issn = {1873-4235},
mesh = {*SARS-CoV-2/genetics/isolation &amp; purification ; *COVID-19/diagnosis/virology ; Humans ; *CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Point-of-Care Systems ; *Biosensing Techniques/instrumentation ; Gold/chemistry ; *Molecular Diagnostic Techniques/instrumentation ; *COVID-19 Nucleic Acid Testing/instrumentation ; Point-of-Care Testing ; Lab-On-A-Chip Devices ; Equipment Design ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {The rapid and precise detection of nucleic acids is crucial for effective disease diagnosis and management at the point-of-care (POC) level. Here we report a palm-sized plasmonic photothermal platform for real-time on-chip recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection. An ultrathin photothermal nanoplasmonic cavity (PNC) of Au nanoislands (AuNIs) and an aluminum reflector delivers uniform and efficient photothermal heating under white LED illumination. The configuration drives isothermal amplification and CRISPR-mediated cleavage in a single microchamber while a fluorescence microlens array (FMLA) camera records real-time emission. The compact platform detects the SARS-CoV-2 E gene in 25 min at 25.7 copies per cartridge and achieves 100 % concordance with RT-qPCR across 42 clinical samples. This all-in-one platform can offer a robust and cost-effective solution for molecular diagnostics, facilitating scalable and real-time testing of infectious diseases in decentralized POC settings.},
}
@article {pmid41242302,
year = {2025},
author = {Ishihara, K and Kitagawa, S and Adachi, N and Akutsu, M and Senda, T and Inanaga, H and Numata, T},
title = {Cryo-EM structure of Archaeoglobus fulgidus type III-B CRISPR-Cas effector and intermediate crRNA processing during effector assembly.},
journal = {Biochemical and biophysical research communications},
volume = {792},
number = {},
pages = {152978},
doi = {10.1016/j.bbrc.2025.152978},
pmid = {41242302},
issn = {1090-2104},
mesh = {*Archaeoglobus fulgidus/metabolism/genetics/chemistry/ultrastructure ; *CRISPR-Cas Systems ; Cryoelectron Microscopy ; *Archaeal Proteins/chemistry/metabolism/ultrastructure/genetics ; *RNA, Archaeal/metabolism/chemistry/ultrastructure/genetics ; *CRISPR-Associated Proteins/chemistry/metabolism/ultrastructure/genetics ; Models, Molecular ; },
abstract = {Type III CRISPR-Cas effectors recognize target RNAs complementary to the crRNA guide, activating diverse downstream antiviral responses. In contrast to type III-A systems, the architecture of the type III-B effector (Cmr), comprising six proteins (Cmr1-Cmr6) and a crRNA, remains incompletely defined. Moreover, although maturation of the 3' region of type III crRNA has been attributed to polynucleotide phosphorylase (PNPase), an alternative maturation pathway has been suggested but remains to be elucidated. Here we determined the cryo-EM structure of the Cmr1-lacking Archaeoglobus fulgidus Cmr (AfCmrΔ1) bound to a target analog at 3.4 Å resolution. The complex forms a continuous basic channel that accommodates a crRNA-target heteroduplex. Comparative interface analysis explains why the previously reported cross-species Cmr assembly retains activity, revealing interface flexibility that enables compatible Cmr3-Cmr4 and Cmr2-Cmr5 interactions. Furthermore, we show the cooperative, site-specific processing of an intermediate crRNA that requires both AfCmrΔ1 and AfCmr1 and proceeds without divalent cations. In addition to identifying the cleavage site within the intermediate crRNA, mutational analysis of AfCmr1 reveals residues critical for the reaction. These findings suggest an alternative pathway for crRNA maturation during type III effector assembly that complements PNPase-mediated trimming of the intermediate crRNA, thereby expanding the mechanistic landscape of type III CRISPR-Cas systems.},
}
@article {pmid41242303,
year = {2025},
author = {Dong, M and Zhang, J and Long, L and Wang, H and Zhang, A and Zeng, W and Li, J},
title = {Establishment of a liver-specific Albumin-Cre recombinase transgenic golden hamster model using gRosa26-Targeted dual-fluorescent reporter system for hepatocyte-specific genetic manipulation.},
journal = {Biochemical and biophysical research communications},
volume = {792},
number = {},
pages = {152912},
doi = {10.1016/j.bbrc.2025.152912},
pmid = {41242303},
issn = {1090-2104},
mesh = {Animals ; *Integrases/genetics/metabolism ; *Mesocricetus/genetics ; *Hepatocytes/metabolism ; Cricetinae ; *Genes, Reporter ; Animals, Genetically Modified ; *Liver/metabolism ; *Albumins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *RNA, Untranslated/genetics ; Male ; Mice ; Organ Specificity ; },
abstract = {BACKGROUND AND AIMS: Despite its superior physiological relevance to human liver metabolism and diseases, the absence of reliable tissue-specific gene manipulation systems has considerably limited genetic research in the Syrian golden hamster (Mesocricetus auratus). The Cre/loxP-based Rosa26-targeted dual-fluorescent reporter system (mT/mG) is widely utilized in murine models for accurate lineage tracing and conditional gene editing. However, such powerful genetic tools are not available for golden hamsters. Herein, we established a functional Rosa26-targeted mT/mG reporter system and a hepatocyte-specific albumin-Cre Recombinase (Alb-Cre) driver line in golden hamsters, providing an important platform for hepatocyte-specific gene manipulation.<br><br>METHODS: The Rosa26 homolog (gRosa26) was identified in the golden hamster genome. Then, a CRISPR/Cas9-based two-cell microinjection system was developed to precisely knock-in gRosa26. The dual-fluorescent Cre reporter (mT/mG) was inserted into gRosa26. Thereafter, a liver-specific Alb-Cre transgenic line was developed using murine Alb enhancer/promoter elements. Finally, Cre-mediated recombination efficiency was evaluated in double transgenic hamsters.<br><br>RESULTS: The gRosa26 locus exhibited >70&#xa0;% sequence conservation with mice. Golden hamsters with the mT/mG reporter exhibited ubiquitous tdTomato expression, with efficient EGFP activation after Cre-mediated recombination. Furthermore, Alb-Cre transgenic golden hamsters exhibited hepatocyte-specific Cre expression. In double transgenic hamsters, robust hepatocyte-specific tdTomato-to-EGFP recombination was observed by postnatal days 21 and 42. This validated the functionality of the gRosa26-targeted mT/mG system and Alb-driven Cre expression.<br><br>CONCLUSIONS: We reported the development of a functional Rosa26-targeted mT/mG dual-reporter system and a liver-specific Alb-Cre driver in Syrian golden hamsters for the first time. This platform facilitates precise spatiotemporal gene editing in hepatocytes and serves as an essential genetic tool for liver research in this emerging model organism.},
}
@article {pmid41242543,
year = {2026},
author = {Hillary, VE and Rajan, V and Ceasar, SA},
title = {CRISPR's impact on cancer: From fundamental models to clinical solutions.},
journal = {Life sciences},
volume = {384},
number = {},
pages = {124087},
doi = {10.1016/j.lfs.2025.124087},
pmid = {41242543},
issn = {1879-0631},
mesh = {*Neoplasms/genetics/therapy ; Humans ; *CRISPR-Cas Systems/genetics ; Animals ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Tumor Microenvironment ; },
abstract = {CRISPR is a programmable genome editing method that has transformed genome engineering and advanced cancer research. Various engineering iterations of CRISPR, including base and prime editing, facilitate more accurate and adaptable genomic manipulations, opening new frontiers in understanding and combating cancer. This review emphasizes CRISPR's significant influence in cancer biology, from its ability to accurately recreate somatic mutations in cellular and animal models to its application in developing precise diagnostic tools. We also examine advancements in cancer therapies, particularly through the ex vivo engineering of robust chimeric antigen receptor (CAR) T cells that perform effectively in restrictive tumor microenvironments. Lastly, we consider the existing limitations of CRISPR-Cas systems and outline future directions that could further enhance the application of this powerful technology against cancer.},
}
@article {pmid41242662,
year = {2026},
author = {Zhang, N and Zhou, X and Jiao, X and Liang, Z and Jiang, W and Liu, S and Wu, P},
title = {Field-deployable CRISPR-Dx for BmNPV and Nosema bombycis: DNA-extraction-free one-pot RPA-Cas12a and Cas12a/Cas13a dual-gene assays with handheld devices.},
journal = {Insect biochemistry and molecular biology},
volume = {186},
number = {},
pages = {104449},
doi = {10.1016/j.ibmb.2025.104449},
pmid = {41242662},
issn = {1879-0240},
mesh = {Animals ; *Nosema/isolation &amp; purification/genetics ; *Nucleopolyhedroviruses/isolation &amp; purification/genetics ; *Bombyx/virology/microbiology ; *CRISPR-Cas Systems ; },
abstract = {Simple, accurate, sensitive, and rapid pathogen diagnosis is crucial for effective control of silkworm diseases. Although CRISPR-based nucleic acid detection systems show great potential for on-site detection of silkworm pathogens, their practicality is hindered by complex workflows and reagent-storage constraints. To address these limitations and enhance field suitability, we developed a DNA extraction-free one-pot RPA-CRISPR/Cas12a (DEORC) system and a dual-gene assay for detecting Bombyx mori nucleopolyhedrovirus (BmNPV) and Nosema bombycis using a handheld device. The DEORC assay detects BmNPV in hemolymph samples as early as 6 h post-infection (hpi) and N. bombycis at 10[3] spores/mL in spore suspensions. The entire process from sampling to visual readout is completed in approximately 70 min without requiring sophisticated equipment. To further enable off-grid deployment, we lyophilized the Cas12a detection reagents using 1 M betaine as a lyoprotectant, which retained measurable activity for at least one month at 4 °C under our test conditions, facilitating short-term refrigerated transport and field storage. Additionally, the dual-gene assay detects 10[3] copies/μL of a double-reference plasmid and simultaneously detects both BmNPV and N. bombycis in a single tube from midgut samples at 48 hpi; when combined with extraction-free techniques, it enables simultaneous detection of both pathogens in hemolymph samples at 72 hpi. Collectively, these advancements provide sensitive and portable tools for on-site sericulture disease management, offering faster and more practical workflows than two-step single-gene and traditional approaches.},
}
@article {pmid41242874,
year = {2025},
author = {Akter, T and Baishnab, SM and Damtie, M and Dumpati, S and Eshete, TB and Irfan, S and Kumar, M and Sara, M and Urmi, UL and Willcox, M},
title = {A review of technological advances in the management of bacterial, fungal, viral, and Acanthamoeba keratitis.},
journal = {Clinical &amp; experimental optometry},
volume = {},
number = {},
pages = {1-11},
doi = {10.1080/08164622.2025.2579169},
pmid = {41242874},
issn = {1444-0938},
abstract = {Microbial keratitis is a major cause of monocular blindness worldwide. Trauma and contact lens wear are major risk factors. Bacteria are a major cause, but fungi, viruses, and Acanthamoeba can also cause the disease. Therapeutic options are dwindling with the rise of antimicrobial resistance in all of these microbes, but especially bacteria. Infection with resistant strains results in worse clinical outcomes. New antimicrobial therapies are being developed but most have yet to be either used to treat ocular infections or become commercially available. For bacterial keratitis, antimicrobial peptides (AMPs) are a potential new option, as are bacteriophages and even bacteria that eat other bacteria or maggot secretions. For fungal keratitis, ocular natamycin, with oral ketoconazole or voriconazole treatments, offer improved outcomes for severe cases. For viral keratitis, AMPs again offer hope as new therapeutics, as do RNA interference and CRISPR-Cas systems. The recent release of a commercially available polyhexamethylene biguanide eye drop is a step forward, but new therapies that can kill both Acanthamoeba trophozoites and cysts are still needed. Beyond antibiotics, photoactivated chromophores for keratitis-corneal collagen cross-linking and photodynamic therapy have been successfully used. Therapies for large ulcers or scarring include corneal gluing, tissue scaffolds such as amniotic membrane or conjunctival flaps, and tissue replacement through penetrating or lamellar keratoplasty.},
}
@article {pmid41243026,
year = {2025},
author = {Menelih, A and Girma, A and Assamere, A},
title = {Mechanisms and applications of epigenome editing in plants: current status, challenges and future perspectives.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {243},
pmid = {41243026},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Epigenome ; CRISPR-Cas Systems ; *Epigenesis, Genetic ; *Plants/genetics ; DNA Methylation ; *Genome, Plant ; Epigenome Editing ; },
abstract = {Epigenome editing has become a leading-edge technology of programmable, heritable and reversible control of gene expression in plants without changing the DNA sequence. CRISPR/dCas9 systems along with transcription activator-like effectors (TALEs) and zinc finger systems have made it possible to manipulate DNA methylation, histone modifications, and RNA epigenetic marks in a precise and locus-specific fashion. These tools have been used on major regulatory genes of flowering time, stress adjustment, and yield maximization in model and crop plants. This review synthesizes the current status of plant epigenome editing advances and highlights mechanistic innovations including SunTag, CRISPRoff/on and RNA m6A editing. It also emphasizes new paradigm shifts in chromatin reprogramming, including transcription-resistive chromatin states, locus-specific H3K27me3 demethylation, and nanobody-mediated chromatin targeting. Furthermore, it considers the consequences of these shifts in the context of trait stability and epigenetic inheritance. Moreover, the relative evaluation of dCas9-, TALE-, and ZFP-based platforms indicated that there are still enduring problems in the performance of delivery, off-target effects, and transgenerational stability. The review concludes with a conceptual framework connecting epigenome editing to climate-smart crop improvement and outlines future research priorities focused on combinatorial multi-omics integration and the development of environmentally responsive editing platforms.},
}
@article {pmid41247434,
year = {2026},
author = {Liu, F and Ge, D and Lian, G and Han, Z and Chai, J},
title = {Unraveling plant immunity: from pathogen perception to resistance engineering.},
journal = {Science China. Life sciences},
volume = {69},
number = {3},
pages = {779-793},
pmid = {41247434},
issn = {1869-1889},
mesh = {*Plant Immunity/genetics ; *Disease Resistance/genetics/immunology ; *Plant Diseases/immunology/microbiology ; Receptors, Pattern Recognition/metabolism/immunology ; NLR Proteins/metabolism/genetics ; Signal Transduction ; *Plants/immunology/genetics ; Calcium Signaling ; Plant Proteins/genetics/metabolism ; Host-Pathogen Interactions/immunology ; CRISPR-Cas Systems ; },
abstract = {Unlike animals, which rely on circulatory systems and mobile immune cells, each plant cell must autonomously detect and respond to pathogenic threats. Plant immunity operates through two major layers: pattern-triggered immunity (PTI), initiated by cell-surface pattern recognition receptors (PRRs), and effector-triggered immunity (ETI), primarily mediated by intracellular nucleotide-binding leucine-rich repeat (NLR) receptors. Recent advances have substantially enhanced our understanding of PTI and ETI signaling. Notably, some NLRs following activation by specific recognition of pathogen effectors form higher-order oligomeric complexes termed resistosomes that act as Ca[2+]-permeable channels to trigger immune signaling. Increasing evidence points to extensive crosstalk and mutual potentiation between PTI and ETI, with Ca[2+] functioning as a pivotal second messenger in both pathways. Elucidating the molecular basis of these pathways, combined with emerging tools like CRISPR/Cas9, offers new strategies for engineering durable disease resistance in crops. This review highlights current insights into PTI and ETI, with an emphasis on the central role of Ca[2+] signaling and key challenges in engineering NLR receptors.},
}
@article {pmid41247804,
year = {2026},
author = {Li, A and Yang, C and Zhao, Z and Lu, T and Yin, C and Xu, C and Lv, C and Liu, Y and Zhan, Y and Li, S and Cao, C},
title = {Strategic engineering of DNA aptamers as precision modulators in CRISPR-driven oncogene control.},
journal = {International journal of surgery (London, England)},
volume = {112},
number = {2},
pages = {3015-3027},
doi = {10.1097/JS9.0000000000003837},
pmid = {41247804},
issn = {1743-9159},
mesh = {*Aptamers, Nucleotide/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; Mice ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Oncogenes/genetics ; Cell Line, Tumor ; *Neoplasms/genetics/therapy ; *CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; *Genetic Therapy/methods ; },
abstract = {OBJECTIVE: The goal of this study was to address the limited availability of intracellular protein-binding aptamers that restrict the potential of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based signal conductor systems. We aimed to develop a programmable CRISPR-SaCas9 platform using DNA aptamers to achieve precise and dynamic gene regulation for cancer therapy.<br><br>METHODS: We identified and characterized five DNA aptamers with high specificity for the SaCas9 protein. Functional assays were performed to evaluate their effects on the transcriptional regulatory activity of SaCas9. Some aptamers were engineered to associate with promoter elements, enabling transcription factor-responsive control of downstream gene expression. To test therapeutic efficacy, the system was delivered in vivo using In vivo-jetPEI reagents, and tumor progression and metastasis were monitored.<br><br>RESULTS: The identified aptamers exhibited distinct functionalities - some partially inhibited SaCas9 activity, while others enabled precise gene expression control in response to endogenous transcription factors. The aptamer-CRISPR complexes formed ternary assemblies with SaCas9 and single-guide RNAs, enabling logic-gated responses. In vivo , the system significantly suppressed tumor growth and metastasis.<br><br>CONCLUSION: We developed a modular CRISPR signal conductor platform that leverages SaCas9-binding DNA aptamers for transcription factor-mediated logic operations and precise gene control. This system expands the toolkit for genetic circuit design and offers a promising strategy for targeted cancer gene therapy.},
}
@article {pmid41248011,
year = {2025},
author = {Dereki, I and Chondrou, V and Lagoumintzis, G and Sgourou, A},
title = {Epigenetic Engineering of K562 Cells: Dual-Vector Episomal Strategy for Stable Targeted DNA Methylation using dCas9-DNMT3A and -HDAC1 Fusion Proteins.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {224},
pages = {},
doi = {10.3791/69328},
pmid = {41248011},
issn = {1940-087X},
mesh = {Humans ; *DNA Methylation/genetics ; K562 Cells ; DNA Methyltransferase 3A ; *DNA (Cytosine-5-)-Methyltransferases/genetics/biosynthesis ; Plasmids/genetics ; *Histone Deacetylase 1/genetics/metabolism ; Genetic Vectors/genetics ; Recombinant Fusion Proteins/genetics/biosynthesis ; *Epigenesis, Genetic ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/genetics ; CRISPR-Cas Systems ; },
abstract = {Investigating the precise role of DNA methylation in gene transcriptional regulation and developing therapies targeting specific gene methylation patterns presents significant challenges due to the scarcity of versatile tools capable of inducing site-specific and long-term epigenetic modifications for modulating gene expression. The study aimed to develop and validate an innovative episomal-based system to facilitate stable DNA methylation at a targeted gene locus, potentially useful for both basic epigenetic research and therapeutic applications. To achieve this, the K562 cell line was co-transfected with two distinct episomal vectors. Both vector types were engineered to express guide RNAs (gRNAs) targeting a 367 bp unmethylated region within the CpG island 326, located upstream of the ZBTB7A gene. Each vector encoded a deactivated form of endonuclease Cas9 (dead or dCas9) fused to either the catalytic domain of DNA methyltransferase DNMT3A (dCas-DNMT3A-CD) or the full-length histone deacetylase HDAC1 (dCas-HDAC1). The dCas sequence included two Nuclear Localization Signals (NLS) to ensure the protein's nuclear import. This dual system expression cassette promotes a chromatin state potentially conducive to long-term epigenetic silencing, promising robust and durable epigenetic results. This intervening approach to the host epigenome via utilization of self-replicating episomal vectors offers several advantages: maintenance and expression of vectors at low copy numbers throughout multiple cell divisions without integration into the host genome, hence minimizing off-target effects and preserving genome integrity. We report the precise and significant increase of DNA methylation at the targeted ZBTB7A CpG island 326. The findings validate that the engineered episomal CRISPR/dCas systems can elicit durable, site-specific DNA methylation. Therefore, this system is a valuable research tool for evaluating the functional effects of targeted methylation changes and a promising platform for developing future epigenetic treatments.},
}
@article {pmid41248420,
year = {2026},
author = {Long, SA and Todd, H and Goodhart, G and Chang, WH and Amparo, AM and Bridgens, R and Dilly, J and Park, SJ and Beal, RM and Shehadeh, SM and Satyadi, MA and Trivedi, VK and Ackermann, SE and Mukherjee, R and Goodwin, CM and Edwards, AC and Stalnecker, CA and Greis, KD and Aguirre, AJ and Hobbs, GA and Bryant, KL and Ahmad, SA and Cox, AD and Der, CJ and Waters, AM},
title = {CRISPR-Cas9 Screening Identifies Resistance Mechanisms to KRAS Inhibition in Pancreatic Cancer.},
journal = {Cancer research},
volume = {86},
number = {4},
pages = {1035-1053},
pmid = {41248420},
issn = {1538-7445},
support = {K22 CA276632/CA/NCI NIH HHS/United States ; IRG-23-1141524//American Cancer Society (ACS)/ ; U01CA199235//National Cancer Institute (NCI)/ ; 15-90-25-DER//Pancreatic Cancer Action Network (PCAN)/ ; K22CA276632//National Cancer Institute (NCI)/ ; P01CA203657//National Cancer Institute (NCI)/ ; T32CA244125//National Cancer Institute (NCI)/ ; P50CA257911//National Cancer Institute (NCI)/ ; R35CA232113//National Cancer Institute (NCI)/ ; S10OD026717//National Institutes of Health (NIH)/ ; 22-WG_DERB//Pancreatic Cancer Action Network (PCAN)/ ; P50CA196510//National Cancer Institute (NCI)/ ; P50 CA257911/CA/NCI NIH HHS/United States ; R01CA42978//National Cancer Institute (NCI)/ ; R37CA251877//National Cancer Institute (NCI)/ ; PF-23-1072348-01-CDP//American Cancer Society (ACS)/ ; R25CA261610//National Cancer Institute (NCI)/ ; W81XWH2110692//U.S. Department of Defense (DOD)/ ; 23-MF-DILL//Pancreatic Cancer Action Network (PCAN)/ ; },
mesh = {Humans ; *Pancreatic Neoplasms/genetics/drug therapy/pathology ; *Proto-Oncogene Proteins p21(ras)/antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; *Drug Resistance, Neoplasm/genetics ; *Carcinoma, Pancreatic Ductal/genetics/drug therapy/pathology ; Cell Line, Tumor ; Mutation ; *Protein Kinase Inhibitors/pharmacology ; Erlotinib Hydrochloride/pharmacology ; },
abstract = {UNLABELLED: KRAS inhibitors (KRASi) targeting various KRAS mutations have entered clinical trials for pancreatic cancer. Despite promising preliminary clinical responses, most patients relapse due to intrinsic or acquired resistance. Thus, combination treatments are essential to extend the efficacy of KRAS-targeted therapies. To further determine the genetic mechanisms of KRASi resistance, we performed KRASi-anchored CRISPR-Cas9 loss-of-function screens in KRASG12D-, KRASG12C-, KRASG12R-, and KRASQ61H-mutant pancreatic ductal adenocarcinoma (PDAC) cell lines, using six KRASi, to identify genes that modulate sensitivity to KRAS inhibition. Several hits from the screens, including EGFR, CK2, p110α, p110γ, and YAP, were validated by combining targeted inhibitors with KRASi. KRASQ61H-mutant PDAC cell lines were intrinsically less dependent on KRAS for survival than other KRAS mutational subtypes. Furthermore, the EGFR inhibitor erlotinib synergized with the RAS(ON) multiselective inhibitor RMC-7977 in KRASQ61H-mutant PDAC cell lines and in cell lines with highly active EGFR by mitigating ERK rebound activity. KRASi-resistant cell lines featured sustained ERK/MAPK dependence despite decreased ERK activity. Together, these findings enhance the understanding of intrinsic and acquired resistance to KRASi and identify therapeutic vulnerabilities that can potentially be exploited for KRASi combination therapies in patients with pancreatic cancer.<br><br>SIGNIFICANCE: A comprehensive assessment of genetic modulators of KRAS inhibitor sensitivity identifies combination approaches to increase the efficacy of KRAS inhibitors and demonstrates the limited response of KRASQ61H-mutant cancer cells to KRAS inhibition.},
}
@article {pmid41248489,
year = {2025},
author = {Sinan, S and Kooistra, RM and Rajaraman, K and Islam, Z and Madan, D and Nalefski, EA and Finkelstein, IJ},
title = {One-Pot Isothermal Linear Amplification and Cas12a-Based Nucleic Acid Detection.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4714-4722},
doi = {10.1021/acssynbio.5c00463},
pmid = {41248489},
issn = {2161-5063},
mesh = {*Nucleic Acid Amplification Techniques/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Human papillomavirus 16/genetics/isolation &amp; purification ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; Papillomavirus Infections/diagnosis/virology ; DNA, Viral/genetics ; Uterine Cervical Neoplasms/virology/diagnosis ; DNA, Single-Stranded/genetics ; DNA-Directed DNA Polymerase/metabolism ; },
abstract = {CRISPR-based nucleic acid diagnostics are a promising class of point-of-care tools that could dramatically improve healthcare outcomes for millions worldwide. However, these diagnostics require nucleic acid preamplification, an additional step that complicates deployment to low resource settings. Here, we developed CATNAP (Cas trans-nuclease detection of amplified products), a method that integrates isothermal linear DNA amplification with Cas12a detection in a single reaction. CATNAP uses a nicking enzyme and DNA polymerase to continuously generate single-stranded DNA, activating Cas12a's trans-cleavage activity without damaging the template. We optimized enzyme combinations, buffer conditions, and target selection to achieve high catalytic efficiency. CATNAP successfully distinguished between high- and low-risk HPV strains and detects HPV-16 in crude cell lysates of cervical cancer cells with minimal equipment, offering advantages over PCR-based approaches. We conclude that CATNAP bridges the sensitivity gap in CRISPR diagnostics while maintaining simplicity, making accurate disease detection more accessible in resource-limited settings.},
}
@article {pmid41249169,
year = {2025},
author = {Ramadoss, GN and Namaganda, SJ and Kumar, MM and Hamilton, JR and Sharma, R and Chow, KG and Workley, LA and Macklin, BL and Sun, M and Ha, AS and Liu, JC and Fellmann, C and Watry, HL and Dierks, PH and Bose, RS and Jin, J and Perez, BS and Sandoval Espinoza, CR and Matia, MP and Lu, SH and Judge, LM and Shy, BR and Nussenzweig, A and Adamson, B and Murthy, N and Doudna, JA and Kampmann, M and Conklin, BR},
title = {Characterizing and controlling CRISPR repair outcomes in nondividing human cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {9883},
pmid = {41249169},
issn = {2041-1723},
support = {R01 NS119678/NS/NINDS NIH HHS/United States ; R35 GM138167/GM/NIGMS NIH HHS/United States ; K99 GM143461/GM/NIGMS NIH HHS/United States ; R21 HL173710/HL/NHLBI NIH HHS/United States ; U19 AI135990/AI/NIAID NIH HHS/United States ; L30 TR002983/TR/NCATS NIH HHS/United States ; U19 NS132303/NS/NINDS NIH HHS/United States ; R00 GM118909/GM/NIGMS NIH HHS/United States ; F32 AG081085/AG/NIA NIH HHS/United States ; R35 GM143124/GM/NIGMS NIH HHS/United States ; HHSN261201500003I/CA/NCI NIH HHS/United States ; R01 AG072052/AG/NIA NIH HHS/United States ; U54 AI170792/AI/NIAID NIH HHS/United States ; K08 CA273529/CA/NCI NIH HHS/United States ; UM1 AI164559/AI/NIAID NIH HHS/United States ; P01 HL146366/HL/NHLBI NIH HHS/United States ; UH3 AI150552/AI/NIAID NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; R01 MH125979/MH/NIMH NIH HHS/United States ; R01 HL130533/HL/NHLBI NIH HHS/United States ; },
mesh = {Humans ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Gene Editing/methods ; *DNA Repair/genetics ; *CRISPR-Cas Systems/genetics ; *Neurons/metabolism/cytology ; Myocytes, Cardiac/metabolism ; DNA Damage ; T-Lymphocytes/metabolism ; },
abstract = {Genome editing is poised to revolutionize treatment of genetic diseases, but poor understanding and control of DNA repair outcomes hinders its therapeutic potential. DNA repair is especially understudied in nondividing cells like neurons, limiting the efficiency and precision of genome editing in many clinically relevant tissues. Here, we address this barrier by using induced pluripotent stem cells (iPSCs) and iPSC-derived neurons to examine how postmitotic human neurons repair Cas9-induced DNA damage. CRISPR editing outcomes differ dramatically in neurons compared to genetically identical dividing cells: neurons take longer to fully resolve this damage, and upregulate non-canonical DNA repair factors in the process. Manipulating this response with chemical or genetic perturbations allows us to direct DNA repair toward desired editing outcomes in nondividing human neurons, cardiomyocytes, and primary T cells. By studying DNA repair in clinically relevant cells, we reveal unforeseen challenges and opportunities for precise therapeutic editing.},
}
@article {pmid41249545,
year = {2025},
author = {Warkad, S and Kumar, A and Gampa, M and Goswami, S and T, V and Kumar, S and Dalal, M and Mishra, D and Jha, GK and C, V and Kumar, RR},
title = {Small RNAs big impact: a review on microRNA-mediated tolerance in wheat under terminal heat.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {245},
pmid = {41249545},
issn = {1438-7948},
support = {CABin//Indian Council of Agricultural Research/ ; NICRA//Indian Council of Agricultural Research/ ; },
mesh = {*Triticum/genetics/physiology ; *MicroRNAs/genetics/metabolism ; Gene Expression Regulation, Plant ; *Thermotolerance/genetics ; *Heat-Shock Response/genetics ; *RNA, Plant/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {MicroRNAs (miRNAs) are key regulators of gene expression in plant responses to abiotic stresses, including heat stress. High temperatures during the critical developmental stages of wheat (Triticum aestivum L.) drastically limit growth and production. Recent research has found that specific miRNAs regulate molecular complexes and physiological responses by targeting transcription factors, heat shock proteins, and signaling components, thereby modulating heat stress tolerance pathways. This review highlights current knowledge about heat-responsive miRNAs in wheat, including their validated targets and functional involvement in thermotolerance. In addition, we summarized the potential CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats /CRISPR associated protein 9) genome editing tool for precise alteration of miRNA genes or their targets, degradome profiling, the secondary structure of miRNA, and how interplay of miRNAs with HSFs and HSPs in target gene regulation to improve heat resilience. A comprehensive understanding of miRNA-regulated networks presents novel possibilities for developing climate-resilient wheat varieties, thereby ensuring food security in the face of global warming.},
}
@article {pmid41249581,
year = {2025},
author = {Turgeman-Grott, I and Golan, N and Neri, U and Naki, D and Altman-Price, N and Eizenshtein, K and Choudhary, DK and Levy, R and Navok, S and Cohen, L and Shalev, Y and Singla, H and Reshef, L and Gophna, U},
title = {A previously undescribed archaeal virus suppresses host immunity.},
journal = {EMBO reports},
volume = {26},
number = {24},
pages = {6159-6178},
pmid = {41249581},
issn = {1469-3178},
support = {787514//EC | European Research Council (ERC)/ ; 1599/24//Israel Science Foundation (ISF)/ ; },
mesh = {*Archaeal Viruses/genetics/physiology ; *Haloferax/virology/genetics/immunology/growth &amp; development ; *Host-Pathogen Interactions/immunology ; Gene Expression Regulation, Archaeal ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Extremophilic archaea can have chronic viral infections that are well-tolerated by the hosts and may potentially protect against more lethal infections. Here we show that a natural Haloferax strain (48N), is chronically infected by a lemon-shaped virus. This viral infection is not cleared spontaneously, despite the multiple defense systems of the host. Curing 48N of its virus led to radical changes in the gene expression profile of 48N and a dramatic improvement in its growth rate. Remarkably, the cured 48N is the fastest-growing haloarchaeon reported to date, with a generation time of ~107 min at 45 °C, and faster than any known Haloferax species at this temperature. The virus subverts host defenses by reducing its transcription, including the CRISPR spacer acquisition machinery. Nonetheless, even in the virus-cured background, spacer acquisition is very low, indicating that another genetic element is disrupting CRISPR activity. Our results suggest that the slow growth of some halophilic archaea could be due to the effects of proviruses within their genomes that consume resources and alter the gene expression of their hosts.},
}
@article {pmid41250994,
year = {2025},
author = {Bereiter, R and Manjunath, A and Beal, PA},
title = {Clickable RNA via 4'-C-Ethynyl Cytidine─A Novel Design for Metabolically Stable Guide RNAs in RNA Editing.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {12},
pages = {2637-2644},
doi = {10.1021/acs.bioconjchem.5c00480},
pmid = {41250994},
issn = {1520-4812},
mesh = {*Cytidine/chemistry/analogs &amp; derivatives ; Click Chemistry ; Humans ; *RNA Editing ; *RNA, Guide, CRISPR-Cas Systems/chemistry/genetics/metabolism ; Alkynes/chemistry ; Cycloaddition Reaction ; Triazoles/chemistry ; },
abstract = {Chemical modifications in RNA therapeutics have addressed major challenges by enhancing metabolic stability, cellular uptake, and biological activity─regardless of their mechanism of action. Here, we report on the synthesis of 4'-C-ethynyl cytidine (4'-C-EthC) and its 2'-O-methylated derivative (4'-C-EthC-2'-OMe) as phosphoramidite building blocks and their subsequent incorporation into oligonucleotides. These ribose C4-terminal alkyne modifications provide a click handle directly within oligonucleotides. The novel modification is accessible via copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and serves as a universal 4'-C-ribose modifier on the oligonucleotide level. We identified both aromatic and aliphatic triazole residues that increase the thermodynamic stability in A-form RNA duplexes. Furthermore, 4'-C-triazole-modified oligonucleotides exhibit high resistance to nuclease-mediated degradation in metabolic stability assays. Finally, we introduced the novel modification and its substituted triazoles into guide RNAs (gRNAs) for site-directed A-to-I editing in mammalian cells and compared their performance with phosphorothioate-modified gRNAs.},
}
@article {pmid41251053,
year = {2026},
author = {Shrestha, M and Kim, Y and Park, S and Jiang, HL and Lee, JY and Kim, D and Jeong, JH},
title = {eVLP-Mediated Cas9 Delivery for Preventing IBMIR in Islet Transplantation.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {1},
pages = {e08128},
pmid = {41251053},
issn = {1613-6829},
support = {//National Research Foundation of Korea/ ; RS-2025-02303064//Korean government/ ; RS-2024-02507183//Ministry of Health and Welfare/ ; 22A0205L1//Ministry of Health and Welfare/ ; 23A0205L1//Ministry of Health and Welfare/ ; 21A0202L1//Ministry of Health and Welfare/ ; 21A0202L1//Ministry of Science and ICT, South Korea/ ; },
mesh = {Animals ; *Islets of Langerhans Transplantation/adverse effects/methods ; Thromboplastin/metabolism/genetics ; Plasminogen Activator Inhibitor 1/metabolism/genetics ; Rats ; Diabetes Mellitus, Experimental/therapy ; Male ; *Inflammation/prevention &amp; control ; *CRISPR-Associated Protein 9/metabolism ; Mice ; Islets of Langerhans ; *CRISPR-Cas Systems/genetics ; },
abstract = {Islet transplantation is a promising strategy for effective β-cell replacement in patients with type 1 diabetes. However, its success is hindered significantly by instant blood-mediated inflammatory reaction (IBMIR), which leads to rapid graft loss. IBMIR is triggered when the transplanted islets come in contact with blood, activating the coagulation cascade, complement pathways, and innate immune responses. Tissue factor (TF), abundantly expressed on the islet surface, initiates the coagulation cascade, leading to thrombin formation, platelet activation, and neutrophil infiltration. Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in IBMIR by inhibiting fibrinolysis and causing ischemic injury in the graft. TF and PAI-1 contribute significantly to IBMIR, thus making them critical targets for genetic interventions to prevent IBMIR. In this study, an engineered virus-like particle (eVLP)-mediated Cas9 nuclease is employed to knock out TF and PAI-1 genes in rat islets. TF and PAI-1 expression are effectively downregulated without inducing any off-target effects or without compromising the viability and functionality of the islets. Streptozotocin-induced diabetic mice transplanted with TF- and PAI-1-knockout islets exhibited improved glycemic control and a significant reduction in the plasma levels of thrombin-antithrombin (TAT) complex and complement component 3a (C3a), indicating the successful inhibition of IBMIR post-transplantation.},
}
@article {pmid41251373,
year = {2025},
author = {Pérez Antón, E and Dujeancourt-Henry, A and Rotureau, B and Glover, L},
title = {A CRISPR-based diagnostic tool to survey drug resistance in human African trypanosomiasis.},
journal = {Antimicrobial agents and chemotherapy},
volume = {69},
number = {12},
pages = {e0093325},
pmid = {41251373},
issn = {1098-6596},
support = {ANR-PRC 2021 SherPa//Agence Nationale de la Recherche/ ; LabEx IBEID//Labex/ ; },
mesh = {Humans ; *Trypanosomiasis, African/drug therapy/parasitology/diagnosis ; *Trypanocidal Agents/pharmacology ; *Drug Resistance/genetics ; *Trypanosoma brucei gambiense/drug effects/genetics ; Pentamidine/pharmacology/therapeutic use ; Melarsoprol/pharmacology/therapeutic use ; *CRISPR-Cas Systems/genetics ; Mutation ; Protozoan Proteins/genetics ; },
abstract = {The World Health Organization aims to eliminate human African trypanosomiasis caused by Trypanosoma brucei gambiense (gHAT) by 2030. With the decline of reported cases, maintaining active surveillance is essential, including for the potential emergence of drug-resistant parasites. We have developed new highly specific diagnostic tools, using the Cas13a-based Specific High-Sensitivity Reporter Enzymatic UnLOCKing (SHERLOCK) technology, for the detection of drug-resistant genotypes that (i) are already circulating, such as the AQP2/3(814) chimera providing resistance to pentamidine and melarsoprol or (ii) could emerge, such as the TbCPSF3 (N[232]H) mutation, associated with acoziborole resistance under laboratory conditions. The AQP2/3(814) SHERLOCK assay detected RNA from both cultured parasites and field strains isolated from gHAT patients who relapsed following melarsoprol or pentamidine treatment. The CPSF3(SNV) SHERLOCK assay discriminated between wild-type CPSF3 RNA and CPSF3 bearing a single A-C mutation that confers resistance to acoziborole in vitro. These SHERLOCK assays are amenable for use as a high-throughput screening method to monitor for drug-resistant-associated mutations in Trypanosoma brucei, providing a new molecular tool for epidemiological surveillance during the gHAT elimination phase.},
}
@article {pmid41252186,
year = {2025},
author = {Calhoun, CCS and Capps, MES and Muya, K and Gannaway, WC and Martina, V and Conklin, CL and Klein, MC and Webster, JM and Torija-Olson, EG and Thyme, SB},
title = {Removal of developmentally regulated microexons has a minimal impact on larval zebrafish brain morphology and function.},
journal = {eLife},
volume = {13},
number = {},
pages = {},
pmid = {41252186},
issn = {2050-084X},
mesh = {Animals ; *Zebrafish/genetics/growth &amp; development ; *Brain/anatomy &amp; histology/growth &amp; development/physiology ; Larva/growth &amp; development/genetics ; *Exons/genetics ; *Zebrafish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Gene Expression Regulation, Developmental ; *Alternative Splicing ; Phenotype ; },
abstract = {Microexon splicing is a vertebrate-conserved process through which small, often in-frame, exons are differentially included during brain development and across neuron types. Although the protein sequences encoded by these exons are highly conserved and can mediate interactions, the neurobiological functions of only a small number have been characterized. To establish a more generalized understanding of their roles in brain development, we used CRISPR/Cas9 to remove 45 microexons in zebrafish and assessed larval brain activity, morphology, and behavior. Most mutants had minimal or no phenotypes at this developmental stage. Among previously studied microexons, we uncovered baseline and stimulus-driven phenotypes for two microexons (meA and meB) in ptprd and reduced activity in the telencephalon in the tenm3 B0 isoform. Although mild neural phenotypes were discovered for several microexons that have not been previously characterized, including in ppp6r3, sptan1, dop1a, rapgef2, dctn4, vti1a, and meaf6. This study establishes a general approach for investigating conserved alternative splicing events and prioritizes microexons for downstream analysis.},
}
@article {pmid41252434,
year = {2026},
author = {Hirata, M and Namula, Z and Lin, Q and Nagahara, M and Gan, S and Ju, X and Takebayashi, K and Nakai, A and Wittayarat, M and Otoi, T and Tanihara, F},
title = {IL2RG deficient X-SCID pigs generated via electroporation-mediated gene editing using CRISPR/Cas9.},
journal = {Journal of animal science},
volume = {104},
number = {},
pages = {},
pmid = {41252434},
issn = {1525-3163},
mesh = {Animals ; *Gene Editing/veterinary/methods ; Male ; *Interleukin Receptor Common gamma Subunit/genetics/deficiency ; Female ; *Electroporation/veterinary ; *CRISPR-Cas Systems ; Swine/genetics ; *X-Linked Combined Immunodeficiency Diseases/genetics/veterinary ; Animals, Genetically Modified ; Gene Knockout Techniques/veterinary ; },
abstract = {Interleukin 2 receptor gamma (IL2RG) knockout (KO) is frequently used to generate X-linked severe combined immunodeficiency (X-SCID) models. Humans and pigs have anatomical and physiological similarities; hence, X-SCID pigs are being actively developed. IL2RG KO X-SCID pigs are typically achieved through somatic cell nuclear transfer using genetically modified somatic cells as nuclear donors. Therefore, we investigated the possibility of generating IL2RG KO pigs through electroporation-mediated zygotic gene editing using CRISPR/Cas9. First, we designed and selected an efficient guide RNA targeting the porcine IL2RG gene. Subsequently, we obtained one mosaic mutant male piglet carrying a frame-shift mutation (-19 bp) in IL2RG without off-target events, after the transfer of gene-edited zygotes. We also confirmed that F1 female pigs successfully inherited the -19bp mutation (XKOX). Subsequently, we obtained 30 offspring, including 10 IL2RG-KO male pigs (XKOY), from three F1 XKOX litters. The XKOY pigs showed thymic tissue hypoplasia, and flow cytometry analysis revealed absence of cytotoxic T lymphocytes, helper T, and natural killer cells. Typically, X-linked disorders predominantly affect males. Therefore, female carriers pigs are usually maintained and mated with WT males, generating animal models of the disease. In this study, we successfully generated IL2RG-modified F0 male hemizygous pigs rescued by mosaic genotype via electroporation-mediated zygotic gene editing and subsequently generated IL2RG KO pigs by mating F0 pigs with WT females. Our results demonstrated the feasibility of using zygote electroporation to generate IL2RG-deficient pigs. Furthermore, while obtaining a fertile IL2RG mosaic male pig capable of transmitting the mutation was serendipitous, it presents a potential alternative approach for generating X-linked immunodeficiency models.},
}
@article {pmid41252463,
year = {2025},
author = {Fehrenbach, A and Mitrofanov, A and Alkhnbashi, OS and Backofen, R and Baumdicker, F},
title = {An evolutionary approach to predict the orientation of CRISPR arrays.},
journal = {PLoS computational biology},
volume = {21},
number = {11},
pages = {e1013706},
pmid = {41252463},
issn = {1553-7358},
mesh = {*CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Evolution, Molecular ; Computational Biology/methods ; Archaea/genetics ; Bacteria/genetics ; Models, Genetic ; },
abstract = {CRISPR-Cas is a defense system of bacteria and archaea against phages. Parts of the foreign DNA, called spacers, are incorporated into the CRISPR array which constitutes the immune memory. The orientation of CRISPR arrays is crucial for analyzing and understanding the functionality of CRISPR systems and their targets. Several methods have been developed to identify the orientation of a CRISPR array. To predict the orientation, different methods use different features such as the repeat sequences between the spacers, the location of the leader sequence, the Cas genes, or PAMs. However, those features are often not sufficient to predict the orientation with certainty, or different methods disagree. Remarkably, almost all CRISPR systems have been found to insert spacers in a polarized manner at the leader end of the array. We introduce CRISPR-evOr, a method that leverages the resulting patterns to predict the acquisition orientation for (a group of) CRISPR arrays by reconstructing and comparing the likelihood of their evolutionary history with respect to both possible acquisition orientations. The new method is independent of Cas type, leader existence and location, and transcription orientation. CRISPR-evOr is thus particularly useful for arrays that other CRISPR orientation tools cannot predict confidently and to verify or resolve conflicting predictions from existing tools. CRISPR-evOr currently confidently predicts the orientation of 28.3% of the arrays in the considered subset of CRISPRCasdb, which other tools like CRISPRDirection and CRISPRstrand cannot reliably orient. As our tool leverages evolutionary information we expect this percentage to grow in the future when more closely related arrays will be available. Additionally, CRISPR-evOr provides confident decisions for rare subtypes of CRISPR arrays, where knowledge about repeats and leaders and their orientation is limited.},
}
@article {pmid41252908,
year = {2026},
author = {Zhou, F and Zhao, X and Wang, Y and Huang, Y},
title = {Cooperation of CRISPR/Cas12a and exonuclease III-assisted cascade cycling amplification for ultrasensitive electrochemical detection of ciprofloxacin.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129131},
doi = {10.1016/j.talanta.2025.129131},
pmid = {41252908},
issn = {1873-3573},
mesh = {*Ciprofloxacin/analysis ; *Exodeoxyribonucleases/metabolism/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *CRISPR-Cas Systems ; Limit of Detection ; *Nucleic Acid Amplification Techniques ; *Anti-Bacterial Agents/analysis ; Food Contamination/analysis ; *CRISPR-Associated Proteins/metabolism ; DNA Probes/chemistry/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Antibiotic residues have been a serious public health concern worldwide, while sensitive and reliable detection of antibiotic residues is significant to control antibiotic contamination, ensure food safety, and safeguard human health. Herein, an ultrasensitive electrochemical biosensor is engineered for the detection of ciprofloxacin (CIP) based on the cooperation of CRISPR/Cas12a and exonuclease III (Exo III)-assisted cascade cycling amplification. The presence of CIP induces the conformational change of DNA probes and further triggers Exo III to catalyze the cascade cycling amplification, enabling propagation and ongoing accumulation of DNA fragments which act as the target strands to activate the trans-cleavage activity of CRISPR/Cas12a. Consequently, the activated CRISPR/Cas12a initiates its trans-cleavage activity to swiftly cleave the signal probes on the surface of electrode, bringing about remarkable change of electrochemical signal and eventually realizing the ultrasensitive detection of CIP. The exceptional enzymatic cycle amplification of Exo III incorporated with the superior trans-cleavage activity of CRISPR/Cas12a synergistically facilitates considerable improvement of analytical performance, resulting in a limit of detection as low as 0.022 ng mL[-1]. Benefiting from the effective amplification capacity, high fidelity and programmability of the designed detection system, the biosensor shows good precision and specificity along with robust stability for CIP detection. Moreover, the proposed electrochemical biosensor dispensing with complicated probe construction is label-free and convenient-operated, which contributes to the credible application for CIP detection in real food samples with satisfactory results, indicating promising practicability in food safety monitoring.},
}
@article {pmid41253931,
year = {2025},
author = {Ni, J and Gong, J and Ran, Y and Bai, R and Huang, P and Zheng, Z and Zhou, M and Lan, F and Gu, W and Liu, X},
title = {Optimization of gene knockout approaches and sgRNA selection in hPSCs with inducible Cas9 expression.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40461},
pmid = {41253931},
issn = {2045-2322},
support = {JCYJ20220531091615034//Shenzhen Fundamental Research Program/ ; },
mesh = {Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Knockout Techniques/methods ; Gene Editing/methods ; *Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Associated Protein 9/genetics/metabolism ; Cell Line ; INDEL Mutation ; },
abstract = {CRISPR/Cas9 has been extensively used for gene knockout, enabling functional studies of genetic loss-of-function in human pluripotent stem cells (hPSCs). However, commonly used Cas9 systems usually exhibit limited and variable efficiencies, and identifying single-guide RNAs (sgRNAs) with high cleavage activity-while avoiding ineffective ones-remains a major challenge. To address these issues, we generated a doxycycline-inducible spCas9-expressing hPSCs (hPSCs-iCas9) line and developed it into an optimized gene knockout system through systematically refining critical parameters. Through this optimization, the system achieved stable INDELs (Insertions and Deletions) efficiencies of 82-93% for single-gene knockouts, over 80% for double-genes knockouts, and up to 37.5% homozygous knockout efficiency for large DNA fragment deletions. Moreover, using this optimized system, we precisely evaluated three widely used gRNA scoring algorithms and integrated Western blotting to rapidly identify the ineffective sgRNA. As a result, among the tested algorithms, Benchling provided the most accurate predictions. Notably, we identified an ineffective sgRNA targeting exon 2 of ACE2, where the edited cell pool exhibited 80% INDELs but retained ACE2 protein expression. Together, these findings provide a robust framework for improving gene knockout efficiency in hPSCs and offer practical guidance for reliable sgRNA selection in gene editing experiments.},
}
@article {pmid41254174,
year = {2026},
author = {Thomson, T and Li, G and Strilchuk, A and Cui, H and Wang, B and Li, B},
title = {Harnessing artificial intelligence to advance CRISPR-based genome editing technologies.},
journal = {Nature reviews. Genetics},
volume = {27},
number = {3},
pages = {212-230},
pmid = {41254174},
issn = {1471-0064},
mesh = {*Gene Editing/methods ; Humans ; *Artificial Intelligence ; *CRISPR-Cas Systems/genetics ; Animals ; Machine Learning ; Deep Learning ; },
abstract = {CRISPR-based genome editing technologies, including nuclease-based editing, base editing and prime editing, have revolutionized biological research and modern medicine by enabling precise, programmable modification of the genome and offering new therapeutic strategies for a wide range of genetic diseases. Artificial intelligence (AI), including machine learning and deep learning models, is now further advancing the field by accelerating the optimization of gene editors for diverse targets, guiding the engineering of existing tools and supporting the discovery of novel genome-editing enzymes. In this Review, we summarize key AI methodologies underlying these advances and discuss their recent noteworthy applications to genome editing technologies. We also discuss emerging opportunities, such as AI-powered virtual cell models, which can guide genome editing through target selection or prediction of functional outcomes. Finally, we identify key directions where the integration of AI methods is poised to have a substantial impact going forward.},
}
@article {pmid41254256,
year = {2025},
author = {Wang, Q and Wang, Z and Liu, H and Lv, Y and Zhou, C and Li, C and Fan, H and Ouyang, X and Tao, L and Pei, X and Xie, T},
title = {PAM-readID is a rapid, simple, and accurate PAM determination method for CRISPR-Cas enzymes in mammalian cells.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1601},
pmid = {41254256},
issn = {2399-3642},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; HEK293 Cells ; *CRISPR-Associated Protein 9/metabolism/genetics ; Animals ; DNA Breaks, Double-Stranded ; },
abstract = {One CRISPR-Cas enzyme's recognized protospacer adjacent motif (PAM) profile always shows intrinsic differences between assays with different working environments, such as in vitro, in bacterial cells, or in mammalian cells. The developed methods in mammalian cells are technically complex and not readily amenable to be broadly adopted, highlighting the urgent need for a well-established PAM-determining method in mammalian cells. In this study, we construct a rapid, simple, and accurate method for determining the PAM recognition profile of CRISPR-Cas nucleases in mammalian cells. The developed method is termed PAM-readID, PAM REcognition-profile-determining Achieved by Double-stranded oligodeoxynucleotides Integration in DNA double-stranded breaks. Using PAM-readID, the PAM recognition profiles of SaCas9, SaHyCas9, Nme1Cas9, SpCas9, SpG, SpRY, and AsCas12a in mammalian cells are well produced. An accurate PAM preference for SpCas9 can be identified by analysis with extremely low sequence depth (500 reads). PAM-readID can also define a PAM recognition profile of Cas9 based on Sanger sequencing with a significantly lower cost of time and price than that of high-throughput sequencing. We present an easy-to-use method for comprehensively revealing functional PAM of CRISPR-Cas nucleases in mammalian cells, which can contribute towards accelerating the advancement of exploiting novel genome editing nucleases.},
}
@article {pmid41254269,
year = {2025},
author = {Hernández-Huertas, L and Moreno-Sánchez, I and Crespo-Cuadrado, J and Vargas-Baco, A and da Silva Pescador, G and Zhang, Y and Wen, Z and Florens, L and Santos-Pereira, JM and Bazzini, AA and Moreno-Mateos, MA},
title = {CRISPR-RfxCas13d screening uncovers Bckdk as a post-translational regulator of maternal-to-zygotic transition in teleosts.},
journal = {The EMBO journal},
volume = {44},
number = {23},
pages = {7021-7059},
pmid = {41254269},
issn = {1460-2075},
support = {RyC-2017-23041//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; Margarita Salas Postdoctoral contract//EC | NextGenerationEU (NGEU)/ ; PRE2019-087721//Ministerio de Ciencia e Innovaci&#xf3;n (MCIN)/ ; R01 GM136849/GM/NIGMS NIH HHS/United States ; CNS2022-135564//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; UPO-1380590//Junta de Andaluc&#xed;a (Andalusian Board)/ ; NIHR01GM136849//HHS | National Institutes of Health (NIH)/ ; R21 OD034161/OD/NIH HHS/United States ; PGC2018-097260-B-I00//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; NIH R21OD034161//HHS | National Institutes of Health (NIH)/ ; MDM-2016-0687//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; PID2021-127535NB-I00//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; EMC21_00188//Junta de Andaluc&#xed;a (Andalusian Board)/ ; CEX2020-001088-M//MEC | Agencia Estatal de Investigaci&#xf3;n (AEI)/ ; PREDOC_01569//Junta de Andaluc&#xed;a (Andalusian Board)/ ; PRTR-C17.I1//EC | NextGenerationEU (NGEU)/ ; P20_00866//Junta de Andaluc&#xed;a (Andalusian Board)/ ; },
mesh = {Animals ; *Zebrafish/genetics/embryology/metabolism ; *Zygote/metabolism ; *Zebrafish Proteins/genetics/metabolism ; *Protein Processing, Post-Translational ; Gene Expression Regulation, Developmental ; CRISPR-Cas Systems ; Female ; Phosphorylation ; },
abstract = {The maternal-to-zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screen, in which we targeted mRNAs encoding kinases and phosphatases or proteins regulating them in zebrafish. This screen identified branched-chain ketoacid dehydrogenase kinase, Bckdk, as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA, and expression of a phospho-mimetic mutant of Phf10 rescued the developmental defects observed after bckdk mRNA depletion, as well as restored H3K27ac levels. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation.},
}
@article {pmid41254867,
year = {2026},
author = {Podkowik, M and Tillman, A and Takats, C and Carion, H and Putzel, G and McWilliams, J and See, B and Wang, G and Munoz-Gomez, S and Otto, C and Drlica, K and Marraffini, L and Pironti, A and Hochman, S and Kerantzas, C and Shopsin, B},
title = {CRISPR-Cas-associated SCCmec Variants in Methicillin-resistant Staphylococcus aureus Evade Rapid Diagnostic Detection.},
journal = {The Journal of infectious diseases},
volume = {233},
number = {2},
pages = {e534-e540},
pmid = {41254867},
issn = {1537-6613},
support = {U01CK000590/CC/CDC HHS/United States ; AI137336/NH/NIH HHS/United States ; AI140754/NH/NIH HHS/United States ; T32 HL007974/HL/NHLBI NIH HHS/United States ; //NYULH Antimicrobial-Resistant Pathogens Program/ ; },
mesh = {*Methicillin-Resistant Staphylococcus aureus/genetics/isolation &amp; purification ; Humans ; *Staphylococcal Infections/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Bacterial Proteins/genetics ; Molecular Diagnostic Techniques/methods ; *Genetic Variation ; Rapid Diagnostic Tests ; },
abstract = {Rapid molecular assays guiding treatment of methicillin-resistant Staphylococcus aureus detect SCCmec (Xpert) or the SCCmec-orfX junction (BCID2). Sequence variation in this region can disrupt primer binding, yielding false-negative results. Investigation of a missed bloodstream infection linked escape to a CRISPR-Cas-associated SCCmec variant, leading to identification of 64 variants from 45 patients-2% of 2432 screened. Misdiagnosis was restricted to clonal complex 5, a hospital-associated lineage; 11 of 40 SCCmec/junctions evaded detection by BCID2 or Xpert. Variants had mecA instability and circulated in healthcare settings. Our findings reveal a unique escape mechanism and underscore a threat to diagnostic accuracy.},
}
@article {pmid41254880,
year = {2026},
author = {Zhang, L and Xiang, C and Zheng, N and Kang, H and Kong, L and Huang, W and Peng, H and Lian, Y and Lu, W and Gao, M and Wang, J and Meksem, K and Peng, D and Yu, F and Liu, S},
title = {GmSHMT08-Mediated Soybean Cyst Nematode Resistance Is Negatively Modulated by Two Heat Shock Protein 70s in Soybean.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {3},
pages = {1214-1228},
doi = {10.1111/pce.70296},
pmid = {41254880},
issn = {1365-3040},
support = {//This work was supported by the Joint Fund for S&amp;T Research and Development Programme of Henan, China (grant no. 222301420028), the China Postdoctoral Science Foundation (grant no. 2023M741140), the Postdoctoral Fellowship Programme of CPSF (grant no. GZC20230772), the National Natural Science Foundation of China (grant no. 31972248), and the National Key Research and Development Programme of China (grant no. 2023YFD1400400)./ ; //This work was supported by the National Key Research and Development Programme of China(grant no. 2023YFD1400400), the China Postdoctoral Science Foundation (grant no. 2023M741140), the Postdoctoral Fellowship Programme of CPSF (grant no. GZC20230772), the Joint Fund for S&amp;T Research and Development Programme of Henan, China (grant no. 222301420028), and the National Natural Science Foundation of China (grant no. 31972248)./ ; },
mesh = {*Glycine max/genetics/parasitology/metabolism/immunology ; *Disease Resistance/genetics ; Animals ; *Plant Diseases/parasitology/immunology/genetics ; *HSP70 Heat-Shock Proteins/metabolism/genetics ; *Plant Proteins/metabolism/genetics ; *Tylenchoidea/physiology ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {The soybean GmSHMT08 (Rhg4) is a major gene conferring resistance to soybean cyst nematode (SCN), which is a devastating pathogen in soybean, yet its underlying resistance mechanism remains elusive. Heat shock protein 70 (HSP70) mainly functions in maintaining protein homoeostasis and regulating plant immunity. In this study, CRISPR/Cas9-mediated knockout of two highly similar GmHSP70s [GmHSP70-13a (Glyma.13g254900) and GmHSP70-15a (Glyma.15g059900)] enhanced, whereas overexpressing either of them suppressed, SCN resistance. Both GmHSP70-13a and GmHSP70-15a interacted with the key one-carbon metabolism enzyme GmSHMT08, and promoted GmSHMT08 degradation via 26S proteasome pathway, but neither of them altered GmSHMT08 transcript levels in soybean. Furthermore, both GmHSP70-13a and GmHSP70-15a were strongly induced in susceptible soybeans, while both of them still remained low in resistant soybeans, after SCN infection. Taken together, GmSHMT08-mediated SCN resistance is shown to be negatively modulated by both GmHSP70-13a and GmHSP70-15a. The regulation is facilitated through interacting with and influencing 26S proteasome system-involved degradation of GmSHMT08. This study elucidates a SCN-resistance mechanism of GmSHMT08, expands functions of HSP70s and provides two GmHSP70s gene-editing targets for soybean SCN resistance breeding.},
}
@article {pmid41255389,
year = {2025},
author = {Pizzoccheri, R and Falchi, FA and Alloni, A and Caldarulo, M and Camboni, T and Zambelli, F and Pavesi, G and Visentin, C and Camilloni, C and Sertic, S and Briani, F},
title = {Pathological PNPase variants with altered RNA binding and degradation activity affect the phenotype of bacterial and human cell models.},
journal = {NAR molecular medicine},
volume = {2},
number = {1},
pages = {ugae028},
pmid = {41255389},
issn = {2976-856X},
abstract = {Human PNPase (hPNPase) is an essential RNA exonuclease located in mitochondria, where it contributes to RNA import from the cytoplasm, degradation of mitochondrial RNA and R-loop homeostasis. Biallelic mutations in the hPNPase PNPT1 gene cause different genetic diseases, ranging from hereditary hearing loss to Leigh syndrome. In this work, we used an Escherichia coli model to test the effects of four pathological PNPT1 mutations associated with diseases of different severity. Moreover, we generated a new human cell model by introducing PNPT1 mutations into 293T cells via CRISPR-Cas editing. Notably, the bacterial cells expressing the different mutant alleles exhibited similar phenotypes consistent with hPNPase loss of function. In contrast, the human cell model responded differently to the two mutations tested, with responses correlating with the severity of the respective pathologies. We interpreted the data derived from both models in the light of the in vitro RNA binding and degradation activity of the wild-type and mutated hPNPase variants. We found that all pathogenic mutations tested caused defects in protein assembly and affected the degradation and RNA binding efficiency to varying degrees. However, the severity of the conditions caused by different mutations did not correlate with the catalytic activity of the mutant proteins.},
}
@article {pmid41256794,
year = {2025},
author = {Zhou, X and Ye, C and Xie, M and Wei, Y and Zhao, Y and Liu, X and Ma, J and Qing, J and Chen, Z},
title = {Advances in the application of CRISPR technology in pathogen detection: amplification-based and amplification-free strategies.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1645699},
pmid = {41256794},
issn = {2235-2988},
mesh = {Humans ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Bacteria/genetics/isolation &amp; purification ; *Communicable Diseases/diagnosis ; },
abstract = {CRISPR technology, with its high specificity and programmability, has become an important tool for the detection of human pathogens. The timely and accurate detection of pathogens is crucial for public health. In recent years, significant progress has been made in the application of CRISPR technology for pathogen detection. However, several challenges remain, including detection sensitivity, specificity, and operational convenience. This review summarizes the latest advances in CRISPR technology for pathogen detection, with a focus on the principles and performance comparisons of amplification-based CRISPR (such as those combined with isothermal amplification techniques like RPA and LAMP) and amplification-free CRISPR (such as cascade CRISPR, sensor technologies, and digital droplet CRISPR). It also discusses their applications in pathogen detection. In addition, the article analyzes the advantages and limitations of CRISPR detection technology and looks forward to future development trends, providing a theoretical basis for the optimization of rapid diagnostic techniques for pathogens.},
}
@article {pmid41257388,
year = {2025},
author = {Gervais, NC and Hendriks, A and Shapiro, RS},
title = {Effective Strategies for Translating CRISPR-dCas Systems to Diverse Microbes.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4624-4635},
doi = {10.1021/acssynbio.5c00537},
pmid = {41257388},
issn = {2161-5063},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Bacteria/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-dCas tools have widespread applications for rapidly manipulating and dissecting gene function across the microbial tree of life. However, despite their theoretical suitability for use in a broad range of species, CRISPR-dCas tools that are often initially optimized for use in model cell lines and model organisms still frequently require extensive modifications to enable their application in specific microbial organisms. Here, we review different iterations of CRISPR-dCas in microbes and the application of these techniques. We further discuss common obstacles faced and troubleshooting approaches while developing and applying CRISPR-dCas systems to a microbial organism. Finally, we suggest enhancements that can be made that may help improve the applicability of a CRISPR-dCas tool developed for nonmodel microbial organisms.},
}
@article {pmid41257956,
year = {2025},
author = {Feng, C and Wang, Y and Liu, C and Dong, F and Guo, M and Liu, F and Li, Y and Zhang, L},
title = {Programmable no-nonspecific genetic analytical system via dual-circle-based rolling circle amplification with an efficient CRISPR/Cas12a biosensing strategy.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {40723},
pmid = {41257956},
issn = {2045-2322},
support = {CSTB2024NSCQ-MSX0785//Natural Science Foundation Project of Chongqing, Chongqing Science and Technology Commission/ ; 2023YQB061//Young Ph.D. Talent Incubation Program of the Second Affiliated Hospital of Army Medical University/ ; CQYC20220303658//Chongqing Science and Technology Innovation Leading Talent Support Program/ ; 2024ZDXM012//Chongqing Municipal Science and Health Joint Medical Research Project Key Project/ ; 82472384//National Natural Science Foundation of China/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; *Biosensing Techniques/methods ; Humans ; DNA, Single-Stranded/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; DNA, Circular/genetics ; Limit of Detection ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Ultrasensitive and specific detection of DNA is highly important for early cancer screening and diagnosis. Nucleic acid amplification technology is the most commonly used method for oncogene detection, but nonspecific amplification may occur. We designed a nicking endonuclease (NEase)-mediated exponential rolling circle amplification (RCA) that avoids nonspecific amplification for the CRISPR/Cas12a preamplification process. The purpose was to construct a NEase-assisted target recycling (NATR)-triggered no-nonspecific exponential RCA (NER) reaction integrated with a CRISPR/Cas12a (NATR-NER/Cas12a) system, enabling ultrasensitive and high-fidelity target detection. Innovatively, two circular single-stranded DNAs (ssDNAs) with NEase recognition sites were designed as the preprimer and template for RCA. In the presence of the target, the endonuclease Nt.BstNBI cleaves the circular preprimers into linear fragments, triggering the NER reaction. This generates many short ssDNA fragments, which are recognized by CRISPR/Cas12a and generates a fluorescence signal. The proposed strategy exhibited a wide linear range (10 fM-1 nM), a low detection limit (0.77 fM), and specifically recognized single mismatched DNA. In serum samples, this method exhibited good agreement with real-time quantitative polymerase chain reaction (qPCR) results at lower cost. The developed NATR-NER/Cas12a system provides a promising tool for the early screening and clinical diagnosis of cancer in resource-limited areas.},
}
@article {pmid41259334,
year = {2025},
author = {Siroosi, M and Ghasemi, F and Jabalameli, F and Emaneini, M and Salehi, M and Beigverdi, R and Amoozegar, MA},
title = {Investigating the impact of type I-E CRISPR-Cas systems and acrEI10 on multidrug-resistance in clinical isolates of Klebsiella pneumoniae.},
journal = {PloS one},
volume = {20},
number = {11},
pages = {e0335756},
pmid = {41259334},
issn = {1932-6203},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; Humans ; *Bacterial Proteins/genetics ; Anti-Bacterial Agents/pharmacology ; Klebsiella Infections/microbiology/drug therapy ; Microbial Sensitivity Tests ; beta-Lactamases/genetics ; },
abstract = {Klebsiella pneumoniae is a pathogen related to nosocomial infections with a high rate of antibiotic resistance. The aim of this study was to understand the impact of the presence of CRISPR-Cas systems and an anti-CRISPR gene on multidrug-resistance in K. pneumoniae isolates. The study analyzed 100 clinical K. pneumoniae isolates obtained from a hospital setting. The investigation involved determining antibiotic resistance profiles, including ESBL production, identifying specific carbapenemase and aminoglycoside resistance genes, detecting the presence of CRISPR-Cas systems, identifying the anti-CRISPR gene acrEI10, and sequencing CRISPR arrays. Correlation analysis between resistance genes and CRISPR-Cas systems was also performed. All isolates in this study were determined to be multidrug-resistant (MDR), with resistance rates exceeding 70% for the majority of antibiotics tested. The most prevalent carbapenemase genes were blaOXA-48 and blaNDM, while aminoglycoside resistance was primarily mediated by aac(6´)-Ia and ant(2")-Ia. Only 7% of the isolates harbored CRISPR-Cas systems and the gene acrEI10, which encodes an anti-CRISPR protein, was detected in one of the CRISPR-Cas positive isolates. Sequencing of the CRISPR array from this isolate showed similarities between the spacers and sequences found in plasmids and K. pneumoniae chromosome. No strong correlation was identified between the antibiotic resistance genes and CRISPR-Cas systems. Findings from this study suggest a complex interplay between these factors in MDR isolates of K. pneumoniae and show that further investigations are needed to have a better understanding of the mechanisms related to the coexistence of these elements and their impact on dissemination of antibiotic resistance genes.},
}
@article {pmid41259745,
year = {2026},
author = {Liu, L and Glaser, A and Isiaku, AI and Fairfax, K and Casolari, DA and Ristovski, A and Murphy, V and van Twest, S and Henrikus, SS and Heraud-Farlow, J and Granger, E and Novakovic, S and Monks O'Byrne, SF and Tsui, V and Conyers, R and Gonda, TJ and Crismani, W and D'Andrea, RJ and Heierhorst, J and Deans, AJ},
title = {Fancl-mutant mice reveal central role of monoubiquitination in Fanconi anemia and a model for therapeutic gene editing.},
journal = {Blood advances},
volume = {10},
number = {3},
pages = {821-836},
pmid = {41259745},
issn = {2473-9537},
mesh = {Animals ; *Fanconi Anemia/genetics/therapy/metabolism/pathology ; Mice ; *Gene Editing/methods ; *Ubiquitination ; Disease Models, Animal ; *Fanconi Anemia Complementation Group L Protein/genetics/metabolism ; *Mutation ; Fanconi Anemia Complementation Group D2 Protein/metabolism ; Humans ; CRISPR-Cas Systems ; },
abstract = {Fanconi anemia (FA) is a rare genetic disorder causing the progressive loss of hematopoietic stem cells (HSCs) and bone marrow failure. Most cases result from deficient monoubiquitination of FANCD2 by the FA core complex. However, given that additional functions for the complex have been proposed, it remains unclear whether loss of FANCD2 monoubiquitination is the sole cause of all FA phenotypes. Here, we generated a murine allele (FanclTATΔ) that mimics an allele from a patient with FA. This 3-bp deletion removes a catalytic cysteine in the E3 RING ligase domain of the FANCL subunit. Biochemical assays show that the mutant FA core complex retains structural integrity but lacks FANCD2 monoubiquitination activity. Homozygous FanclTATΔ/TATΔ mice phenocopy classical human FA features, including infertility, craniofacial anomalies, DNA damage hypersensitivity, and progressive HSC loss with age. Correcting the mutation using CRISPR-Cas9 or prime editing technology restores FANCD2 monoubiquitination and normal DNA damage resistance in myeloid cells. Collectively, our mouse model demonstrates that loss of RING E3 ubiquitin ligase activity of the FA core complex explains developmental defects and hematopoietic failure in FA and provides a new animal model for testing potentially therapeutic gene editing.},
}
@article {pmid41259748,
year = {2025},
author = {Zheng, L and Zhou, X and Zhang, Y and Wang, W and Chen, C and Lin, X and Zheng, Y and Lou, Y},
title = {Rapid Bacterial Identification and Antimicrobial Susceptibility Testing Directly from Urine Samples via an Asymmetric Polymerase Chain Reaction-Cas12a Platform.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26466-26474},
doi = {10.1021/acs.analchem.5c04410},
pmid = {41259748},
issn = {1520-6882},
mesh = {Humans ; Microbial Sensitivity Tests ; *Bacteria/drug effects/isolation &amp; purification/genetics ; *Anti-Bacterial Agents/pharmacology ; *Polymerase Chain Reaction/methods ; CRISPR-Cas Systems ; *CRISPR-Associated Proteins/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; Bacterial Proteins ; },
abstract = {Antimicrobial resistance poses a critical global health challenge, largely due to the prolonged turnaround times of conventional pathogen identification (ID) and antimicrobial susceptibility testing (AST). Here, we present a clinically validated diagnostic platform integrating asymmetric polymerase chain reaction (aPCR) with CRISPR/Cas12a for direct bacterial ID and phenotypic AST from urine samples. Unlike traditional multiplex PCR requiring complex primer sets, our platform employs a singleplex aPCR targeting the V3-V4 region of 16S rDNA to generate single-stranded and double-stranded DNA. This design enables protospacer adjacent motif-free activation of Cas12a when required via the ssDNA fraction generated by aPCR, facilitating species-level multiplex detection of six common uropathogens at 10[3] CFU/mL via programmable CRISPR/Cas12a crRNAs. Phenotypic AST is accomplished within 60 min by quantifying nucleic acid changes following antibiotic exposure, allowing accurate discrimination between susceptible and resistant strains. When validated with 86 clinical urine samples, the aPCR-Cas12a platform achieved complete concordance with culture-based identification among the 45 samples carrying target pathogens and demonstrated high accuracy for AST, confirming its reliability for direct pathogen detection and susceptibility assessment from urine. The complete workflow requires only 5.5 h, significantly reducing the diagnostic time compared to standard methods (>48 h). This rapid, cost-effective, and scalable platform offers a promising solution for infection diagnosis and antimicrobial stewardship, with strong potential for integration into routine clinical microbiology and point-of-care settings.},
}
@article {pmid41260247,
year = {2026},
author = {Han, F and Lu, Y and Zhang, Y and Ma, X and Tong, C and Wei, J and Guo, Y and Liu, C and Wu, Z and Han, W and Wang, Y},
title = {Intrinsic NPRL2 and NPRL3 regulate the sensitivity of B-cell malignancies to CAR-T cell therapy.},
journal = {Journal of genetics and genomics = Yi chuan xue bao},
volume = {53},
number = {4},
pages = {655-666},
doi = {10.1016/j.jgg.2025.11.007},
pmid = {41260247},
issn = {1673-8527},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; Animals ; Mice ; *Receptors, Chimeric Antigen/genetics/immunology ; Cell Line, Tumor ; *Lymphoma, B-Cell/therapy/genetics/immunology/pathology ; CRISPR-Cas Systems ; Mechanistic Target of Rapamycin Complex 1/metabolism ; Xenograft Model Antitumor Assays ; },
abstract = {Although chimeric antigen receptor (CAR) T-cell therapy has markedly improved outcomes for many patients with B-cell malignancies, a subset experiences limited benefit due to primary or secondary resistance. Building on CRISPR/Cas9 genome-wide screening in malignant B-cells, we identify NPRL2 and NPRL3 as key regulators of tumor sensitivity to CAR-T cytotoxicity. This study aims to investigate the impact and mechanisms of tumor-intrinsic NPRL2 and NPRL3 on the efficacy of CAR-T cell therapy. In a tandem CD19/20 CAR-T clinical trial for relapsed/refractory (R/R) B-cell lymphoma (NCT03097770), high tumor NPRL2 or NPRL3 expression correlates with therapeutic resistance in patients. Consistently, in vitro experiments confirm that tumor cells overexpressing NPRL2/NPRL3 exhibit resistance to CAR-T-mediated cytolysis. Mechanistically, NPRL2/NPRL3 suppresses mTORC1 activity within tumor cells, negatively regulating the conjugation between tumor cells and CAR-T cells, consequently impairing CAR-T cell activation and cytotoxic function, ultimately facilitating immune escape. As therapeutic strategies, either genetic ablation of tumor-intrinsic NPRL2/NPRL3 or pharmacological activation of mTORC1 enhances CAR-T cell activation, cytotoxic degranulation, and tumor clearance both in vitro and in vivo. In conclusion, targeting tumor NPRL2/NPRL3 or directly activating mTOR represents a promising combinational strategy to potentiate CAR-T efficacy and overcome resistance in clinical practice.},
}
@article {pmid41260396,
year = {2026},
author = {Zhong, Z and Li, G and Liang, G and Ren, T and Teng, C and Xiong, J and Ji, G and Zheng, M and Pan, Y and Qin, Y and Ouyang, K and Yin, Y and Chen, Y and Huang, W and Wei, Z},
title = {Establishment of a nucleic acid detection method for foot-and-mouth disease virus serotype O utilizing RPA-CRISPR/Cas12a technology.},
journal = {Journal of virological methods},
volume = {340},
number = {},
pages = {115304},
doi = {10.1016/j.jviromet.2025.115304},
pmid = {41260396},
issn = {1879-0984},
mesh = {*Foot-and-Mouth Disease Virus/genetics/isolation &amp; purification/classification ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems ; *Foot-and-Mouth Disease/diagnosis/virology ; Animals ; Serogroup ; Recombinases/metabolism/genetics ; DNA Primers/genetics ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {This study aimed to develop a rapid and visually interpretable nucleic acid detection assay for Foot-and-Mouth Disease Virus serotype O (FMDV-O) by integrating recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Specific RPA primers and CRISPR RNA (crRNA) sequences were designed and optimized based on the conserved 3D gene region of FMDV-O. An assay combining RPA pre-amplification with Cas12a-mediated cleavage was subsequently established. The sensitivity and specificity of the RPA-CRISPR/Cas12a method were systematically evaluated, and its diagnostic utility was further assessed using clinical samples. The results demonstrated that the primer set RPA-F1/R1 paired with crRNA1 constituted the optimal combination, with an ideal reaction system comprising 50 nM Cas12a protein and 200 nM crRNA. This system exhibited a detection limit of 2.60 × 10[2] copies/μL for target plasmid DNA following a 20-minute incubation at 37°C. Specificity analysis confirmed positive detection exclusively for FMDV-O plasmids, with no cross-reactivity observed with other tested pathogens. When applied to clinical samples, the proposed method demonstrated a superior detection rate relative to conventional PCR. In conclusion, a novel diagnostic platform for FMDV-O was successfully developed based on RPA-CRISPR/Cas12a. This method is characterized by its rapidity, operational simplicity, high sensitivity, and excellent specificity, holding significant promise for application in clinical diagnostics, epidemiological surveillance, and field-based testing.},
}
@article {pmid41260510,
year = {2025},
author = {Jiang, W and Yang, A and Ma, J and Lv, D and Liu, M and Wang, C and Chen, S and Fang, H and Chu, Y and He, Z and Li, W and Liu, Y and Zhao, Y and Zhou, Z and Long, G and Jiang, H},
title = {Genetic determinants of efficacy of antiviral drugs revealed by genome-wide CRISPR screens.},
journal = {Antiviral research},
volume = {244},
number = {},
pages = {106309},
doi = {10.1016/j.antiviral.2025.106309},
pmid = {41260510},
issn = {1872-9096},
mesh = {*Antiviral Agents/pharmacology ; Animals ; Humans ; *CRISPR-Cas Systems ; Mice ; Virus Replication/drug effects ; Cell Line, Tumor ; Ribavirin/pharmacology ; Adenosine Monophosphate/analogs &amp; derivatives/pharmacology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Nucleoside and nucleobase analog antiviral drugs are pivotal in antiviral therapy, but comprehensive methods to understand their cellular response mechanisms and genetic regulators are still lacking. Here, we show that Eμ-Myc; Arf[-/-] mouse lymphoma cells, which are highly apoptosis-prone, enabled genome-wide CRISPR-Cas9 screening on such drugs to identify genes that modulate their efficacy. Using retroviral sgRNA libraries and MAGeCK analysis, we uncovered key regulators of drug transport, activation, and inactivation for these drugs. For ribavirin, adenosine kinase (ADK) and adenylsuccinate synthase (ADSS) were critical for nucleotide metabolism and bioactivation. Remdesivir uptake and activation depended on the transporter SLC29A3 and phosphoamidase HINT1, whereas favipiravir resistance was linked to NT5C2-mediated dephosphorylation. Viral replication assays in Huh7 cells validated that knockout of SLC29A3, HINT1, or NT5C2 significantly altered antiviral efficacy. This study delineates the genetic network governing nucleotide analog response, providing mechanistic insights and potential biomarkers for personalized antiviral therapy.},
}
@article {pmid41260544,
year = {2026},
author = {Gupta, A and Beg, MA and Badhwar, S and Srivastava, S and Srivastava, R and Puri, N and Saxena, A and Abdin, MZ and Selvapandiyan, A},
title = {Nucleoside diphosphate kinase (LdNDK2): A metacyclogenesis-regulating kinase essential for Leishmania parasite survival within eukaryotic host cells.},
journal = {Microbial pathogenesis},
volume = {210},
number = {},
pages = {108192},
doi = {10.1016/j.micpath.2025.108192},
pmid = {41260544},
issn = {1096-1208},
mesh = {*Nucleoside-Diphosphate Kinase/metabolism/genetics ; *Leishmania donovani/enzymology/growth &amp; development/genetics ; Humans ; Protozoan Proteins/metabolism/genetics ; THP-1 Cells ; Adenosine Triphosphate/metabolism ; CRISPR-Cas Systems ; *Eukaryotic Cells/parasitology ; Cloning, Molecular ; Recombinant Proteins/metabolism/genetics ; },
abstract = {Nucleoside diphosphate kinase (NDK) transfers phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphates (NDPs) via a ping-pong mechanism, benefiting both prokaryotes and eukaryotes. In Leishmania donovani, we identified a putative NDK2 (LdNDK2), hypothesized to play a crucial role in nucleotide metabolism and cellular energy regulation. To investigate its function and enzymatic properties, we cloned, expressed, and purified recombinant LdNDK2, confirming enzymatic activity via the ADP-Glo assay. The secondary structure and thermal stability were analyzed using circular dichroism spectroscopy, while intrinsic tryptophan fluorescence assays revealed that ATP's gamma phosphate is first transferred to rLdNDK2, forming an intermediate phospho-enzyme complex. We further evaluated the role of rLdNDK2 in ATP-mediated cytolysis of LPS-activated THP-1 cells by measuring lactate dehydrogenase release. Using the CRISPR-Cas9 method, we fluorescently tagged LdNDK2, localizing it to distinct regions of the endomembrane system. In vitro growth studies of LdNDK2 deleted (LdNDK2[-/-]) procyclic promastigotes, through CRISPR-Cas9, revealed that these mutants exhibit a longer flagellum, a reduced cell body, an altered cell division cycle, and increased growth compared to wild-type parasites. LdNDK2[-/-] parasites were arrested at the metacyclic stage, contrasting with continuous differentiation in parental counterparts. Morphological alterations and growth defects were confirmed by re-expressing LdNDK2 in knockouts. Infection with LdNDK2[-/-] parasites significantly reduced the viability and recovery of mast cells and THP-1 macrophages and flow cytometry indicated increased apoptosis in host cells, likely due to parasites inability to utilize NDK enzyme for preventing cytolysis. Our findings highlight LdNDK2's crucial role in metacyclogenesis and immune evasion, underscoring its therapeutic potential for leishmaniasis.},
}
@article {pmid41261109,
year = {2025},
author = {Zhang, T and Zheng, Y and Sheng, M and Wang, Q and Jin, Z and Li, Z and Chen, W and Huang, J and Yang, X},
title = {Electrochemical Biosensing Platform Based on MOF Carrier Signal Probes and CRISPR/Cas12a Cleavage for Sensitive Detection of MicroRNAs.},
journal = {Analytical chemistry},
volume = {97},
number = {47},
pages = {26265-26273},
doi = {10.1021/acs.analchem.5c05821},
pmid = {41261109},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis ; *Metal-Organic Frameworks/chemistry ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; Humans ; *CRISPR-Cas Systems ; DNA, Single-Stranded/chemistry ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism ; Methylene Blue/chemistry ; Limit of Detection ; Bacterial Proteins ; },
abstract = {Dysregulation of microRNA (miRNA) expression is associated with a variety of human diseases, including cancers, and the development of highly sensitive miRNA detection is important for the early diagnosis of cancer. In this study, we developed an electrochemical biosensing platform for miRNA detection through the integration of a DNA-gated metal-organic framework (MOF) signal probe, duplex-specific nuclease (DSN)-assisted signal amplification, and a CRISPR/Cas12a system. The zirconium-based MOF UiO-66-NH2 was engineered as a nanocarrier for methylene blue (MB) entrapment, and single-stranded DNA (ssDNA) was capped on the MOF surface as a "gatekeeper" that allowed the controlled release of MB molecules, resulting in a DNA-gated MOF electrochemical signal probe. In the presence of target miRNA, target-initiated DSN cyclic amplification and digestion of the CRISPR/Cas12a initiator DNA sequence blocked the collateral cleavage activity of Cas12a toward ssDNA, thus preventing the release of MB and generating a high electrochemical signal. Conversely, the CRISPR/Cas12a system would be activated to cleave ssDNA in the absence of the target, leading to the release of a signal molecule and a low response. With the usage of the proposed biosensing strategy, sensitive detection of miRNA let-7a, a biomarker associated with nonsmall cell lung cancer, has been achieved. Therefore, this work expands the application scope of a MOF as a nanocarrier to prepare electrochemical signal probes and provides a valuable biosensing method for clinical diagnosis.},
}
@article {pmid41261131,
year = {2025},
author = {Pierce, SE and Erwood, S and Oye, K and An, M and Krasnow, N and Zhang, E and Raguram, A and Seelig, D and Osborn, MJ and Liu, DR},
title = {Prime editing-installed suppressor tRNAs for disease-agnostic genome editing.},
journal = {Nature},
volume = {648},
number = {8092},
pages = {191-202},
pmid = {41261131},
issn = {1476-4687},
support = {P01 CA065493/CA/NCI NIH HHS/United States ; R35 GM118062/GM/NIGMS NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; Animals ; *RNA, Transfer/genetics/metabolism ; Mice ; Codon, Nonsense/genetics ; Cystic Fibrosis/genetics/therapy ; Codon, Terminator/genetics ; Female ; Male ; CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Cystic Fibrosis Transmembrane Conductance Regulator/genetics ; HEK293 Cells ; },
abstract = {Precise genome-editing technologies such as base editing[1,2] and prime editing[3] can correct most pathogenic gene variants, but their widespread clinical application is impeded by the need to develop new therapeutic agents for each mutation. For diseases that are caused by premature stop codons, suppressor tRNAs (sup-tRNAs) offer a more general strategy. Existing approaches to use sup-tRNAs therapeutically, however, require lifelong administration[4,5] or show modest potency, necessitating potentially toxic overexpression. Here we present prime editing-mediated readthrough of premature termination codons (PERT), a strategy to rescue nonsense mutations in a disease-agnostic manner by using prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA. Iterative screening of thousands of variants of all 418 human tRNAs identified tRNAs with the strongest sup-tRNA potential. We optimized prime editing agents to install an engineered sup-tRNA at a single genomic locus without overexpression and observed efficient readthrough of premature termination codons and protein rescue in human cell models of Batten disease, Tay-Sachs disease and cystic fibrosis. In vivo delivery of a single prime editor that converts an endogenous mouse tRNA into a sup-tRNA extensively rescued disease pathology in a model of Hurler syndrome. PERT did not induce detected readthrough of natural stop codons or cause significant transcriptomic or proteomic changes. Our findings suggest the potential of disease-agnostic therapeutic genome-editing approaches that require only a single composition of matter to treat diverse genetic diseases.},
}
@article {pmid41261174,
year = {2025},
author = {Seker-Polat, F and Rogozinska, M and Ban, Y and Abdula, F and Buyukcelebi, K and Xie, P and Fan, J and Abbaszadeh, N and Kingham, Y and Paylakhi, SZ and Zhang, B and Adli, M},
title = {Druggable genome CRISPR screening identifies the KEAP1/NRF2 axis as a mediator of PD-L1 expression.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1610},
pmid = {41261174},
issn = {2399-3642},
support = {R01 CA267544/CA/NCI NIH HHS/United States ; U54 CA268084/CA/NCI NIH HHS/United States ; R01CA267544//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; U54CA268084//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {*B7-H1 Antigen/genetics/metabolism ; Humans ; *Kelch-Like ECH-Associated Protein 1/metabolism/genetics ; *NF-E2-Related Factor 2/metabolism/genetics ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Animals ; Female ; Mice ; CRISPR-Cas Systems ; Pancreatic Neoplasms/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Signal Transduction ; Ovarian Neoplasms/genetics ; },
abstract = {Cancer cells rapidly induce PD-L1 expression in response to inflammatory cytokines such as IFNγ from cytotoxic T cells. Increased surface PD-L1 is a primary mechanism of cancer cells evading cytotoxic T-cell-mediated immune clearance. Identifying how cancer cells increase PD-L1 expression may yield clinically relevant immune checkpoint regulators. However, the key regulators and molecular mechanisms mediating rapid PD-L1 induction are yet to be understood entirely. To identify targetable mechanisms controlling cytokine-induced PD-L1 expression, we performed functional CRISPR gene KO screening with a custom-designed sgRNA library that targets "druggable" genes. We performed the screening in 6 different cancer lines: 3 ovarian (OVCAR4, CaOV3, and SKOV3) and three pancreatic cancer (MiaPaca2, ASPC1 and KP4) cell lines. The screening recovered the known regulators of PD-L1 expression and uncovered several novel regulators of PD-L1 that control its expression in all cell lines or in a cancer-type-specific fashion. For example, while genetic or pharmacological depletion of CSNK1A1 results in reduced PD-L1 expression in ovarian cancer cells, CDK1 depletion modulates PD-L1 in pancreatic cancer cell lines. Significantly, we discovered that KEAP1 depletion or pharmacological inhibition diminishes PD-L1 in all cell lines tested (n = 6). Mechanistically, KEAP1 depletion-mediated reduced PD-L1 is due to transcriptional repression of the PD-L1 gene by NRF2 activation. As such, depletion of NRF2 restores PD-L1 expression, while its overexpression leads to diminished PD-L1 expression. Supporting this, pharmacological NRF2 activation resulted in significant antitumor immunity with increased cytotoxic effector T cell infiltration and reduced exhausted T cells, resulting in smaller xenografted tumors. These findings establish the KEAP1/NRF2 axis as a novel and potentially druggable mechanism of IFNγ-meditated PD-L1 expression in cancer cells.},
}
@article {pmid41261774,
year = {2026},
author = {Sun, W and Zhu, Y and Zhang, X and Meng, X and Xia, K and Zhang, X and Han, X and Pan, W and Guo, J and Li, J and Zhang, H},
title = {A mortise-tenon joint system facilitates precise targeted DNA insertion and replacement in rice.},
journal = {Molecular plant},
volume = {19},
number = {1},
pages = {36-47},
doi = {10.1016/j.molp.2025.11.006},
pmid = {41261774},
issn = {1752-9867},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Mutagenesis, Insertional/methods ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; DNA, Plant/genetics ; },
abstract = {Precise and scarless DNA insertion and replacement represent two of the major challenges in plant genome editing. Numerous tools have been developed, including homology-directed repair-dependent tools and prime-editing-mediated systems. However, none has fully resolved these challenges. In this study, we develop the "mortise-tenon joint system" (MT), a novel strategy that enables precise and efficient targeted DNA insertion and replacement. By leveraging the APOBEC-Cas9-uracil DNA glycosylase/(apurinic or apyrimidinic site) lyase within our previously reported APOBEC-Cas9 fusion-induced deletion system, which performs single-strand cleavage on the non-target strand and double-strand cleavage on the target strand, we generated a unique "mortise" structure, consisting of double-strand breaks with single or double non-complementary 5' overhangs. We further designed "tenons," double-stranded DNA donors containing 5' sticky ends precisely matching the 5' overhangs of the mortises. The end-capture interaction between mortises and tenons facilitates precise targeted insertion and replacement, achieving frequencies of 16.30%-59.47% across seven tested targets using 21-85 bp donor inserts in rice. If long DNA donors with sticky ends complementary to the mortise structure can be generated, the MT system may enable highly precise targeted insertion and replacement of large DNA fragments.},
}
@article {pmid41261856,
year = {2025},
author = {Xiao, Y and Zhao, R and Bao, Y and Lu, B and Jiang, Y and Tang, Y and Li, B},
title = {Cas12a-assisted split crRNA complex for analysis and detection of diverse entities.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41261856},
issn = {1362-4962},
support = {SKL202402017//The Science and Technology Development Plan Project of Jilin Province/ ; SKL202302030//The Science and Technology Development Plan Project of Jilin Province/ ; 20240101005JJ//Jilin Province Science Fund for Distinguished Young Scholars/ ; 23GZZ03//Major Project of Changchun State Key Laboratory/ ; 20230203193SF//Key R&amp;D Program of Jilin Province/ ; 22525405//National Nature Science Foundation of China/ ; 22504138//National Nature Science Foundation of China/ ; 22474135//National Nature Science Foundation of China/ ; 22374142//National Nature Science Foundation of China/ ; },
mesh = {*CRISPR-Associated Proteins/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *CRISPR-Cas Systems ; MicroRNAs/genetics/analysis ; DNA, Single-Stranded/genetics ; DNA/genetics ; *Bacterial Proteins/genetics/metabolism ; *Biosensing Techniques/methods ; },
abstract = {The Cas12a-crRNA system possesses inherent sequence-specific recognition of double-stranded DNA/single-stranded DNA (dsDNA/ssDNA) coupled with trans-cleavage activity toward ssDNA, making it a powerful tool for nucleic acid diagnostics. However, its application beyond nucleic acid targets remains challenging, limiting its potential as a universal detection platform. In this study, we systematically explore the key parameters governing the activation of a Cas12a-split crRNA system and established a comprehensive set of design guidelines. Building on these findings, we developed CASCADE (Cas12a-Assisted Split crRNA Complex for Analysis and Detection of Diverse Entities), an adaptable detection platform that extends Cas12a's application beyond nucleic acids. Using microRNA as a model, we validated the system's sensitivity, specificity, and mismatch discrimination capability. Additionally, we successfully demonstrated its capability for non-nucleic acid target detection by detecting tobramycin, kanamycin, biotin, and tetracycline repressor protein, confirming its sensitivity and specificity. Finally, by integrating a lateral flow assay (LFA), we enhanced the portability of CASCADE, enabling user-friendly, on-site detection. This work expands the application scope of the Cas12a system and offers a promising strategy for point-of-care diagnostics or environmental monitoring.},
}
@article {pmid41262251,
year = {2025},
author = {Stelcer, E and Wozniak, A and Magner, D and Zeyland, J},
title = {Genetically modified pigs with α1,3-galactosyltransferase knockout and beyond: a comprehensive review of xenotransplantation strategies.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1663246},
pmid = {41262251},
issn = {1664-3224},
mesh = {Animals ; *Transplantation, Heterologous/methods ; *Galactosyltransferases/genetics/deficiency ; *Animals, Genetically Modified ; Swine ; *Graft Rejection/immunology/genetics/prevention &amp; control ; Gene Knockout Techniques ; Humans ; Heterografts/immunology ; },
abstract = {Xenotransplantation holds promise to eliminate the shortage of organs intended for humans in need. Pigs constitute the most suitable organ xenograft donor due to the fact that their organ anatomy physiological metabolism and immune system resemble those of humans. However, swine organs rapidly cause hyperacute rejection (HAR) and acute humoral xenograft rejection (AHXR) after transplantation. HAR and AHXR are caused by the presence of xenoreactive natural immunoglobulins directed toward a galactose alpha1-3-galactose (alpha-Gal) epitope on porcine vascular endothelium. In order to suppress both types of rejection, pigs with alpha1,3-galactosyltransferase gene knockout (GT-KO) and other genetic modifications (like simultaneous expression of the human complementary regulatory proteins) are intensively investigated. This review highlights the usefulness of GT-KO pig - derived organs such as kidney, heart, corneal, and lung in xenotransplantation. To obtain transgenic pigs researchers can use several techniques based on pronuclear and cytoplasmic microinjection, somatic cell nuclear transfer (SCNT), viral transduction of DNA and DNA transposable element -based technology, site specific nucleases and modifications of the CRISPR/Cas bacterial immune system. Some additional strategies like targeted immunosuppression or tolerance induction of B and T cells will be essential for sustained survival of xenografts. Although xenotransplantation with the use of pigs is a very rapidly evolving field, more research is needed to create perfectly compatible with the human immune system organs.},
}
@article {pmid41262327,
year = {2026},
author = {Jia, Y and Horvath, K and Rananaware, SR and Jain, PK and Sampath, J},
title = {Exploring the temperature stability of CRISPR-Cas12b using molecular dynamics simulations.},
journal = {Molecular systems design &amp; engineering},
volume = {11},
number = {2},
pages = {195-207},
pmid = {41262327},
issn = {2058-9689},
support = {R35 GM147788/GM/NIGMS NIH HHS/United States ; },
abstract = {The thermal stability of CRISPR-Cas nucleases is a critical factor for their successful application in 'one-pot' diagnostic assays that utilize high-temperature isothermal amplification. To understand the atomistic mechanism of stabilization in a previously engineered variant of the thermostable BrCas12b protein, we performed all-atom molecular dynamics (MD) simulations on the wild-type and mutant forms of apo BrCas12b. High-temperature simulations reveal a small structural change along with greater flexibility in the PAM-interacting domain of the mutant BrCas12b, with marginal structural and flexibility changes in the other mutated domains. Comparative essential dynamics analysis between the wild-type and mutant BrCas12b at both ambient and elevated temperatures provides insights into the stabilizing effects of the mutations. Our findings offer comprehensive insights into the important protein motions induced by these mutations. These results provide insights into thermal stability mechanisms in BrCas12b that may inform the future design of CRISPR-based tools.},
}
@article {pmid41262458,
year = {2025},
author = {Gao, Z and Gao, Y and Wang, S and Li, X and Cao, W and Deng, W and Yao, L and Wei, X and Zhang, Z and Wang, S and Zhang, Y and Li, M and Xie, Y},
title = {Application progress and biosafety challenges of gene editing and synthetic biotechnology in diagnosis, treatment and prevention of infectious diseases.},
journal = {Biosafety and health},
volume = {7},
number = {5},
pages = {312-322},
pmid = {41262458},
issn = {2590-0536},
abstract = {Global infectious disease prevention faces escalating challenges due to the continual emergence of novel pathogens and rapid viral mutations. Synthetic biology has revolutionized this field by enabling precise diagnostics, innovative vaccine platforms, and targeted therapeutics, yet it simultaneously raises concerns regarding dual-use potential, biosafety, and ethical governance. This systematic review (2015-2025, PubMed, Web of Science, Scopus) focuses on CRISPR-based diagnostics, synthetic vaccines, and engineered probiotics. CRISPR/Cas systems such as DETECTR (Cas12a) and SHERLOCK (Cas13a) demonstrate high sensitivity and rapid pathogen detection (e.g., SARS-CoV-2, Ebola), but their misuse could enhance pathogen virulence or enable bioweapon development. mRNA and viral vector vaccines offer flexible and rapid responses to emerging infections but encounter limitations in molecular stability, delivery system toxicity, and ecological safety. Engineered probiotics, designed as "living therapeutics," can detect pathogens and modulate immune responses, yet pose potential risks of horizontal gene transfer and host-specific variability. Overall, while synthetic biology provides transformative tools for infectious disease control, it necessitates robust global regulatory frameworks, standardized biosafety practices, and ethical oversight to ensure responsible and sustainable application.},
}
@article {pmid41263111,
year = {2026},
author = {Feng, X and Li, Y and Zheng, J and Chen, X and Yang, S and Chen, Y and Li, SC},
title = {MicrobialScope: an integrated genomic resource with rich annotations across bacteria, archaea, fungi, and viruses.},
journal = {Nucleic acids research},
volume = {54},
number = {D1},
pages = {D842-D851},
pmid = {41263111},
issn = {1362-4962},
support = {C2004-23Y//Young Collaborative Research/ ; JCYJ20220818101201004//Shenzhen Science and Technology Program/ ; 32300527//National Natural Science Foundation of China/ ; 32470695//National Natural Science Foundation of China/ ; 2022A1515110784//Guangdong Basic and Applied Basic Research Foundation/ ; 2023B0303040004//Key-Area Research and Development Program of Guangdong Province/ ; TC2024JC43//Basic Research Programs of Taicang, 2024/ ; //Shenzhen-Hong Kong Institute of Brain Science/ ; //SIAT-HKUST Joint Laboratory of Brain Science/ ; },
mesh = {*Molecular Sequence Annotation ; Archaea/genetics ; *Databases, Genetic ; Fungi/genetics ; Genome, Viral ; Genome, Archaeal ; Bacteria/genetics ; Viruses/genetics ; *Genomics/methods ; *Software ; Genome, Fungal ; Genome, Bacterial ; Computational Biology/methods ; Metagenome ; },
abstract = {Microorganisms, including bacteria, archaea, fungi, and viruses, are the most taxonomically diverse and ecologically dominant life forms on Earth, playing critical roles in ecosystems, human health, and industrial applications. While existing microbial databases such as BV-BRC and IMG archive both monoisolate and metagenome-assembled genomes (MAGs) across domains, challenges remain in standardized, multi-level annotations and interactive tools for all microbial groups. Here, we present MicrobialScope (https://microbial.deepomics.org/), a comprehensive microbial genomic platform that integrates large-scale genome collections, multilevel annotations, and interactive visualizations. MicrobialScope harbors 2 411 503 bacterial, 24 472 archaeal, 20 203 fungal, and 188 267 viral genomes derived from both monoisolate assemblies and MAGs. Integrating 15 state-of-the-art bioinformatics tools and 10 specialized databases, MicrobialScope provides extensive annotations encompassing basic genomic features, genomic element prediction (e.g., genes, tRNAs, tmRNAs, CRISPR-Cas and anti-CRISPR elements, secondary metabolite biosynthetic clusters, signal peptides, and transmembrane proteins), and functional and structural annotations. This includes 1 072 114 935 proteins with diverse annotations, 24 640 186 tRNAs and tmRNAs, 140 888 CRISPR-Cas systems, 173 256 anti-CRISPR elements, 105 121 secondary metabolite biosynthetic clusters, 13 235 096 signal peptides, and 50 811 729 transmembrane proteins. In addition, MicrobialScope offers unrestricted access to all data resources, interactive visualization tools, and built-in online analytical modules for intuitive exploration and comparative analysis. With its extensive genome collection, comprehensive annotations, and user-friendly interface, MicrobialScope serves as a scalable platform to advance genome research across diverse microbial domains.},
}
@article {pmid41264498,
year = {2025},
author = {Guo, Y and Guo, W and Wu, Z and Xu, H and Zhang, X and Zou, X and Sun, Z},
title = {A Microfluidic Chip-Based Electrochemical Biosensor Coupled with CRISPR/Cas12a for Simultaneous Detection of Foodborne Pathogens.},
journal = {Analytical chemistry},
volume = {97},
number = {49},
pages = {27266-27275},
doi = {10.1021/acs.analchem.5c05232},
pmid = {41264498},
issn = {1520-6882},
mesh = {*Biosensing Techniques/methods ; *Electrochemical Techniques/instrumentation ; *Staphylococcus aureus/isolation &amp; purification/genetics ; Gold/chemistry ; *Salmonella/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Food Microbiology ; Metal Nanoparticles/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; *Lab-On-A-Chip Devices ; Limit of Detection ; Food Contamination/analysis ; Pyrroles/chemistry ; *Bacterial Proteins/genetics/metabolism ; Endodeoxyribonucleases ; Polymers ; },
abstract = {Staphylococcus aureus (S. aureus) and Salmonella frequently cocontaminate food products, posing significant health threats. We developed a microfluidic electrochemical biosensor that enables simultaneous detection of both pathogens within 65 min. The chip integrates sample loading, recombinase polymerase amplification (RPA), and CRISPR/Cas12a-based recognition into the upper layer. Reaction products enter a detection chamber with a single-walled carbon nanohorns-polypyrrole-gold nanoparticles (SWCNHs-ppy-AuNPs)-modified three-electrode system, where ssDNA probes act as signal reporters. Upon target recognition, Cas12a cleaves the probes, releasing electroactive molecules and reducing the current. The sensor exhibits linear responses for S. aureus (1.06 × 10[1]-1.06 × 10[7] CFU/mL) and Salmonella (1.04 × 10[1]-1.04 × 10[7] CFU/mL), with detection limits of 3 CFU/mL. This platform offers a rapid, sensitive, and accurate tool for on-site detection of foodborne pathogens in food products.},
}
@article {pmid41264695,
year = {2025},
author = {Rönspies, M and Khosravi, S and Helia, O and Valisi, A and Fajkus, J and Fojtová, M and Houben, A and Puchta, H},
title = {CRISPR-Cas-mediated heritable chromosome fusions in Arabidopsis.},
journal = {Science (New York, N.Y.)},
volume = {390},
number = {6775},
pages = {843-848},
doi = {10.1126/science.adz8505},
pmid = {41264695},
issn = {1095-9203},
mesh = {*Arabidopsis/genetics ; *Chromosomes, Plant/genetics ; *CRISPR-Cas Systems ; Gene Editing ; *Genome, Plant ; Karyotype ; Meiosis ; Recombination, Genetic ; Gene Fusion ; },
abstract = {The genome of Arabidopsis thaliana consists of 10 chromosomes. By inducing CRISPR-Cas-mediated breaks at subcentromeric and subtelomeric sequences, we fused entire chromosome arms, obtaining two eight-chromosome lines. In one line, both arms of chromosome 3 were fused to chromosome 1. In another line, the arms were transferred to chromosomes 1 and 5. Both chromosome number-reduced lines were fertile. Phenotypic and transcriptional analyses revealed no differences compared with wild-type plants. After crossing with the wild type, the progeny showed reduced fertility. The meiotic recombination patterns of the transferred chromosome arms were substantially changed. Directed chromosome number changes in plants may enable new breeding strategies, redefining linkage groups and establishing genetic barriers. Moreover, our data indicate that plants are highly robust to engineered karyotype changes.},
}
@article {pmid41264989,
year = {2026},
author = {Liu, X and Yang, M and Sun, D and Lu, C and Ma, Y and Jiang, Y and Ouyang, R and Miao, Y},
title = {Integrating amplification strategies and functional nanomaterials for advanced electrochemical biosensing of MicroRNA.},
journal = {Bioelectrochemistry (Amsterdam, Netherlands)},
volume = {168},
number = {},
pages = {109170},
doi = {10.1016/j.bioelechem.2025.109170},
pmid = {41264989},
issn = {1878-562X},
mesh = {*MicroRNAs/analysis/genetics ; *Biosensing Techniques/methods ; *Electrochemical Techniques/methods ; *Nanostructures/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Humans ; Nucleic Acid Hybridization ; },
abstract = {MicroRNAs (miRNAs) are crucial disease biomarkers, yet their short length, low abundance, and high sequence homology pose significant challenges for sensitive detection. Electrochemical biosensing presents a promising alternative, though effective signal amplification remains essential. This review summarizes recent advances in amplification strategies for electrochemical miRNA detection, covering nucleic acid-based techniques-such as hybridization chain reaction (HCR), rolling circle amplification (RCA), and catalytic hairpin assembly (CHA)-as well as nanomaterial-assisted approaches using metal-organic frameworks and transition metal dichalcogenides. Key mechanisms, advantages, and limitations of each method are discussed, along with performance metrics (e.g., detection limit and linear range) and emerging hybrid systems like RCA-CRISPR/Cas. Current challenges, including probe complexity and nanomaterial aggregation, are also addressed. Finally, the review highlights future directions involving multi-mechanism integration and clinical translation, offering insights for the development of highly sensitive and reliable electrochemical biosensors to advance precision medicine.},
}
@article {pmid41265135,
year = {2026},
author = {Wright, M and Choi, W and Deng, Z and Kim, D and Lee, K},
title = {CRISPR/Cas9 Gene Editing of Turkey Cells Using Adenoviral Delivery Running Head: RESEARCH NOTE.},
journal = {Poultry science},
volume = {105},
number = {1},
pages = {106096},
pmid = {41265135},
issn = {1525-3171},
mesh = {Animals ; *Gene Editing/veterinary/methods ; *CRISPR-Cas Systems ; *Turkeys/genetics ; *Adenoviridae/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Cell Line ; Receptors, Leptin/genetics ; Genetic Vectors ; },
abstract = {Poultry, including birds such as chicken, duck, turkey, geese, and other game birds is the most widely consumed animal protein globally, with per capita poultry consumption steadily rising in the United States over the past several decades. To meet the demand for poultry, many avenues of improving poultry production have been explored including genetic modification solutions. While genetic modification in many poultry species has been well explored, gene editing in turkey has no research into its application. This study investigated the feasibility of using CRISPR/Cas9 mediated gene editing using adenoviral delivery in vitro, a method that has been proven successful for other species of poultry but never turkey. Three guide RNAs (gRNAs) were designed, targeting the leptin receptor (LEPR) gene matching the gene sequences of both chicken and turkey. Following the assessment of each gRNA's editing efficiency in turkey embryonic fibroblast (TEF) cells and commercially available chicken DF-1 cells via liposome-mediated transfection of the CRISPR vector, the most effective gRNA was chosen for recombinant adenovirus type 5 production. From there, both DF-1 and TEF cells were transduced using the adenovirus and the genome editing efficiency was assessed. The results show that turkey cells can be genetically modified in vitro with indel frequencies comparable to those observed in chicken cells, using both CRISPR plasmids and adenoviral vectors. These findings can lay the groundwork for future experiments and adaptation to in vivo modification of turkey in the future.},
}
@article {pmid41265176,
year = {2026},
author = {Mao, S and Guo, Y and Dong, C and Wang, D and Wang, X and Weng, L and Yang, Y and Li, Y and Niu, T and Wu, Q and Zheng, Z and Shan, Z and Tan, X and Gao, Y and Jin, J and Wang, P and Ge, X and Shen, B and Yao, X and Fang, L},
title = {Loss of cyclin C drives resistance to anti-TIGIT therapy by upregulating CD155-mediated immune evasion.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {84},
number = {},
pages = {101318},
doi = {10.1016/j.drup.2025.101318},
pmid = {41265176},
issn = {1532-2084},
mesh = {Humans ; *Receptors, Immunologic/antagonists &amp; inhibitors/immunology ; Killer Cells, Natural/immunology ; Cell Line, Tumor ; *Drug Resistance, Neoplasm/immunology/genetics ; Up-Regulation ; *Neoplasms/immunology/drug therapy/genetics ; CRISPR-Cas Systems ; T-Lymphocytes/immunology ; Gene Expression Regulation, Neoplastic ; Immune Evasion ; Immune Checkpoint Inhibitors/pharmacology ; Tumor Escape ; Receptors, Virus ; },
abstract = {AIMS: CD155 is an immune checkpoint protein expressed in tumor cells that interacts with its ligand T cell immunoreceptor with immunoglobulin and ITIM domain (TIGIT) on natural killer (NK) cells and T cells, mediating inhibitory regulation on immune cells. Blockade of the CD155-TIGIT interaction has demonstrated clinical benefits in patients with advanced cancers. The transcriptional and post-translational mechanisms governing CD155 expression remain largely unknown.<br><br>METHODS: To identify regulators of CD155, we conducted a genome-wide CRISPR-Cas9 screen in cancer cells. Surface CD155 protein levels were analyzed via flow cytometry. The role of candidate regulators was validated through loss- and gain-of-function experiments with flow cytometry, Western blot, quantitative PCR, and chromatin immunoprecipitation (ChIP) assays. Additionally, ubiquitination assay was performed to examine post-translational modifications. Functional studies, including NK and T cell cytotoxicity assays, were conducted to assess the immune modulatory effects of CD155 regulation. Clinical relevance was evaluated by analyzing Cyclin C (CCNC) and CD155 expression in datasets of cancer patients who underwent immune checkpoint blockade therapy.<br><br>RESULTS: The CRISPR-Cas9 screen identified CCNC as a transcriptional suppressor of CD155. CCNC knockout led to increased surface CD155 expression in cancer cell lines. Mechanistically, CCNC inhibited CD155 transcription by suppressing the activity of the transcription factor FOSL2. Furthermore, CCNC was found to be ubiquitinated and degraded by the E3 ubiquitin ligase FBXO11, suggesting a post-translational regulatory mechanism. Functionally, loss of CCNC promoted CD155 upregulation, thereby enhancing tumor immune evasion from NK and T cell-mediated responses. Clinically, CCNC expression was negatively correlated with CD155 levels in cancer patients, particularly those receiving immune checkpoint blockade therapy.<br><br>CONCLUSION: This study identifies a previously unrecognized master regulator CCNC that functions as a suppressor of CD155-mediated cancer immune evasion. The findings of this study suggest that tumors with low CCNC expression may be resistant to monotherapy and highlight a combination immunotherapy (TIGIT/PD-1 co-blockade) as a promising anti-cancer therapeutic strategy to overcome immune evasion in CCNC-deficient tumors.},
}
@article {pmid41265249,
year = {2025},
author = {Papadopoulou, M and Ramachandran, H and Binder, S and Hildebrandt, B and Rossi, A and Krutmann, J},
title = {CRISPR/Cas9-mediated editing of XPA in induced pluripotent stem cells: A model for investigating Xeroderma Pigmentosum and NER dysfunction.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103869},
doi = {10.1016/j.scr.2025.103869},
pmid = {41265249},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *CRISPR-Cas Systems/genetics ; *Xeroderma Pigmentosum/genetics/pathology/metabolism ; *Xeroderma Pigmentosum Group A Protein/genetics/metabolism ; *DNA Repair/genetics ; *Gene Editing ; Cell Differentiation ; Cell Line ; },
abstract = {Xeroderma pigmentosum group A (XPA) is caused by defects in the nucleotide excision repair (NER) pathway, which is essential for repairing UV-induced DNA damage. Mutations in XPA impair lesion recognition and repair, resulting in mutation accumulation, genomic instability, and a high risk of skin cancers. In this study, we generated a CRISPR/Cas9-engineered human induced pluripotent stem cell (iPSC) line, WTSIi018-B-30, carrying a homozygous single nucleotide variant in exon 3 of XPA. The edited iPSCs retained normal morphology, expressed pluripotency markers, and differentiated into all three germ layers. This mutant iPSC line provides a robust isogenic model to dissect the molecular consequences of XPA deficiency and to explore therapeutic strategies for XPA-associated diseases.},
}
@article {pmid41265250,
year = {2025},
author = {Ramachandran, H and Becker, A and Dobner, J and Hildebrandt, B and Distelmaier, F and Rossi, A and Anand, R},
title = {CRISPR/Cas9-mediated editing of MIC13 in human induced pluripotent stem cells: A model for mitochondrial hepato-encephalopathy.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103870},
doi = {10.1016/j.scr.2025.103870},
pmid = {41265250},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; *Mitochondrial Encephalomyopathies/genetics/pathology/metabolism ; *Mitochondrial Proteins/genetics/metabolism ; Cell Line ; },
abstract = {MIC13 is essential for cristae formation and functions as a key component of the large mitochondrial multi subunit MICOS complex. Mutations in MIC13 causes severe mitochondrial disease called mitochondrial hepato-encephalopathy. In this study, we describe the generation of a human induced pluripotent stem cell (iPSC) line carrying a patient-specific MIC13 mutation, introduced using a CRISPR/Cas knock-in approach. The resulting iPSC line will provide a valuable model to study the pediatric severe mitochondrial disease and to determine the pathological mechanisms as well as to facilitate the identification of potential therapeutic targets in the future.},
}
@article {pmid41265646,
year = {2026},
author = {Bao, W and Ji, P and Xi, W and Chimed, G and Liu, Z and Ping, Y},
title = {Genome editing of Spp1 by inhalable CRISPR/Cas9 formulation for treating pulmonary fibrosis.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {389},
number = {},
pages = {114424},
doi = {10.1016/j.jconrel.2025.114424},
pmid = {41265646},
issn = {1873-4995},
mesh = {Animals ; *Gene Editing/methods ; *Osteopontin/genetics ; *CRISPR-Cas Systems ; Polylactic Acid-Polyglycolic Acid Copolymer/chemistry ; Administration, Inhalation ; Nanoparticles/chemistry/administration &amp; dosage ; Humans ; Mice ; Mice, Inbred C57BL ; *Pulmonary Fibrosis/therapy/genetics ; RNA, Guide, CRISPR-Cas Systems/administration &amp; dosage/genetics ; *Idiopathic Pulmonary Fibrosis/therapy/genetics ; Bleomycin ; Male ; Genetic Therapy/methods ; Lung/metabolism/pathology ; Calcium Phosphates ; },
abstract = {Secreted phosphoprotein 1 (Spp1) encoding osteopontin (OPN), a matrix cell protein with pro-inflammatory and pro-necrotic tissue properties, plays a crucial role in the onset and progression of idiopathic pulmonary fibrosis (IPF). In order to treat IPF by taking advantage of Spp1, we herein developed an inhalable system composed of calcium phosphate/ poly (lactic-co-glycolic acid) (PLGA) core-shell nanoparticles which are loaded with CRISPR/Cas9 system targeting Spp1 to investigate its therapeutic potential. Specifically, the plasmid encoding Cas9 and single-guide RNA (sgRNA) selectively targeting Spp1 gene was first condensed by calcium phosphate to form Cas9 complexes, which was then encapsulated by PLGA to formulate into a gene-editing inhalable delivery system (termed CaP/Cas9/PLGA). Interestingly, the aerosolized inhaled delivery of CaP/Cas9/PLGA nanoparticles results in the effective traverse of mucosal barriers to fibrotic lungs, where they are internalized by lung cells without inducing noticeable cytotoxicity. Following endo/lysosomal escape and gene expression of CRISPR system, the disruption of Spp1 gene by Cas9/sgRNA induces the mutation frequency exceeding 30 %, resulting in efficient down-regulation of OPN level. In a bleomycin-induced pulmonary fibrosis mouse model, the inhalation of aerosolized CaP/Cas9/PLGA complexes significantly attenuates fibrosis development and improves lung function with undetectable systemic toxicity. This current study defines an innovative inhalable gene-editing formulation and offers a promising gene therapy modality for treating IPF.},
}
@article {pmid41266199,
year = {2026},
author = {Wu, X and Wang, M and Luo, S and Zhou, Z and Wang, Y and Du, G and Chen, J and Liu, X},
title = {Dual enhancement of mycoprotein nutrition and sustainability via CRISPR-mediated metabolic engineering of Fusarium venenatum.},
journal = {Trends in biotechnology},
volume = {44},
number = {3},
pages = {745-765},
doi = {10.1016/j.tibtech.2025.09.016},
pmid = {41266199},
issn = {1879-3096},
mesh = {*Metabolic Engineering/methods ; *Fusarium/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Fungal Proteins/genetics/metabolism ; Animals ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {Mycoprotein (MP) production represents a promising environmentally sustainable strategy to address global protein deficit. To enhance the nutritional profile and production efficiency of MP, we employed CRISPR/Cas9-mediated scarless gene knockout and obtained a Fusarium venenatum strain (designated FCPD), which exhibited a 32.9% increase in essential amino acid index (EAAI) through targeted truncation of competitive metabolic pathways and regulation of amino acid metabolism or biosynthesis. FCPD achieved a 44.3% reduction in substrate consumption while improving MP production rate by 88.4% compared with the wild type (WT) strain. The cradle-to-gate life cycle assessment (LCA) shows that FCPD could reduce environmental impacts such as global warming potential (GWP) by 4-61.3% under production scenarios in six representative countries. Comparative environmental performance demonstrated the superiority of FCPD-MP over cell-cultured meat and chicken meat. These findings establish CRISPR/Cas technology and metabolic engineering as the dual-purpose tool for both nutritional enhancement and environmental impact mitigation in alternative protein production.},
}
@article {pmid41266327,
year = {2025},
author = {Corda, L and Volpe, E and Dallali, H and Di Tommaso, E and Colantoni, A and Guarracino, A and Chittoor, SS and Capulli, M and Tassone, E and Giunta, S},
title = {Cell line-matched reference enables high-precision functional genomics.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11194},
pmid = {41266327},
issn = {2041-1723},
support = {#101078838//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; #25189//Associazione Italiana per la Ricerca sul Cancro (Italian Association for Cancer Research)/ ; },
mesh = {Humans ; *Genomics/methods/standards ; *Genome, Human/genetics ; Cell Line ; Haplotypes ; Centromere/genetics ; Gene Editing ; CRISPR-Cas Systems ; Kinetochores/metabolism ; },
abstract = {Comparative analyses of newly available human genome assemblies highlight extensive variation that peaks at centromeres. Reliance on a single generic reference genome can thus hinder whole-genome analysis of sequencing data derived from laboratory cell lines and limit their accurate genomic manipulation. Here, we demonstrate that using an "isogenomic" diploid reference genome - specific for the experimental cell line - substantially improves the accuracy of genomic, epigenomic, transcriptomic analyses and genome editing compared to a non-matched reference. Using our recently generated reference genome of the widely used diploid human cell line RPE-1, we uncover haplotype-specific genetic and epigenetic divergence across all centromeres. Mapping quality of RPE-1 data - DNA- and RNA-seq reads, improves both genome-wide and at highly divergent loci when using the matched RPE1v1.1 reference, resolving haplotype-specific enrichment. For genome engineering experiments, centromeric CRISPR guide RNA efficiency and chromosome specificity are best achieved using the RPE-1 reference. Leveraging high-confidence CUT&amp;RUN read mapping using the matched reference, we define the site of the human kinetochore and identify a wide variation in the position, size and structural organization between haplotypes and chromosomes. This work establishes matched-reference genomics as a powerful framework for high-precision cell biology, calling for the systematic assembly of experimentally relevant cell line genomes.},
}
@article {pmid41266370,
year = {2025},
author = {Giddins, M and Kratz, AF and De Los Santos, MB and Forget, A and Tiwari, R and Jang, G and Blazejewski, T and Qin, C and Huang, Y and Lao, YH and Falconer, T and Leong, KW and Krogan, N and Staller, M and Wang, H and Wei, L and Chavez, A},
title = {Combinatorial protein engineering identifies potent CRISPR activators with reduced toxicity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11114},
pmid = {41266370},
issn = {2041-1723},
support = {DP2NS131566-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2 NS131566/NS/NINDS NIH HHS/United States ; 1R01EB031935//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; S10 OD020056/OD/NIH HHS/United States ; P30 CA013696/CA/NCI NIH HHS/United States ; R01 EB031935/EB/NIBIB NIH HHS/United States ; HR0011-19-2-0009//United States Department of Defense | Defense Advanced Research Projects Agency (DARPA)/ ; U54 CA274502/CA/NCI NIH HHS/United States ; },
mesh = {*Protein Engineering/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; HEK293 Cells ; Protein Domains ; Recombinant Fusion Proteins/genetics/metabolism ; },
abstract = {Current protein engineering methods are inadequate to explore the combinatorial potential offered by nature's vast repertoire of protein domains-limiting our ability to create optimal synthetic tools. To overcome this barrier, we develop an approach to create and test thousands of chimeric proteins and employ it to probe an expansive combinatorial landscape of over 15,000 multi-domain CRISPR activators. Our findings indicate that many activators produce substantial cellular toxicity, often unrelated to their capacity to regulate gene expression. We also explore the biochemical features of activation domains and determine how their combinatorial interactions shape activator behavior. Finally, we identify two potent CRISPR activators, MHV and MMH, and demonstrate their enhanced activity across diverse targets and cell types compared to the gold-standard MCP activator, synergistic activation mediator (SAM).},
}
@article {pmid41266743,
year = {2025},
author = {Zhang, W and Shi, J and Wang, B and Qu, H and Wu, X and Wang, X},
title = {An integrated Aptamer-CRISPR-Cas12a method for rapid and sensitive detection of carbendazim.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41119},
pmid = {41266743},
issn = {2045-2322},
support = {22A310016//Henan Province Higher Education Key Scientific Research Project/ ; },
mesh = {*Carbamates/analysis ; *Aptamers, Nucleotide/genetics/chemistry ; *CRISPR-Cas Systems ; *Benzimidazoles/analysis ; Limit of Detection ; *Biosensing Techniques/methods ; *Pesticide Residues/analysis ; Reproducibility of Results ; },
abstract = {Concerns over carbendazim (CBZ) pesticide residues in agricultural products and medicinal herbs have intensified due to their potential health and environmental risks. While existing detection techniques offer distinct advantages, they are often limited by complex procedures, specialized equipment, and high costs. To address these challenges, we developed a novel Aptamer-CRISPR/Cas12a assay, which combines the specificity of aptamers with the high sensitivity and precision of the CRISPR/Cas12a system. This assay achieves a linear detection range of 10-5000 ng/mL (R[2] = 0.9639 at 15 min and R[2] = 0.9774 at 30 min) and a limit of detection (LOD) of 10 ng/mL. In real samples, the average recovery rate of CBZ ranges from 92.10% to 102.86%, demonstrating robust accuracy and reliability. Notably, the method is user-friendly, requires minimal equipment, and delivers results in about 40 min, making it suitable for field applications. Furthermore, the crRNA serves as a universal sequence, enabling the detection of different targets by simply replacing the aptamer and complementary strand, while keeping the CRISPR/Cas12a system intact. This streamlined approach enhances flexibility and broad applicability. In conclusion, the Aptamer-CRISPR/Cas12a assay offers a practical solution for monitoring agricultural products, Chinese herbal medicine, and environmental safety.},
}
@article {pmid41267399,
year = {2026},
author = {Dua, PH and Simon, BMJ and Marley, CBE and Feliciano, CM and Watry, HL and Cowan, QT and Steury, D and Abraham, A and Gilbertson, EN and Ramey, GD and Capra, JA and Conklin, BR and Judge, LM},
title = {Haplotype editing with CRISPR-Cas9 as a therapeutic approach for dominant-negative missense mutations in NEFL.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1633-1651},
pmid = {41267399},
issn = {1525-0024},
support = {R01 NS119678/NS/NINDS NIH HHS/United States ; U01 ES032673/ES/NIEHS NIH HHS/United States ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Induced Pluripotent Stem Cells/metabolism/cytology ; *Mutation, Missense ; *Haplotypes ; *Charcot-Marie-Tooth Disease/genetics/therapy ; Motor Neurons/metabolism ; Polymorphism, Single Nucleotide ; Alleles ; Genetic Therapy/methods ; Phenotype ; },
abstract = {Inactivation of disease alleles by allele-specific editing is a promising approach to treat dominant-negative genetic disorders, provided the causative gene is haplosufficient. We previously edited a dominant NEFL missense mutation causing Charcot-Marie-Tooth type 2E (CMT2E) with inactivating frameshifts and rescued disease-relevant phenotypes in induced pluripotent stem cell (iPSC)-derived motor neurons. However, a multitude of different NEFL missense mutations cause CMT2E. Here, we addressed this challenge by targeting common single-nucleotide polymorphisms in cis with NEFL disease mutations for gene excision. We validated this haplotype editing approach in two iPSC lines with different missense mutations and demonstrated phenotypic rescue in iPSC-motor neurons. Surprisingly, our analysis revealed that gene inversion, a frequent by-product of excision editing, failed to reliably disrupt mutant allele expression. We deployed novel molecular assays to optimize our approach and achieve therapeutic levels of editing in immature iPSC-motor neurons. Finally, population genetics analysis demonstrated the power of haplotype editing to enable therapeutic development for the greatest number of patients. Our data serve as an important case study for many dominant genetic disorders amenable to this approach.},
}
@article {pmid41267649,
year = {2025},
author = {Matsubara, K and Hirota, M and Kajiwara, K and Senga, H and Matsui, S and Marutani, M and Seki, Y},
title = {Lineage-specific enhancer insertions regulate Prdm14 to drive the rapid transition from naïve to formative pluripotency in rodents.},
journal = {Development (Cambridge, England)},
volume = {152},
number = {24},
pages = {},
doi = {10.1242/dev.204886},
pmid = {41267649},
issn = {1477-9129},
support = {18H02422//Japan Society for the Promotion of Science/ ; //Kwansei Gakuin University/ ; },
mesh = {Animals ; Mice ; *Transcription Factors/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Gene Expression Regulation, Developmental ; *Cell Lineage/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; CRISPR-Cas Systems/genetics ; Humans ; Cell Differentiation/genetics ; DNA-Binding Proteins ; RNA-Binding Proteins ; },
abstract = {The network of transcription factors is dynamically reorganized during the transition from naïve- to formative-pluripotency. In mice, Prdm14 is expressed in naïve pluripotent cells but rapidly downregulated upon exit from the naïve state. In contrast, PRDM14 expression persists throughout pluripotency transitions in non-rodent mammals, including pigs and humans. Here, we investigate the molecular mechanisms underlying the rodent-specific expression of Prdm14. Using CRISPR/Cas9-mediated deletions, we demonstrated that POU5F1 and TFCP2L1 recognition sequences within Muroidea-specific cis-regulatory elements located downstream of Prdm14 are essential for its transcriptional upregulation in naïve embryonic stem cells. Loss of these enhancers attenuates the upregulation of Prdm14, leading to reduced Pramel7 induction and impaired degradation of UHRF1, which consequently diminished global DNA demethylation under 2iL conditions. Moreover, deletion of PRDM14-binding motifs in Muroidea-specific enhancers disrupts its negative feedback loop, resulting in a delayed transition from the naïve to formative pluripotent state. Our findings reveal that rodent-specific enhancer insertions endow Prdm14 with a dynamic regulatory architecture, enabling both activation and repression that collectively ensure the timely exit from naïve pluripotency during early embryogenesis.},
}
@article {pmid41270157,
year = {2025},
author = {Li, Y and Wu, Y and Zheng, Z and Wu, Y and Zhang, Y and Zhang, J and Quan, F and Zhao, W and Xu, R and Li, Y and Gao, H and Zhang, K},
title = {Renal clearable CRISPR nanosensor targeting mitochondrial DNA mutation for noninvasive monitoring of tumor progression and metastasis.},
journal = {Science advances},
volume = {11},
number = {47},
pages = {eadz4594},
pmid = {41270157},
issn = {2375-2548},
mesh = {*DNA, Mitochondrial/genetics/metabolism/urine ; Mutation ; CRISPR-Cas Systems ; *Biomarkers, Tumor/genetics/metabolism/urine ; *Biosensing Techniques/methods ; Carcinogenesis/genetics ; *Cell-Free Nucleic Acids/genetics/metabolism/urine ; Liquid Biopsy/methods ; *Lung Neoplasms/diagnosis/genetics/pathology/urine ; Disease Progression ; Mitochondria/genetics/pathology ; Renal Elimination ; Cell Line ; Humans ; Animals ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasms, Experimental/genetics/metabolism/pathology/urine ; Female ; },
abstract = {Mitochondrial DNA (mtDNA) mutations are emerging as important molecular features of tumorigenesis. Liquid biopsies, involving analysis of cell-free mtDNA, enable early cancer detection but suffer from low sensitivity due to scarce analytes. Here, we developed a CRISPR/Cas12a-mediated urinary biomarker, termed CasUber, for in vivo monitoring of tumor progression and metastasis. Our results demonstrate that CasUber can deliver a CRISPR detection system into tumor cell mitochondria, leverage the single-nucleotide variant recognition ability and trans-cleavage activity of Cas12a to convert tumor-specific mtDNA mutations into renal-clearable fluorescent biomarkers, and exocytosed along with the natural efflux pathway of damaged mtDNA. As a result, CasUber enables discrimination of ultrasmall tumor lesions (~1 cubic millimeter) and detection of lung tumor nodules earlier than bioluminescence imaging in a blood-lung metastasis model. This renal clearable nanosensor allows in situ recognition of specific gene mutation to generate amplified signals, overcoming the limitation of low mtDNA abundance and enabling noninvasive and ultrasensitive monitoring of tumor progression and metastasis via a simple urine test.},
}
@article {pmid41270297,
year = {2025},
author = {Kraiczy, J and Yu, B},
title = {Human fallopian tube epithelial organoids with TP53 mutation recapitulate features of serous tubal intraepithelial carcinoma (STIC).},
journal = {Gynecologic oncology},
volume = {203},
number = {},
pages = {198-208},
pmid = {41270297},
issn = {1095-6859},
support = {K08 CA222385/CA/NCI NIH HHS/United States ; R01 AI139189/AI/NIAID NIH HHS/United States ; },
mesh = {Humans ; Female ; *Tumor Suppressor Protein p53/genetics ; *Organoids/pathology ; *Fallopian Tubes/pathology ; *Fallopian Tube Neoplasms/genetics/pathology ; Mutation ; *Carcinoma in Situ/genetics/pathology ; *Ovarian Neoplasms/genetics/pathology ; DNA Copy Number Variations ; *Cystadenocarcinoma, Serous/genetics/pathology ; CRISPR-Cas Systems ; Gene Editing ; },
abstract = {OBJECTIVE: Serous tubal intraepithelial carcinoma (STIC) is the immediate precursor lesion for high-grade serous ovarian carcinoma (HGSOC) and harbors universal TP53 mutations. The lack of an appropriate in vitro model for STIC presents a major challenge in studying its pathogenesis. We aimed to develop a human in vitro model that mimics STIC lesions.<br><br>METHODS: Using CRISPR-Cas9 gene editing, we generated human fallopian tube epithelial organoids with TP53 loss-of-function mutations (TP53[-/-] FTOs). We characterized TP53[-/-] FTOs on a cellular and molecular level using immunofluorescence confocal imaging, copy number variation (CNV) analysis, and RNA sequencing.<br><br>RESULTS: TP53[-/-] FTOs recapitulated key features of STIC lesions. They exhibited increased proliferation and nuclear abnormalities, including nuclear enlargement and atypical mitotic figures. Copy number variation analysis revealed aneuploidy in some TP53[-/-] FTOs. Compared to unedited controls, TP53[-/-] FTOs demonstrated significant transcriptomic changes, including the downregulation of DNA repair genes and upregulation of epithelial-mesenchymal transition (EMT) pathways. Similar to STIC lesions, TP53[-/-] FTOs showed a marked reduction in ciliated cells and ciliogenesis-associated gene expression.<br><br>CONCLUSIONS: These findings suggest that p53 loss in FTOs promotes a proliferative and genomically unstable state that is conducive to carcinogenesis. The TP53[-/-] FTO model we have generated provides a valuable tool for studying early events in ovarian carcinogenesis and for developing new strategies for the early detection and prevention of ovarian cancer.},
}
@article {pmid41270510,
year = {2026},
author = {Li, J and Li, X and Li, X and Xu, W and Yuan, L and Shi, H and Lei, Z and Li, N and Wei, Y and Hua, J},
title = {CRISPR/Cas9-generated CD46-knockout spermatogonial stem cells reveal mechanisms of BVDV-induced reproductive dysfunction in male livestock.},
journal = {Veterinary microbiology},
volume = {312},
number = {},
pages = {110807},
doi = {10.1016/j.vetmic.2025.110807},
pmid = {41270510},
issn = {1873-2542},
mesh = {Animals ; Male ; Goats ; Cattle ; CRISPR-Cas Systems ; *Diarrhea Viruses, Bovine Viral/physiology/pathogenicity ; *Bovine Virus Diarrhea-Mucosal Disease/virology ; *Adult Germline Stem Cells/virology ; Reproduction ; Gene Knockout Techniques ; *Spermatogonia/virology ; },
abstract = {Bovine viral diarrhea virus (BVDV) is a major viral pathogen that affects ruminants, resulting in significant economic losses due to issues such as immunosuppression, reproductive disorders, and growth retardation. Bulls infected with this virus may become infertile within a few months and can transmit the virus to susceptible cattle during mating. However, the mechanism of BVDV impairing the reproductive function of male livestock is not clear, as there is no suitable cell model. This study used spermatogonial stem cells(SSCs) from cattle and goats as research materials to explore the mechanism by which BVDV affects the reproductive function of male livestock. The results of this study indicate that both cytopathic (cp) and noncytopathic (ncp) BVDV can replicate in SSCs and that SSCs are capable of producing infectious BVDV. Giemsa staining showed significant changes in the morphology of SSCs after BVDV infection. Western blot and mRNA analysis showed that proliferation-related genes (PCNA, CCND1, CDK2) and SSC functional genes (Lin28A, OCT4, SOX2) were down regulated after infection. In addition, BVDV infection can induce ferroptosis in SSCs. Furthermore, CRISPR-Cas9 mediated editing of CD46 in goat SSCs resulted in a decrease in BVDV infection rate and alleviated the negative impact of the virus on cell survival and proliferation. This study provides new insights into the mechanism of reduced reproductive function in male livestock infected with BVDV, and lays the foundation for developing targeted disease resistant breeding strategies.},
}
@article {pmid41270550,
year = {2026},
author = {Hu, Q and Liu, J and Zhang, W and Liao, X and Guo, Y and Lu, Q and Zhang, R and Yang, B and Zhang, T and Zhai, X and Luo, Q},
title = {Rapid nucleic acid detection of Mycoplasma synoviae using dual-mode RAA-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {105},
number = {1},
pages = {106126},
pmid = {41270550},
issn = {1525-3171},
mesh = {Animals ; *Mycoplasma synoviae/isolation &amp; purification ; *Mycoplasma Infections/veterinary/diagnosis/microbiology ; *Poultry Diseases/diagnosis/microbiology ; *Chickens ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; Ducks ; Columbidae ; },
abstract = {Mycoplasma synoviae is a significant avian pathogen implicated in diverse clinical manifestations, including air sacculitis, infectious synovitis with arthritis, and eggshell apex abnormalities, which significantly threaten the poultry industry. To enable the rapid and accurate detection of M. synoviae, we developed a dual-mode diagnostic platform that combines recombinant enzyme-assisted amplification (RAA) with CRISPR/Cas12a-based fluorescence and lateral flow dipsticks (LFD). The Qsep100 automatic nucleic acid analysis system was used to screen for RAA primers by evaluating the amplicon size and concentration. Fluorescence monitoring revealed that the optimal reaction time for the RAA-CRISPR/Cas12a system was 28 min. After optimizing the concentrations of LbaCas12a, crRNA, and the ssDNA reporter, the assay achieved a sensitivity of 5.2 copies/µL. Cross-reactivity testing with seven common avian pathogens confirmed the high specificity of the established method for detecting M. synoviae. In the clinical validation, the method perfectly matched the results of quantitative real time polymerase chain reaction (qPCR). Furthermore, an epidemiological investigation revealed that chickens had the highest positivity rate for M. synoviae among the chickens, ducks, and pigeons. In summary, we developed a rapid, accurate, and portable diagnostic platform for M. synoviae detection, which provides a valuable tool for disease prevention and control in resource-limited settings.},
}
@article {pmid41270608,
year = {2026},
author = {Zhou, Y and Zhai, J and Chen, H and Qu, Y and Fang, Z and Chen, B and Bao, Z and Chen, D},
title = {Dz-SiG CRISPR: A DNAzyme-Switched G-quadruplex-lock CRISPR system for isothermal and rapid detection of lead ions.},
journal = {Talanta},
volume = {299},
number = {},
pages = {129139},
doi = {10.1016/j.talanta.2025.129139},
pmid = {41270608},
issn = {1873-3573},
mesh = {*DNA, Catalytic/chemistry/metabolism/genetics ; *Lead/analysis ; *G-Quadruplexes ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {Lead (Pb[2+]) poses serious risks to health and ecosystems, necessitating rapid, ultrasensitive detection. CRISPR/Cas12a systems offers exceptional specificity and intrinsic signal amplification. Nevertheless, their adaptation to Pb[2+] is hindered by the lack of programmable interfaces to convert small-molecule binding into Cas12a activation. Herein, we report a DNAzyme-Switched G-quadruplex-locked CRISPR (Dz-SiG CRISPR) strategy, enabling ultrasensitive, femtomolar-level detection of Pb[2+]. A RNA G-quadruplex (RG4) structure is conjugated to the 5' end of the crRNA, serving as a conformational lock that suppresses Cas12a's trans-cleavage activity. Upon Pb[2+] binding, the GR-5 DNAzyme catalyzes a hydrolytic cleavage that acts as a molecular switch, releasing the RG4 domain and unleashing active crRNA to trigger Cas12a-mediated cleavage of a fluorogenic reporter, yielding a sharp "off-to-on" fluorescent signal. The Dz-SiG CRISPR system achieves an ultralow limit of detection of 18.91 fM for Pb[2+] and demonstrates outstanding performance in real water and soil samples, with recovery rates ranging from 94.44 % to 99.03 %. The assay can be completed within 30 min, making it highly suitable for rapid on-site lead ion detection. Importantly, the modular Dz-SiG CRISPR framework can be readily reprogrammed for other small molecules by simply substituting the DNAzyme module, offering a generalizable strategy for rapid, ultrasensitive environmental monitoring.},
}
@article {pmid41270733,
year = {2025},
author = {Wei, Z and Lan, Y and Meng, L and Wang, H and Li, L and Li, Y and Zhang, N and Lu, R and Cui, Z and Song, Y and Wang, Y and Li, Y and Yue, Z and Fan, G and Li, Q and Gu, Y and Liu, S and Qian, PY and Meng, L and Shao, C},
title = {Hologenomic insights into the molecular adaptation of deep-sea coral Bathypathes pseudoalternata.},
journal = {Cell host &amp; microbe},
volume = {33},
number = {12},
pages = {2017-2029.e5},
doi = {10.1016/j.chom.2025.10.020},
pmid = {41270733},
issn = {1934-6069},
mesh = {Animals ; *Anthozoa/microbiology/genetics/physiology ; Symbiosis ; *Adaptation, Physiological/genetics ; Phylogeny ; Bacteria/genetics/metabolism/classification ; Ecosystem ; },
abstract = {Deep-sea coral ecosystems support biodiversity and nutrient cycling through interactions with symbionts. However, their molecular mechanisms remain unexplored. Here, hologenomic analyses of Bathypathes pseudoalternata are applied to uncover molecular adaptations underpinning host-symbiont interactions. Genomic evidence reveals that B. pseudoalternata exhibits adaptations in nutrient transport, immune response, and lysosomal digestion, reflecting its genomic adjustments for a stable symbiosis. Candidatus Nitrosopumilus bathypathes (78.43% ± 3.65%) is inferred to oxidize host-derived ammonia to synthesize amino acids and vitamins to provision the host. The presence of CRISPR-Cas and restriction-modification (R-M) systems suggests that Ca. Bathyplasma bathypathes and Ca. Thalassoplasma bathypathes (10.68% ± 2.99%) may protect the host from viral infections. Ca. Bathybacter bathypathes (8.39% ± 1.53%) is hypothesized to synthesize heme, lipoic acid, and glutathione, which serve dual functions as antioxidants and nutrients. These findings collectively provide insights into how the hologenome contributes to the survival of B. pseudoalternata in the extreme environment.},
}
@article {pmid41270742,
year = {2026},
author = {Sun, J and Atiş, IS and Empke, SLL and Khokha, MK and Breslow, DK},
title = {A microscopy-based CRISPR screening platform enables organellar functional genomics and illuminates ciliary biology.},
journal = {Developmental cell},
volume = {61},
number = {3},
pages = {687-705.e9},
pmid = {41270742},
issn = {1878-1551},
support = {S10 OD023651/OD/NIH HHS/United States ; R35 GM137956/GM/NIGMS NIH HHS/United States ; S10 OD018034/OD/NIH HHS/United States ; R01 HD102186/HD/NICHD NIH HHS/United States ; S10 OD030363/OD/NIH HHS/United States ; },
mesh = {*Cilia/metabolism/genetics ; Humans ; Animals ; *Genomics/methods ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Microscopy, Fluorescence/methods ; Xenopus ; },
abstract = {Microscopy offers an indispensable technique for visualizing biological processes and for defining cytological abnormalities characteristic of disease. However, combining microscopy with the power of pooled CRISPR screening presents considerable technical challenges, hindering application of systematic genetic analysis to imaging-defined phenotypes. Here, we establish a fluorescence microscopy-based CRISPR screening platform that combines ease of implementation with flexible analysis of live-cell or antibody-based molecular markers, including post-translational modifications. Applying this methodology, we systematically identify regulators of primary cilium structure and function in human cells through targeted and genome-wide screens. We further show that integration of screens focused on distinct ciliary phenotypes yields multi-dimensional profiles that delineate precise gene functions. Among the identified hits, TZMP1 (SMIM27) encodes a microprotein at the ciliary transition zone that is required for ciliogenesis in human cells and for ciliary function in Xenopus embryos. More broadly, our approach provides a technological and conceptual strategy for microscopy-based functional genomics.},
}
@article {pmid41270744,
year = {2025},
author = {Pei, Y and Li, S and Garipler, G and Kamimoto, K and Mazzoni, EO},
title = {Stem cell-based approach to identify regulatory TFs during mammalian cell differentiation.},
journal = {Stem cell reports},
volume = {20},
number = {12},
pages = {102716},
pmid = {41270744},
issn = {2213-6711},
support = {F30 HD117663/HD/NICHD NIH HHS/United States ; },
mesh = {*Cell Differentiation/genetics ; Animals ; Humans ; Mice ; *Transcription Factors/metabolism/genetics ; Motor Neurons/cytology/metabolism ; *Pluripotent Stem Cells/metabolism/cytology ; Transcriptome ; Single-Cell Analysis ; CRISPR-Cas Systems ; },
abstract = {Cell differentiation is regulated by transcription factors (TFs), but specific TFs needed for mammalian differentiation pathways are not fully understood. For example, during spinal motor neuron (MN) differentiation, 1,370 TFs are transcribed, yet only 55 have reported functional relevance. We developed a method combining pluripotent stem cell differentiation, single-cell transcriptomics, and a CRISPR-based TF loss-of-function screen and applied it to MN differentiation. The CRISPR screen identified 245 genes important for mouse MN differentiation, including 116 TFs. This screen uncovered important genes not showing differential transcription and identified a regulatory hub at the MN progenitor (pMN) stage. A secondary human screen of 69 selected candidates revealed a conservation between mouse pMN and human pMN and ventral pMN (vpMN) regulations. The validation of three hits required for efficient human MN differentiation supported the effectiveness of our approach. Collectively, our strategy offers a framework for identifying important TFs in various differentiation pathways.},
}
@article {pmid41271111,
year = {2026},
author = {Huang, Q and Zhao, T and Su, W and Li, S and Liu, J and Ge, Z and Zhang, B and Ren, X and Zhang, X and Wei, J},
title = {Screening of monoclonal vaccine strains based on real-time live-cell imaging technology.},
journal = {Journal of virological methods},
volume = {340},
number = {},
pages = {115305},
doi = {10.1016/j.jviromet.2025.115305},
pmid = {41271111},
issn = {1879-0984},
mesh = {Animals ; *Vaccinia virus/genetics/immunology/isolation &amp; purification ; *Viral Vaccines/immunology/genetics/isolation &amp; purification ; Viral Plaque Assay/methods ; Vaccines, Attenuated/immunology/genetics ; Antibodies, Viral/blood/immunology ; Humans ; Green Fluorescent Proteins/genetics ; CRISPR-Cas Systems ; Vaccines, Synthetic/immunology/genetics ; Antibodies, Neutralizing/blood ; },
abstract = {The plaque purification is a critical step in the screening of traditional live-attenuated vaccines and recombinant viral vaccines, aiming to acquire vaccine clones with homogeneous characteristics and desirable immunogenicity to address outbreaks of emerging diseases such as monkeypox, chikungunya fever, and dengue fever. The traditional plaque purification process to screen out a vaccine strain with genetically consistent stability from a mixed pool of viral clones generally requires laborsome work. We utilized live-cell imaging technique enabling us to isolate monoclonal vaccine strains to simplify and improve the efficiency of this process. Here, we genetically engineered the vaccinia virus TianTan (VTT) using CRISPR/Cas9 system to generate recombinant VTT viruses (VTT-WS01-EGFP) that expressed enhanced green fluorescent protein (EGFP). Initially, we performed 9 rounds of plaque purification using traditional plaque assay, yielding 50 candidate clones. The Incucyte Live-Cell Imaging and Analysis system was subsequently performed to conduct a rigorous, high-resolution screening of these candidates in a more automated, sensitive and high-throughput way. Through this screening process, we ultimately obtained 31 pure viral clones that were free of parental strain contamination, followed by the analysis of plaque formation, fluorescent plaque size, and plaque morphology, and 11 candidate clones were selected for immunological evaluation. Furthermore, we found that clone 49 induced a relatively high titer of anti-VTT neutralizing antibodies and elicited the production of cross-reactive IgG against monkeypox virus antigens, thereby validating its potential as a candidate strain as a monkeypox virus vaccine. Taken together, our data demonstrates that live-cell imaging technique significantly accelerates the screening process for the isolation of monoclonal viral clones as recombinant viral vaccines, and holds considerable potential in attenuated strain selection as well as investigations into biological characteristics of viruses, including viral replication.},
}
@article {pmid41271253,
year = {2025},
author = {Li, L and Xiong, Y and Guo, Y and Duan, H and Leng, Y and Huang, X and Chen, G and Xiong, Y},
title = {G-Quadruplex-Enhanced DNA Silver Nanoclusters Enable CRISPR/Cas12a System for Ultrasensitive Detection of Salmonella typhimurium.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {49},
pages = {31603-31610},
doi = {10.1021/acs.jafc.5c13828},
pmid = {41271253},
issn = {1520-5118},
mesh = {G-Quadruplexes ; *CRISPR-Cas Systems ; *Silver/chemistry ; *Salmonella typhimurium/genetics/isolation &amp; purification ; Metal Nanoparticles/chemistry ; *Biosensing Techniques/methods/instrumentation ; Limit of Detection ; Milk/microbiology ; Animals ; *DNA/chemistry/genetics ; Bacterial Proteins/genetics/metabolism ; CRISPR-Associated Proteins/genetics/metabolism ; Endodeoxyribonucleases ; },
abstract = {DNA-templated silver nanoclusters (tDNA-AgNCs) show considerable promise as fluorescence reporters for the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system because of their ease of synthesis, strong resistance to photobleaching, and large Stokes shift. However, the weak luminous intensity and low cleavage efficiency of tDNA-AgNCs limit the sensitivity of CRISPR assays. In this study, we developed a novel approach by introducing activator DNA (aDNA) with a G-quadruplex structure to enhance the luminous intensity of the tDNA-AgNCs. As a result of the high cleavage efficiency of free aDNA by activated CRISPR/Cas12a, the G-quadruplex-enhanced tDNA-AgNCs (GED-AgNCs) were integrated into recombinase polymerase amplification and CRISPR/Cas12a system for the ultrasensitive detection of Salmonella typhimurium. By optimizing the synthesis of tDNA-AgNCs and GED-AgNCs, our developed G-quadruplex-enhanced DNA-AgNC CRISPR assay (G-DACA) platform enabled the sensitive determination of S. typhimurium with a detection limit as low as 1 CFU/mL and a wide dynamic range of 10-10[8] CFU/mL. Moreover, our proposed method demonstrated good accuracy and reliability for the quantitative analysis of S. typhimurium in real pasteurized milk samples, with recovery rates ranging from 81.06% to 102.33% and relative standard deviations between 7.17% and 14.84%. Overall, our innovative G-DACA platform offers an economical and versatile solution for food safety and clinical diagnostics.},
}
@article {pmid41271362,
year = {2025},
author = {Rahman, MA and Akter, S and Ashrafudoulla, M and Jung, SJ and Rapak, MT and Ha, SD},
title = {CRISPR-Cas systems as emerging tools for precision biofilm control for food safety: Mechanisms and applications.},
journal = {Food research international (Ottawa, Ont.)},
volume = {222},
number = {Pt 2},
pages = {117803},
doi = {10.1016/j.foodres.2025.117803},
pmid = {41271362},
issn = {1873-7145},
mesh = {*Biofilms/growth &amp; development ; *CRISPR-Cas Systems ; *Food Safety/methods ; Gene Editing/methods ; *Food Microbiology/methods ; },
abstract = {Biofilms on food-contact surfaces pose persistent challenges to sanitation, safety, and product quality within food processing. Traditional cleaning methods and broad-spectrum antimicrobials often fail to disrupt the resilient matrix and multispecies communities characteristic of these biofilms. Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) systems offer a transformative approach to enhancing food safety, enabling precise modulation of microbial gene networks with applications in diagnostics, programmable sanitation, and targeted microbial control. This review synthesizes recent advances in CRISPR-Cas technology, encompassing Cas9/Cas12-based gene editing, Cas13-mediated RNA targeting, and dead Cas9 (dCas9)-based transcriptional regulation (CRISPR interference/activation, CRISPRi/a), and evaluates their relevance to biofilm prevention and eradication in food environments. We critically assess delivery platforms, including plasmids, nanocarriers, phagemids, and conjugative systems, for their efficiency in complex biofilm settings. The review highlights innovations such as multiplexed repression of redundant pathways, activation of latent antibiofilm functions. These genetic strategies are increasingly being integrated with omics-based analytics (e.g., transcriptomics, proteomics, metabolomics) to reveal systems-level cellular responses and regulatory shifts triggered by biofilm-targeted interventions. We also address the practical limitations, such as delivery barriers, off-target effects, regulatory hurdles, and ethical considerations specific to food applications. Ultimately, we propose a framework for translating CRISPR-Cas technology into scalable, safety-compliant tools for precision control of biofilms in food processing environments. This review aims to guide future research and inform stakeholders on leveraging CRISPR-Cas technology for safe, sustainable, and targeted management of food-associated biofilms.},
}
@article {pmid41271690,
year = {2025},
author = {Huang, Z and Dong, Y and Yang, Y and Han, X and Wang, F and Lyon, CJ and Ding, S and Peng, Y and Zhang, G and Hu, C and Huang, H and Yang, L and Zhao, G and Fan, XY and Lu, S and Hu, T and Wang, J},
title = {Thermally programmed one-pot CRISPR assay for on-site pandemic surveillance.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10286},
pmid = {41271690},
issn = {2041-1723},
support = {31922046//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Pandemics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Point-of-Care Systems ; Sensitivity and Specificity ; Temperature ; },
abstract = {The ongoing monkeypox virus outbreak highlights the need for rapid and accurate diagnostics to enhance epidemic control. CRISPR-based assays hold promise, but clinical translation is hindered by high complexity and low throughput. Here, we describe a thermally regulated asynchronous CRISPR-enhanced (TRACE) assay that rapidly and sensitively detects multiple DNA targets in a streamlined, one-pot format. TRACE exhibits a 2.5 copies/test limit of detection - 40 times lower than a canonical one-pot CRISPR. When applied to clinical samples, it achieves 99.5% accuracy across diverse sample types, and can detect MPXV within 11 minutes. Point-of-care TRACE assays meet ASSURED criteria and deliver comparable performance to qPCR, with a fivefold reduced report time, in outpatient settings. Moreover, TRACE enables simultaneous detection of pathogen and host genes at comparable sensitivity to address a critical limitation of current CRISPR assays, which lack internal controls. TRACE thus enables rapid, on-site surveillance to facilitate bench-to-bedside translation of CRISPR diagnostics.},
}
@article {pmid41271724,
year = {2025},
author = {Elsharkasy, OM and Hegeman, CV and Driedonks, TAP and Liang, X and Lansweers, I and Cotugno, OL and de Groot, IY and de Wit, ZEMNJ and Garcia-Guerra, A and Moorman, NJA and Boonstra, SH and Bosman, EDC and Lefferts, JW and de Voogt, WS and François, JJ and van Wesel, ACW and El Andaloussi, S and Schiffelers, RM and Kooijmans, SAA and Mastrobattista, E and Vader, P and de Jong, OG},
title = {A modular strategy for extracellular vesicle-mediated CRISPR-Cas9 delivery through aptamer-based loading and UV-activated cargo release.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10309},
pmid = {41271724},
issn = {2041-1723},
support = {VI.Veni.192.174//Nederlandse Organisatie voor Wetenschappelijk Onderzoek (Netherlands Organisation for Scientific Research)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Extracellular Vesicles/metabolism ; *Aptamers, Nucleotide/metabolism/genetics ; Humans ; *Gene Editing/methods ; Ultraviolet Rays ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Associated Protein 9/metabolism/genetics ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 gene editing technology offers the potential to permanently repair genes containing pathological mutations. However, efficient intracellular delivery of the Cas9 ribonucleoprotein complex remains a major hurdle in its therapeutic application. Extracellular vesicles (EVs) are biological nanosized membrane vesicles that play an important role in intercellular communication, and have an innate capability of intercellular transfer of biological cargos, including proteins and RNA. Here, we present a versatile, modular strategy for EV-mediated loading and delivery of Cas9. We leverage the high affinity binding of MS2 coat proteins fused to EV-enriched proteins to MS2 aptamers incorporated into guide RNAs, in combination with a UV-activated photocleavable linker domain, PhoCl. Moreover, we demonstrate that Cas9 can readily be exchanged for other variants, including transcriptional activator dCas9-VPR and adenine base editor ABE8e. Taken together, we describe a robust, modular strategy for successful Cas9 delivery, which can be applied for CRISPR-Cas9-based genetic engineering and transcriptional regulation.},
}
@article {pmid41271814,
year = {2025},
author = {Ueno, R and Ito, S and Oyama, T},
title = {A CRISPR/Cas9-induced restoration of bioluminescence reporter system for single-cell gene expression analysis in plants.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41271},
pmid = {41271814},
issn = {2045-2322},
support = {JPMJSP2110//Japan Science and Technology Agency/ ; JPMJAL1108//Japan Science and Technology Agency/ ; JP20K06342//Japan Society for the Promotion of Science/ ; 17KT0022//Japan Society for the Promotion of Science/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Arabidopsis/genetics/metabolism ; *Single-Cell Analysis/methods ; *Genes, Reporter ; Plants, Genetically Modified/genetics ; *Luminescent Measurements/methods ; *Gene Expression Regulation, Plant ; Luciferases/genetics/metabolism ; Single-Cell Gene Expression Analysis ; },
abstract = {Bioluminescence monitoring techniques have greatly contributed to revealing a variety of biological regulatory systems in living organisms, including circadian clocks. In plant science, these techniques are applied to long-term quantitative analyses of gene expression behavior. Transient transfection with a luciferase reporter using the particle bombardment method has been used for bioluminescence observations at the single-cell level. This allows for capturing heterogeneity and temporal fluctuations in cellular gene expression, although bioluminescence could fluctuate according to variation in physiological factors associated with the luciferase reaction. We developed a novel CRISPR/Cas9-induced restoration of bioluminescence reporter system, CiRBS, to monitor cellular bioluminescence from a reporter gene in the genome of transgenic Arabidopsis. In this method, the enzymatic activity of an inactive luciferase mutant, LUC40Ins26bp, which has a 26-bp insertion at the 40th codon, was restored by introducing an indel at the insertion site using CRISPR/Cas9. We succeeded in long-term monitoring of the cellular bioluminescence of Arabidopsis plants expressing LUC40Ins26bp, which was restored by transient transfection with CRISPR/Cas9-inducible constructs using particle bombardment. Recombination events via indels were mostly complete within 24 h of CRISPR/Cas9 induction, and 7.2% of CRISPR/Cas9-transfected cells restored bioluminescence. It was estimated that 94% of the bioluminescence-restored cells carried only one chromosome having the optimal recombination construction. Thus, CiRBS allows for reliable single-cell gene expression analysis of cell-to-cell heterogeneity and temporal fluctuations from a single locus.},
}
@article {pmid41272236,
year = {2025},
author = {Pidishetty, D and Damera, SK and Murugavel, M and Susaimanickam, PJ and Chittajallu, SNSH and Kushawah, G and Sarkar, P and Bharadwaj, SR and Mishra, R and Mariappan, I},
title = {Loss of retinal stem cell reserve and lipofuscin accumulation accelerates cone-rod degeneration and replicates Stargardt disease in abca4b null zebrafish.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {44988},
pmid = {41272236},
issn = {2045-2322},
mesh = {Animals ; Zebrafish/genetics ; *Lipofuscin/metabolism ; *Stargardt Disease/genetics/pathology/metabolism ; *ATP-Binding Cassette Transporters/genetics ; Disease Models, Animal ; *Stem Cells/metabolism/pathology ; *Zebrafish Proteins/genetics/metabolism ; *Retina/pathology/metabolism ; Retinal Cone Photoreceptor Cells/metabolism/pathology ; Retinal Rod Photoreceptor Cells/metabolism/pathology ; Retinal Pigment Epithelium/metabolism/pathology ; Gene Knockout Techniques ; *Cone-Rod Dystrophies/pathology/metabolism/genetics ; CRISPR-Cas Systems ; Mutation ; *Macular Degeneration/genetics/pathology/metabolism ; },
abstract = {Mutations in ABCA4 gene causes Stargardt macular degeneration, which manifests with toxic lipofuscin deposits in the outer retina, gradual atrophy of RPE cells, followed by photoreceptor cell loss. The cone-enriched retina, with macula-like 'area-temporalis' of zebrafish are better models than rodents for studying human macular dystrophies. Here, we generated abca4b knockout zebrafish model using CRISPR/Cas9 editing and evaluated the early and late-stage retinal changes. In adult abca4b[-/-] mutants, the RPE cells exhibited hyperpigmentation, altered retinomotor behaviour and lipofuscin accumulation, but they remained viable. However, the photoreceptors underwent progressive degeneration, with a sequential loss of blue and UV cones, followed by red and green cones and finally the rod cells. This triggered the chronic activation and early depletion of retinal stem cells at the ciliary marginal zone of mutants and resulted in accelerated outer-retinal degeneration and severe visual defects, despite them retaining the Müller glia-dependant retinal repair potential.},
}
@article {pmid41272318,
year = {2025},
author = {Duan, B and Jin, X and An, X and Xiao, Y and Yang, Q and Zhao, H and Huang, Y and Wang, J and Wang, Q and Du, F and Lu, L and Sun, L and Chen, Z and Zhao, B},
title = {Molecular basis of SAM-AMP synthesis and degradation in the type III-B CRISPR-Cas system.},
journal = {Nature chemical biology},
volume = {},
number = {},
pages = {},
pmid = {41272318},
issn = {1552-4469},
support = {2021hwyq36//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
abstract = {Upon sensing nonself target RNA, the CorA-associated type III-B CRISPR-Cas system catalyzes S-adenosyl methionine (SAM) and ATP to synthesize SAM-AMP, which activates the effector CorA and triggers immune responses. SAM-AMP can be degraded by NrN and SAM lyase, potentially deactivating the system. Here we find that the type III-B effector complex from Bacteroides fragilis uses a specific mechanism to recognize nonself target RNA and synthesize SAM-AMP. The 3' anti-tag of nonself target RNA induces conformational changes in the Cmr2 subunit, triggering SAM-AMP synthesis independently of the stalk loop of Cmr3 subunit. SAM-AMP binding induces NrN to transit from an open to a closed conformation, enabling hydrolysis of the 3'-5' phosphodiester bond. SAM lyase forms a triangular trimer that specifically degrades SAM-AMP into 5'-methylthioadenosine-AMP and homoserine lactone. These findings unveil unique mechanisms for SAM-AMP synthesis and degradation and provide deeper insights into the molecular basis of type III CRISPR-Cas signaling.},
}
@article {pmid41272938,
year = {2025},
author = {Salvador, PJ and Lin, S and Chinn, MM and Jauregui-Matos, V and Manjunath, A and Yang, I and Jacobsen, CS and Beal, PA},
title = {Discovery and Tuning of RNA Editing Guides via High-Throughput Screening and Chemical Modification.},
journal = {Chembiochem : a European journal of chemical biology},
volume = {26},
number = {24},
pages = {e202500735},
doi = {10.1002/cbic.202500735},
pmid = {41272938},
issn = {1439-7633},
mesh = {Humans ; *RNA Editing ; *High-Throughput Screening Assays ; HEK293 Cells ; Adenosine Deaminase/metabolism/genetics ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Structure-Activity Relationship ; RNA-Binding Proteins/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry ; },
abstract = {Adenosine deaminases acting on RNA (ADAR) catalyze the deamination of adenosine to inosine in double-stranded RNA. Because inosine is read as guanosine during translation, this process enables programmable A-to-G recoding at the transcript level. ADARs can be harnessed for therapeutic correction of pathogenic mutations through site-directed RNA editing with guide RNAs. To expand the design space of editing-enabling guides, we applied EMERGe, a high-throughput screening platform, to identify motifs targeting a premature termination codon in the MeCP2 transcript associated with Rett syndrome. This uncovered a guide RNA motif that supported efficient ADAR2-mediated editing in vitro, featuring a 5'-GUG-3' sequence predicted to form an asymmetric loop. To enable therapeutic application, structure-activity relationship studies and chemical optimization were performed, yielding a fully modified guide RNA with 2'-O-methyl, 2'-fluoro, and phosphorothioate linkages. This stabilized guide retained the activity of unmodified RNA and showed enhanced nuclease resistance. The optimized guide induces dose-dependent editing at two MECP2 loci in reporter assays in HEK293T cells, demonstrating that EMERGe-selected motifs can be rendered viable in cells through targeted chemical modification. These findings highlight the utility of EMERGe as a discovery platform and establish a pipeline for identifying and optimizing editing-enabling guide RNA features beyond traditional design rules.},
}
@article {pmid41273185,
year = {2025},
author = {Verma, A and Kaur, L and Kandoth, PK},
title = {Agrobacterium rhizogenes-Mediated Hairy Root Transformation for Genome Editing in Recalcitrant Legume Lathyrus sativus.},
journal = {Current protocols},
volume = {5},
number = {11},
pages = {e70256},
doi = {10.1002/cpz1.70256},
pmid = {41273185},
issn = {2691-1299},
mesh = {*Lathyrus/genetics ; *Agrobacterium/genetics ; *Plant Roots/genetics ; *Gene Editing/methods ; *Transformation, Genetic ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Lathyrus sativus, commonly known as the grass pea, is a nutritious legume that is resilient to climate change, allowing it to grow in drought, waterlogged, and saline soils. However, developing effective functional genomic tools for this crop has been challenging, primarily due to the absence of reliable and stable transformation protocols. Agrobacterium rhizogenes-mediated hairy root transformation provides a practical and rapid method for validating gene functions using the CRISPR/Cas system. This method has not been applied to grass pea despite its potential. In this article, we present the first protocol for A. rhizogenes-mediated hairy root transformation and CRISPR/Cas genome editing aimed at the functional characterization of candidate genes in L. sativus. © 2025 Wiley Periodicals LLC. Basic Protocol 1: Designing CRISPR/Cas9 construct for targeted gene editing in L. sativus Support Protocol 1: Escherichia coli competent cell preparation and transformation Support Protocol 2: A. rhizogenes competent cell preparation and transformation Basic Protocol 2: A. rhizogenes-mediated hairy root transformation in L. sativus Basic Protocol 3: Screening of transgenic hairy root lines Support protocol 3: DNA isolation from L. sativus hairy roots.},
}
@article {pmid41273433,
year = {2025},
author = {Schreiber, D and Yang, R and Guan, X and Schalper, KT and Hou, C and Li, Z and Hegde, P and Liu, C},
title = {3D-Printed CRISPR-based detection system powered by a reusable handwarmer.},
journal = {Biomedical microdevices},
volume = {27},
number = {4},
pages = {53},
pmid = {41273433},
issn = {1572-8781},
support = {U01 CA269147/CA/NCI NIH HHS/United States ; U01CA269147/NH/NIH HHS/United States ; UConn Research Excellence Program (REP) award//University of Connecticut/ ; },
mesh = {*Printing, Three-Dimensional ; Humans ; Human papillomavirus 16/genetics ; *CRISPR-Cas Systems/genetics ; *Lab-On-A-Chip Devices ; DNA, Viral/analysis/genetics ; Equipment Reuse ; },
abstract = {Nucleic acid-based molecular diagnostics are essential for the prevention, early detection, and treatment of cancer and infectious diseases. In this study, we developed a 3D-printed, electricity-free detection system for CRISPR-based nucleic acid detection. To eliminate the need for costly electrical heaters, we developed a reusable heating platform powered by a sodium acetate-based handwarmer. To maintain optimal temperatures for the CRISPR reaction, we designed and fabricated a 3D-printed heatsink filled with docosane wax to regulate the temperature. The fully 3D-printed microfluidic chip integrates finger-activated fluid transport via a 3D-printed flexible blister, a CRISPR reaction chamber, and a lateral flow strip for visual readout. We demonstrated the system's analytical performance by detecting HPV-16 DNA with a sensitivity as low as 1 femtomolar. Additionally, we validated its clinical pilot feasibility using clinical cervical samples, achieving results consistent with standard PCR assays. Overall, this low-cost, reusable, and electricity-free detection system offers a practical solution for point-of-care molecular testing, particularly in resource-limited settings.},
}
@article {pmid41273435,
year = {2025},
author = {Yu, SM and Li, TT and Fu, BQ and Zhang, NZ},
title = {Molecular diagnosis of Trichinella spp.: current status and future prospects.},
journal = {Parasitology research},
volume = {124},
number = {11},
pages = {136},
pmid = {41273435},
issn = {1432-1955},
support = {2023YFD1801000//National Key Research and Development Program of China/ ; QYXTZX-RKZ2024-03-3//Science and Technology Project of Tibet Autonomous Region/ ; },
mesh = {Animals ; *Trichinellosis/diagnosis/parasitology/veterinary ; *Trichinella/genetics/isolation &amp; purification ; *Molecular Diagnostic Techniques/methods/trends ; CRISPR-Cas Systems ; Humans ; },
abstract = {Trichinellosis, a significant parasitic zoonotic disease, poses a risk to public health as well as economic implications for the safety of animal feed. Consumption of raw or undercooked meat containing Trichinella larvae can lead to trichinellosis infection. Many molecular diagnostic methods have been developed to identify Trichinella spp. at muscle or intestine stages. However, no molecular diagnostic technique is currently advised for routine testing on Trichinella infection in food animals, particularly at the early stage of infection. Here, the authors review the development of molecular diagnostic techniques of Trichinella spp., such as PCR, RT-PCR, LAMP, RPA and other methods to detect Trichinella DNA. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR-Cas) technology holds great promise for diagnostic testing by providing rapid, sensitive and specific methods for detection. Diagnosis of Trichinella spp. based on CRISPR-Cas system may be a promising method meeting the needs of individual testing.},
}
@article {pmid41273791,
year = {2025},
author = {Espinoza-Erazo, VP and Vela-Chauvin, MG and Collantes-Vela, JC and Zapata-Mena, S and Machado, A},
title = {Biofilms of Salmonella: Implications for Food Safety and Public Health.},
journal = {Foodborne pathogens and disease},
volume = {},
number = {},
pages = {15353141251389597},
doi = {10.1177/15353141251389597},
pmid = {41273791},
issn = {1556-7125},
abstract = {Salmonella entericais a leading cause of foodborne illness worldwide, responsible for an estimated 93.8 million cases and approximately 155,000 deaths annually, according to the World Health Organization. This foodborne pathogen imposes a significant burden on public health and the global economy. A key factor contributing to the persistence and widespread impact of S. enterica is its potential to form biofilms, which may enhance its survival in clinical, industrial, and agricultural environments, making it a major and ongoing public health concern. Biofilms are structured microbial communities encapsulated in a self-produced extracellular matrix that protects against environmental stressors, disinfectants, and antimicrobial agents. This complex phenotype enables Salmonella to colonize food-contact surfaces, medical devices, and host tissues, hampering efforts to eliminate contamination and control transmission. The poultry industry, a key component of the global food supply, is particularly vulnerable to emerging Salmonella strains with increased virulence, stress tolerance, and disinfectant resistance, making biofilm control a top priority.This review aims to provide an updated and comprehensive overview of the mechanisms involved in Salmonella biofilm formation, its implications for food safety, and recent advances in detection and control strategies. Emerging technologies such as CRISPR-Cas systems are receiving particular attention due to their potential as precise molecular tools for investigating genes implicated in biofilm formation.By integrating current findings, this review underscores the urgent need for novel and effective strategies for biofilm control. It highlights the importance of a One Health approach that links human, animal, and environmental health to address the risks posed by Salmonella biofilms in the food production and public health sectors.},
}
@article {pmid41273852,
year = {2025},
author = {Gallucci, FP and de Camargo, JA and Viana, NI and Pimenta, RCA and Guimarães, VR and Candido, P and Leite, KRM and Nahas, WC and Dos Reis, ST},
title = {CRISPR/Cas9-mediated MMP-9 silencing inhibits bladder cancer T24 cell invasion and migration in vitro.},
journal = {Clinics (Sao Paulo, Brazil)},
volume = {80},
number = {},
pages = {100842},
pmid = {41273852},
issn = {1980-5322},
mesh = {Humans ; *Urinary Bladder Neoplasms/pathology/genetics/enzymology ; *Matrix Metalloproteinase 9/genetics/metabolism ; *Cell Movement/genetics ; *CRISPR-Cas Systems/genetics ; Cell Proliferation/genetics ; Cell Line, Tumor ; Neoplasm Invasiveness/genetics ; *Gene Silencing ; Apoptosis/genetics ; Blotting, Western ; Flow Cytometry ; Real-Time Polymerase Chain Reaction ; Gene Expression Regulation, Neoplastic ; },
abstract = {PURPOSE: Bladder Cancer (BCa) ranks as the tenth most common cancer worldwide, with high morbidity and mortality. Matrix Metalloproteinases (MMPs), especially MMP-9, are associated with tumor progression and metastasis. This study aimed to evaluate the effects of MMP-9 silencing using CRISPR-Cas9 in T24-luc bladder carcinoma cells.<br><br>METHODS: Guide RNAs (sgRNA1 and sgRNA2) targeting MMP-9 were cloned into pX-330 plasmids and transfected into T24-luc cells. Gene and protein expression were analyzed via RT-qPCR and Western blotting. Functional assays included flow cytometry for proliferation and apoptosis, colony formation, wound healing, and Matrigel&#x2122; invasion assays.<br><br>RESULTS: sgRNA2 significantly reduced MMP-9 gene expression, while both sgRNAs reduced protein expression. Edited cells showed decreased proliferation and colony formation, increased apoptosis, and reduced migration and invasion capacity.<br><br>CONCLUSION: CRISPR-Cas9-mediated silencing of MMP-9 inhibited cell proliferation, migration, invasion, and increased apoptosis in BCa cells, supporting that MMP-9 has an important effect on the progression of bladder cancer.},
}
@article {pmid41274245,
year = {2025},
author = {Shimura, R and Yamamoto, K and Chang, YH and Otaki, A and Goyama, S},
title = {Development of a CRISPR/Cas9-degron system that enables in vivo specific gene depletion in leukemia models.},
journal = {Biochemical and biophysical research communications},
volume = {793},
number = {},
pages = {153002},
doi = {10.1016/j.bbrc.2025.153002},
pmid = {41274245},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; Humans ; *Leukemia, Myeloid, Acute/genetics/pathology ; Histone-Lysine N-Methyltransferase/genetics ; Mice ; *Gene Editing/methods ; Cell Line, Tumor ; Disease Models, Animal ; CRISPR-Associated Protein 9 ; Degrons ; },
abstract = {The CRISPR/Cas9 system has transformed genome editing, yet precise temporal control of Cas9 activity remains challenging. We developed a Cas9-degron platform that couples degron-tagged Cas9 with a dTAG-based chemical degradation strategy. In the presence of dTAG, Cas9 is rapidly and near-completely degraded, preventing editing; upon dTAG withdrawal, Cas9 activity is restored, enabling precise temporal control. Using this system, we achieved selective in vivo gene depletion in acute myeloid leukemia (AML) models and confirmed that SETDB1, a histone H3K9 methyltransferase, is essential for the in vivo growth of both human (MOLM13) and murine (cSAM) AML cells. By maintaining SETDB1 intact prior to transplantation and depleting it afterward, we avoided culture-induced pre-selection bias inherent to sgRNA transduction and validated its critical role in AML progression within the in vivo context. The Cas9-degron retains activity and delivery efficiency comparable to conventional Cas9 in the absence of dTAG. Thus, this versatile system provides a superior alternative to conventional Cas9 and a powerful platform for in vivo CRISPR screening, gene function studies, and potentially temporally controlled gene therapy.},
}
@article {pmid41274609,
year = {2026},
author = {Lau, CH and Li, X and Liang, QL and Guo, R and Ren, X and Xu, Z and Zhu, Y and Lai, Y and Liu, G and Huang, Y and Wu, W and Zhu, H and Chen, J and Zhang, X},
title = {CRISPR technology for diagnosis and treatment of human brucellosis.},
journal = {Journal of microbiological methods},
volume = {240},
number = {},
pages = {107339},
doi = {10.1016/j.mimet.2025.107339},
pmid = {41274609},
issn = {1872-8359},
mesh = {Humans ; *Brucellosis/diagnosis/therapy/drug therapy/microbiology ; Animals ; *Brucella/genetics/isolation &amp; purification/drug effects ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Livestock/microbiology ; Point-of-Care Testing ; Anti-Bacterial Agents/therapeutic use ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The global burden of Brucella infection in livestock and human health is substantial, particularly in developing countries or rural areas. Currently, brucellosis diagnosis primarily relies on PCR, microbiological culture, and serological tests. However, these approaches have several drawbacks such as long experimental duration, lengthy procedure, low positive detection rates, high variability in results, interspecies cross-reactivity, and require expensive equipment and professional operators. Herein, we review how recent emerging CRISPR/Dx technology can address some of these shortcomings to realize field-deployable detection of Brucella in domestic animals and point-of-care testing (POCT) for human brucellosis. CRISPR technology has been successfully used to treat brucellosis by deleting or inactivating the genes associated with the Brucella replication or survival. Therefore, we also discuss how CRISPR technology can be potentially used to treat brucellosis, as antibiotic therapy may lose efficacy when encountering multidrug-resistant Brucella strains and the treatment is long-lasting in infected individuals to prevent relapse. Lastly, we critically discuss the advances, pitfalls, and future perspectives of CRISPR technology for the diagnosis and treatment of brucellosis in humans and livestock. Ultimately, the continued refinement of CRISPR technology will pave the road for field-deployable pathogen diagnostics and home self-tests of brucellosis to mitigate global Brucella infections.},
}
@article {pmid41275110,
year = {2025},
author = {Yadav, B and Sardar, S and Yadav, A and Kumari, A and Gautam, M and Mandlik, R and Arora, S and Kumar, S and Jewaria, PK and Sonah, H and Deshmukh, R and Chinnusamy, V and Ram, H},
title = {A CRISPR-Cas9 library to target putative redundant gene sets facilitates their functional exploration in grain development in rice.},
journal = {BMC plant biology},
volume = {25},
number = {1},
pages = {1769},
pmid = {41275110},
issn = {1471-2229},
support = {BT/PR53626/BSA/33/96/2024//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {*Oryza/genetics/growth &amp; development ; *CRISPR-Cas Systems/genetics ; *Edible Grain/genetics/growth &amp; development ; Gene Library ; *Genes, Plant ; Gene Editing ; Gene Knockout Techniques ; Seeds/growth &amp; development/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Advent of CRISPR-Cas9 library approach has revolutionized the field of high throughput targeted mutagenesis in plants. By identifying an sgRNA spacer that can target multiple paralogous genes in a genome, higher-order knockout plants can be developed. Using this concept, we developed ten CRISPR-Cas9 pool libraries and generated higher-order knockout plants in rice. Towards this, firstly we identified genome-wide sets of genes which are co-expressed and have high sequence similarity and can be targeted by a single sgRNA. Based on the expression pattern, these genes were divided into ten groups, and subsequently ten CRISPR-Cas9 plasmid libraries were developed. One such library designed against seed-expressed genes was transformed into rice and higher-order knockout plants were developed. Genotyping revealed that around 90% T0 plants had editing, and among the edited plants majority of them were higher-order knockouts. Phenotypic analysis in the next generation discovered functions of several seed specific genes in grain length, width, number and 100-grain weight. By analyzing single and double mutants for two Agenet domain-containing proteins, we have discovered an epistatic interaction between them for grain development. Further application of our approach will help to uncover hidden functions of the targeted genes and accelerate functional genomics research in rice. The CRISPR-Cas9 library is a useful approach to generate higher-order knockout mutants and identify functions of the targeted genes in rice.},
}
@article {pmid41275796,
year = {2025},
author = {Gao, Y and Zhao, L and Shi, J and Wang, B},
title = {NtQPT2 plays critical roles in nicotine biosynthesis and development of tobacco plant.},
journal = {Biochemical and biophysical research communications},
volume = {793},
number = {},
pages = {153030},
doi = {10.1016/j.bbrc.2025.153030},
pmid = {41275796},
issn = {1090-2104},
mesh = {*Nicotiana/growth &amp; development/genetics/metabolism/enzymology ; *Nicotine/biosynthesis ; *Pentosyltransferases/genetics/metabolism ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Plants, Genetically Modified ; },
abstract = {The enzyme quinolinate phosphoribosyltransferase (QPT), encoded by a small gene family in tobacco plant, plays a critical role in the biosynthesis of nicotine, a defensive pyridine alkaloid in Nicotiana species, in addition to its vital function in the NAD(P)(H) synthesis. Previous studies have demonstrated that two NtQPT genes (NtQPT1 and NtQPT2) are present in N. tabacum genome, and it has been believed that NtQPT1 is responsible for NAD(P)(H) synthesis and thus essential for primary metabolism, while NtQPT2 is specifically involved in nicotine biosynthesis. In this study, we generated knockout tobacco lines for NtQPT1 and NtQPT2 respectively using the CRISPR/Cas9-based genome-editing technology and found that knockout of NtQPT2 caused both dramatic reduction of nicotine biosynthesis and a retardation of plant development, indicating that NtQPT2 is important not only to nicotine biosynthesis, but also to the development of tobacco plant. Like NtQPT2, NtQPT1 was also found to contribute to nicotine biosynthesis although to a much lesser extent than NtQPT2. Meanwhile, knockout of NtQPT1 did not significantly affect plant growth. Together with the observation that NtQPT2's expression is remarkably higher than that of NtQPT1 in root, leaf, stem and flower of tobacco plant, it is reasonable to infer that their functional diversification on nicotine biosynthesis and tobacco plant growth may be attributed largely to their markedly different transcript abundance.},
}
@article {pmid41275813,
year = {2026},
author = {Zhang, Z and Zhang, T and Li, Z and Zeng, Z},
title = {CRISPR/Cas13a-based colorimetric biosensing platform for point-of-care detection of viral nucleic acids.},
journal = {Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy},
volume = {348},
number = {Pt 1},
pages = {127221},
doi = {10.1016/j.saa.2025.127221},
pmid = {41275813},
issn = {1873-3557},
mesh = {*Colorimetry/methods ; *SARS-CoV-2/genetics/isolation &amp; purification ; Humans ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *Dengue Virus/genetics/isolation &amp; purification ; *COVID-19/diagnosis/virology ; *Point-of-Care Systems ; *RNA, Viral/analysis/genetics ; G-Quadruplexes ; Limit of Detection ; Point-of-Care Testing ; DNA, Catalytic/chemistry ; Sensitivity and Specificity ; },
abstract = {Rapid and accurate diagnosis is important in preventing and effectively combating infectious disease outbreaks. The CRISPR/Cas13a-based Specific High-sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) platform possesses the advantages of high efficiency, good specificity and sensitivity, and it has been widely adopted in molecular diagnostics. However, the traditional SHERLOCK platform requires dual-labeled RNA probes for fluorescence detection or lateral flow assay, which entail tedious modification procedures and sophisticated optical instruments, limiting its broad applications. Herein, we developed a rapid, sensitive, and label-free point-of-care (POC) platform for colorimetric assays of dengue virus (DV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with the SHERLOCK method. The adoption of the SHERLOCK-mediated guanine-quadruplex (G4)/hemin DNAzyme-based colorimetric strategy produced cascade signal amplification detection with improved analytical performance. Moreover, it exhibited high sensitivity and specificity for detection in cell-cultured DV samples, and DV and SARS-CoV-2 clinical samples, as well as accurate identification of the four DV serotypes. Hence, the proposed colorimetric biosensing platform has great potential for rapid, accurate, and specific POC detection of viral infections in field-deployable assay.},
}
@article {pmid41276041,
year = {2025},
author = {Khan, MF and Javed, M and Kaur, J and Badwal, AK and Singh, S},
title = {CRISPR-Cas mediated targeting of resistance genes for combating ESKAPE pathogen infections: A Review.},
journal = {International journal of biological macromolecules},
volume = {334},
number = {Pt 2},
pages = {149180},
doi = {10.1016/j.ijbiomac.2025.149180},
pmid = {41276041},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems ; Humans ; *Bacterial Infections/microbiology/genetics/therapy ; *Bacteria/genetics/drug effects ; *Drug Resistance, Bacterial/genetics ; Gene Editing/methods ; Bacteriophages/genetics ; *Drug Resistance, Multiple, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; },
abstract = {Advancements in the treatment of antimicrobial infections have highlighted the importance of the CRISPR-Cas system in targeting resistance genes in bacterial pathogens resistant to conventional drugs. Various CRISPR-Cas techniques, such as CRISPR-Cas9, Cas3, dCas9 and the mini-CRISPR system, have been utilized for this purpose in ESKAPE pathogens. Novel strategies like Associates Toxin Antitoxin and CRISPR-Cas to kill multidrug resistant pathogens-CRISPR-regulated toxin antitoxin module (ATTACK-CreTA) and CRISPR interference refine CRISPR-Cas efficacy. This review explores the mechanism of action of resistance genes (e.g., tetM, ermB, VanA, aph-3, aac3, oxa23, blaNDM etc.) prevalent within these pathogens and highlights the notable achievements of CRISPR-Cas technology in targeting these genes, thereby offering a pathway to sensitize resistant bacteria. This article also discusses various delivery approaches for CRISPR components in pathogens, mainly focusing on engineered bacteriophages, including phagemids, temperate phages and virulent phages. Additionally nanoparticles, bacterial conjugation and natural phages hold promise for administering the CRISPR system inside bacteria. Specific targeting of resistance genes in resistant pathogens via CRISPR-Cas based methods would pave a way for combating ESKAPE pathogen infections by reversing the resistance phenotype.},
}
@article {pmid41276526,
year = {2025},
author = {Murray, R and Chowdhury, MR and Botticello-Romero, NR and Desai, K and Chilakapati, SR and Chong, B and Xia, Y and Messana, A and Sobon, H and Rocha, J and Musenge, F and Camblin, A and Ciaramella, G and Sitkovsky, MV and Maldini, CR and Hatfield, SM},
title = {Multiplex gene-editing strategy to engineer allogeneic EGFR-targeting CAR T-cells with improved efficacy against solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11593},
pmid = {41276526},
issn = {2041-1723},
mesh = {Animals ; Humans ; ErbB Receptors/metabolism/genetics/immunology ; *Receptors, Chimeric Antigen/genetics/immunology/metabolism ; Mice ; *Gene Editing/methods ; Tumor Microenvironment/immunology ; *Immunotherapy, Adoptive/methods ; Xenograft Model Antitumor Assays ; Cell Line, Tumor ; *T-Lymphocytes/immunology/metabolism ; CRISPR-Cas Systems/genetics ; Female ; *Lung Neoplasms/therapy/immunology/genetics ; Receptor, Transforming Growth Factor-beta Type II/genetics ; *Neoplasms/therapy/immunology ; Graft vs Host Disease/prevention &amp; control/immunology ; Mice, Inbred NOD ; },
abstract = {Chimeric Antigen Receptor (CAR) T cells have induced remarkable clinical responses in patients with hematological cancers. However, CAR T-cell therapies against solid tumors have not elicited similar outcomes since immunosuppressive barriers in the tumor microenvironment attenuate anti-tumor activity. Here, we describe a multifaceted approach to engineer allogeneic CAR T-cells resistant to both biochemical (hypoxia-adenosinergic) and immunological (PD-L1 and TGF-β) inhibitory signaling using an adenine base editor and a CRISPR-Cas12b nuclease. The resulting EGFR-targeting CAR T-cell product comprised a combination of six gene edits designed to evade allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E) and overcome biochemical (ADORA2A) and immunological (PDCD1, TGFBR2) barriers in solid tumor microenvironment of subcutaneously grown EGFR[+] human lung tumor xenografts. This combinatorial genetic disruption enhances CAR T cell effector function and anti-tumor efficacy leading to improved tumor elimination and survival in xenograft and humanized mouse solid tumor models. Our strategy confers CAR T cells resistance to multiple clinically relevant inhibitory signaling pathways that are amplified in hypoxic tumor areas and may improve the therapeutic potential of CAR T-cells against solid tumors.},
}
@article {pmid41276731,
year = {2025},
author = {Eren Eroglu, AE and Toklu, K and Yasa, &#x130;},
title = {Functional genomics of a food-related thermotolerant Acetobacter oryzifermentans strain AAB5: genetic determinants of stress response, CAZyme repertoire, and CRISPR-Cas system.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {253},
pmid = {41276731},
issn = {1438-7948},
}
@article {pmid41276909,
year = {2026},
author = {Fokin, AI and Lin, Y and Guschin, DY and Chen, HY and James, J and Yan, J and Silberzan, P and Gautreau, AM},
title = {Identification of PKN2 and MOB4 as Coordinators of Collective Cell Migration.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e02907},
pmid = {41276909},
issn = {2198-3844},
support = {ANR-20-CE13-0016//Agence Nationale de la Recherche/ ; ANR-22-CE13-0041//Agence Nationale de la Recherche/ ; ANR-24-CE44-4957//Agence Nationale de la Recherche/ ; 2021 060003815//Fondation ARC pour la Recherche sur le Cancer/ ; INCA_16712//Institut National du Cancer/ ; ANR-11-LABX-0038//Labex Cell(n)Scales/ ; ANR-10-IDEX-0001-02//Labex Cell(n)Scales/ ; //"Institut Pierre-Gilles de Gennes" laboratoire d'excellence/ ; ANR-10-IDEX-0001-02 PSL//"Investissements d'avenir" program/ ; ANR-10-LABX-31//"Investissements d'avenir" program/ ; //French National Cancer Institute/ ; NRF-MSG-2023-0001//National Research Foundation Singapore/ ; },
mesh = {*Cell Movement/physiology/genetics ; Humans ; *Adaptor Proteins, Signal Transducing/metabolism/genetics ; Adherens Junctions/metabolism ; Wound Healing ; CRISPR-Cas Systems ; Protein Kinase C ; },
abstract = {In animals, collective cell migration is critical during development and adult life for repairing organs. It remains, however, poorly understood compared with single-cell migration. The polymerization of branched actin by the RAC1-WAVE-Arp2/3 pathway is established to power membrane protrusions at the front of migrating cells, but also to maintain cell junctions in epithelial monolayers. Here, novel regulators of collective cell migration are identified using a two-pronged approach: candidates are extracted from publicly available RAC1-WAVE-Arp2/3 dependency maps and screened in a second step using CRISPR/Cas9 genetic inactivation. In a wound healing assay, PKN2 knockout (KO) MCF10A cells display decreased collective migration due to destabilization of adherens junctions, whereas MOB4 KO cells display increased collective migration with a loss of migration orientation. Upon wound healing, PKN2 relocalizes to lateral junctions and maintains coordinated migration in the monolayer, whereas MOB4 relocalizes to the front edge of leader and follower cells collectively migrating toward the wound. The role of MOB4 in controlling collective migration requires YAP1, since MOB4 KO cells fail to activate YAP1, and their phenotype is rescued by constitutively active YAP1. Together, these findings reveal two complementary activities required for coordinating cells in collective migration.},
}
@article {pmid41276924,
year = {2026},
author = {Yun, S and Kim, S and Kim, S and Noh, M and Kim, DK and Lee, EJ and Lee, H},
title = {Parallel Genome-Wide CRISPR Screens Reveal SORL1 and ZFYVE19 as Sequential Host Determinants of Salmonella Infection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e15042},
pmid = {41276924},
issn = {2198-3844},
support = {NRF-2022R1A2B5B02002256//National Research Foundation of Korea/ ; NRF-2022R1A4A1025913//National Research Foundation of Korea/ ; NRF-RS-2025-00561488//National Research Foundation of Korea/ ; NRF-RS-2024-00350131//National Research Foundation of Korea/ ; NRF-RS-2025-02219421//National Research Foundation of Korea/ ; RS-2025-16652968//MSIT/ ; },
mesh = {Humans ; *Salmonella Infections/genetics/microbiology/metabolism ; *Host-Pathogen Interactions/genetics ; *Membrane Transport Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Salmonella enterica/pathogenicity/genetics ; CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Salmonella enterica, a major cause of gastroenteritis and typhoid fever, hijacks host machinery to invade cells, and replicate within a specialized niche. While some host factors are known, a comprehensive, temporally-resolved understanding of the host-pathogen interface has been hindered by a lack of suitable genome-wide methodologies. To address this, a parallel CRISPR screening platform is developed to identify host determinants for distinct infection stages. An invasion screen captured factors for bacterial entry, while a fitness screen identified factors governing long-term survival. The screens reveal a temporal switch in host dependency, from endosomal trafficking in early infection to cell cycle and DNA damage response pathways governing host cell fitness in long-term infection. Notably, the approach uncovers two novel host factors with stage-specific roles, SORL1 as a mediator of bacterial invasion and ZFYVE19 as a factor supporting intracellular proliferation. Genetic disruption of SORL1 or ZFYVE19 validate these roles, leading to impaired invasion or replication, respectively. Importantly, antibody-mediated blockade of SORL1 effectively prevented Salmonella entry, highlighting it as a novel host-directed therapeutic target. Together, the screening strategy provides a powerful framework for the temporal dissection of host-pathogen interactions, revealing novel biology and promising therapeutic targets.},
}
@article {pmid41276946,
year = {2026},
author = {Pal, T and Liu, Z and Nair, MG and Chen, J},
title = {CRISPR in MOF Formulation with Enhanced Stability, Activity, and Altered PAM Specificity for Broad-Spectrum Diagnosis of Bacterial Sepsis.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {8},
pages = {e13439},
pmid = {41276946},
issn = {2198-3844},
support = {R35 GM147069/GM/NIGMS NIH HHS/United States ; R35GM147069/GM/NIGMS NIH HHS/United States ; },
mesh = {*Sepsis/diagnosis/microbiology/genetics ; Humans ; *Metal-Organic Frameworks/chemistry ; *CRISPR-Cas Systems/genetics ; RNA, Ribosomal, 16S/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Sepsis is a life-threatening condition caused by polymicrobial infections and remains a global health emergency that requires rapid and broad-spectrum diagnostics. Existing CRISPR-based assays face two major limitations that restrict their application for sepsis: narrow protospacer adjacent motif (PAM) site compatibility and poor enzyme stability under clinical and environmental stresses. A modular diagnostic platform is presented, CRISPR-FLEXMO (CRISPR with flexible PAM in metal-organic framework encapsulation, MOF), which integrates a PAM-relaxed Cas12a variant (K607R) with a manganese-coordinated MOF (Mn-MOF) for stable and specific detection of sepsis-causing bacteria. The system targets a conserved region upstream of the Shine-Dalgarno sequence in the 16S rRNA gene containing a universal TTCC PAM, enabling broad-spectrum detection with a single universal primer pair across Gram-negative and Gram-positive pathogens. The K607R variant shows enhanced cis- and trans-cleavage activity, while Mn-MOF encapsulation maintains enzyme functionality under ambient, thermal, and chaotropic stress. The assay detects as low as 10 CFU mL[-1] in bacterial lysates following amplification and achieves 100% sensitivity and specificity in serum samples from 15 sepsis patients and 3 healthy individuals, with no cross-reactivity to six respiratory viruses. The platform retains over 78% activity after 12 weeks of room-temperature storage, offering a field-deployable CRISPR diagnostic solution for next-generation infectious disease detection.},
}
@article {pmid41277071,
year = {2025},
author = {Hotta, M and Inoue, YU and Asami, J and Hoshino, M and Inoue, T},
title = {Generation of a Triple Tag Knock-In Mouse to Visualize Precise Protein Localization Patterns for Type II Classic Cadherins During Brain Development.},
journal = {Genes to cells : devoted to molecular &amp; cellular mechanisms},
volume = {30},
number = {6},
pages = {e70070},
doi = {10.1111/gtc.70070},
pmid = {41277071},
issn = {1365-2443},
support = {NCNP 3-9//Intramural Research Grant for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry/ ; NCNP 6-9//Intramural Research Grant for Neurological and Psychiatric Disorders of National Center of Neurology and Psychiatry/ ; },
mesh = {Animals ; *Cadherins/metabolism/genetics ; Mice ; *Brain/metabolism/embryology ; Gene Knock-In Techniques ; Gene Expression Regulation, Developmental ; Mice, Transgenic ; CRISPR-Cas Systems ; },
abstract = {Classic cadherin cell-cell adhesion molecules with self-organizing activities play roles in segregating distinct populations of cells at developing brain regions and/or boundaries. However, the protein dynamics of each cadherin subclass in the mouse embryonic brain is poorly described due to the low antigenicity. Here, we generate Cdh6-HA and Cdh8-PA tag knock-in (KI) mice by CRISPR/Cas9-mediated genome editing and establish Cdh6[HA/HA]; Cdh8[PA/PA]; Cdh11[EGFP/EGFP] triple tag KI homo mice with normal viability and fertility. Immunostaining with specific antibodies for these tags reveals differential protein expression profiles almost comparable with mRNA in situ hybridization (ISH) results during embryonic brain development. We can additionally detect considerable levels of immunostaining signals outside the mRNA ISH-positive areas, specifically along the nerve tracts, suggesting physiological accumulation of these type II cadherin proteins along axons. By using super-resolution imaging, we further evaluate cadherin subcellular localization dynamics around the zona limitans intrathalamica to confirm that the prosomere 2/3 compartment boundary at E12.5 is maintained by the distinctive integration of Cdh6 or Cdh11 at apical attachment sites of the ventricular cells. These results highlight the value of the genetic tag KI strategy for proteins with low antigenicity and the functional relevance of type II classic cadherins in brain development.},
}
@article {pmid41277095,
year = {2025},
author = {Thornbury, M and Omran, RP and Kumar, L and Knoops, A and Abushahin, R and Whiteway, M and Martin, VJJ},
title = {Tri-Functional CRISPR Screen Reveals Overexpression of QDR2 and QDR3 Transporters Increase Fumaric Acid Production in Kluyveromyces marxianus.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4907-4922},
doi = {10.1021/acssynbio.5c00689},
pmid = {41277095},
issn = {2161-5063},
mesh = {*Fumarates/metabolism ; *Kluyveromyces/metabolism/genetics ; *Fungal Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Metabolic Engineering/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; *Membrane Transport Proteins/genetics/metabolism ; },
abstract = {Organic acids such as fumaric acid are widely used in the food and beverage industry as acidulants and preservatives, while also serving as versatile precursors for industrially relevant compounds. Fumaric acid is still predominantly produced through petroleum-derived processes. To enhance production efficiency and diversify supply, we are engineering Kluyveromyces marxianus as a biosynthetic platform from renewable feedstocks. In previous work, we have established K. marxianus Y-1190 as a host for lactose valorization based on its high growth rate on lactose and its tolerance for acid conditions. Here, we establish a trifunctional genome-wide library for K. marxianus using CRISPR activation, interference, and deletion to allow identification of gene expression perturbations that enhance tolerance to fumaric acid. We determined that deletion of ATP7, encoding a subunit of the mitochondrial F1F0 ATP synthase, and overexpression of QDR2 and QDR3, two previously uncharacterized members of the 12-spanner H[+] antiporter (DHA1) family in K. marxianus, can enhance fumaric acid tolerance. We also found that integrated overexpression of both QDR2 and QDR3 in a ΔFUM1 background strain improved titers of fumaric acid production from 0.26 to 2.16 g L[-1]. Together, these results highlight roles for membrane transport and mitochondrial function in enabling fumaric acid tolerance and production in K. marxianus.},
}
@article {pmid41277417,
year = {2026},
author = {Choi, MY and Li, C and Choi, JH and Choi, JW},
title = {Intracellular biosensors by functional nanomaterial-integrated CRISPR technologies for real-time molecular sensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {1},
pages = {58-70},
doi = {10.1039/d5cc05016b},
pmid = {41277417},
issn = {1364-548X},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Nanostructures/chemistry ; Gene Editing ; Nanotechnology ; },
abstract = {CRISPR technology, originally developed as a genome-editing tool, has recently emerged as a powerful platform for intracellular biosensing. By harnessing the programmability and target specificity of CRISPR-associated proteins, such as Cas9, Cas12, and Cas13, researchers have engineered biosensors capable of detecting a wide array of intracellular signals, including nucleic acids, non-coding RNAs, and small-molecule metabolites. This review discusses the recent advancements in CRISPR-powered biosensors for real-time, dynamic monitoring of cellular processes and molecular events. Particular focus is given to the integration of nanotechnology, which plays a crucial role in enhancing the delivery efficiency, signal amplification, and sensor stability. Nanomaterials such as gold nanoparticles, quantum dots, DNA nanostructures, and upconversion nanoparticles have been strategically employed to improve the intracellular transport of CRISPR components, facilitate signal readouts, and enable multimodal sensing in complex cellular environments. Additionally, we delve into how CRISPR-nanotechnology hybrids can be adapted for multiplex analysis and single-cell resolution. This review also addresses the current challenges in intracellular biosensing, including precise delivery, biocompatibility, and long-term monitoring, and outlines future directions for the application of these systems in precision medicine, synthetic biology, and advanced therapeutic monitoring. Through the convergence of gene-editing systems and nanotechnology, CRISPR-based intracellular biosensors are anticipated to revolutionize next-generation diagnostic and therapeutic strategies.},
}
@article {pmid41277604,
year = {2026},
author = {Xu, M and Yan, L and Zhu, M and Zhan, Z and Chen, H and Wang, D and Zheng, Z and Zhang, Y and Xiong, L and He, Y},
title = {TKC-MC: An Effective Strategy for Generating Heritable Heterozygous Mutations in Essential Genes in Rice.},
journal = {Plant biotechnology journal},
volume = {24},
number = {4},
pages = {2092-2104},
doi = {10.1111/pbi.70472},
pmid = {41277604},
issn = {1467-7652},
support = {2023ZD04074//Biological Breeding-Major Projects/ ; 32200335//National Natural Science Foundation of China/ ; U21A20207//National Natural Science Foundation of China/ ; Y2023QC39//Youth innovation Program of Chinese Academy of Agricultural Sciences/ ; YBXM2504//Nanfan special project of CAAS/ ; YBXM2505//Nanfan special project of CAAS/ ; CARS-01//Earmarked Fund for China Agriculture Research System/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Heterozygote ; Plants, Genetically Modified/genetics ; *Mutation/genetics ; *Genes, Essential/genetics ; *Genes, Plant/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The CRISPR/Cas9 gene-editing technology has been widely used in defining gene functions and crop improvement. However, some genes are essential for plant growth and development. Loss-of-function homozygous mutations in essential genes lead to plant death or sterility. Mutations in essential genes need to be maintained and propagated in heterozygous plants. CRISPR/Cas9 technology is highly efficient in generating homozygous or bi-allelic mutations at T0 generation in rice, making it difficult to generate useful genetic materials for essential genes using traditional gene editing technology. In this study, we designed Transgene-Killer CRISPR (TKC)-mediated mismatch-spacer targeting (TKC-M) to efficiently generate heritable heterozygous mutations in essential genes in rice. Leveraging our earlier transgenic offspring self-elimination TKC platform, TKC-M relied on timely self-elimination of Cas9 and engineered gRNA-target mismatches to enrich heritable heterozygous or mosaic incomplete-edited T0 mutants and heterozygous progeny. We found that the sensitivity of targets to spacer mismatch(es) varies. A single-base mismatch at gRNA positions 11 or 17 yielded abundant heritable heterozygotes in sensitive targets. For insensitive targets, dual mismatches at positions 8 and 15 maximised heritable heterozygotes. Co-transformation of rice with TKC vectors carrying gRNA without mismatches (G1), gRNA with a mismatch at position 11 (M11) and M8 + M15 spacers, termed TKC-M Cocktail (TKC-MC) significantly increased the incomplete-edited mutant ratio compared with using G1 alone. This work establishes a technical foundation for generating mutant libraries that cover every single gene in a plant genome and for in-depth research on essential genes.},
}
@article {pmid41277686,
year = {2025},
author = {Stohr, AM and Hansen, H and Richards, B and Park, H and Goncalves, AG and Agrawal, A and Blenner, M and Chen, W},
title = {Metabolite-responsive scaffold RNAs for dynamic CRISPR transcriptional regulation.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41277686},
issn = {1362-4962},
support = {MCB2317398//National Science Foundation/ ; GM133803/NH/NIH HHS/United States ; //U.S. Department of Defense/ ; P200A210065//GAANN Fellowship Program/ ; },
mesh = {*CRISPR-Cas Systems ; Theophylline/metabolism/pharmacology ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Tryptophan/metabolism/pharmacology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Transcriptional Activation ; Transcription, Genetic ; *Gene Expression Regulation ; },
abstract = {CRISPR activation is a powerful tool to upregulate a vast array of genes in many different contexts. However, there are few dynamic CRISPR transcriptional programs, which limit its usage in the creation of living biosensors, self-regulating microbial factories, or conditional therapeutics. Here, we address this limitation by embedding a molecular switch directly into a guide RNA to create a combined sensor-actuator called a metabolite-responsive scaffold RNA (MR-scRNA). We demonstrate the regulatory potential for MR-scRNAs by conditionally activating genes in three different kingdoms of life. We create MR-scRNAs responsive to two distinct metabolites, theophylline and tryptophan, by swapping the molecular switch used. MR-scRNAs respond quickly in a dose-dependent manner specifically to their target metabolite and enhance biochemical production when used as a dynamic regulator of pathway enzyme expression. The broad functionality and ease of design of the MR-scRNAs offer a promising tool for dynamic cellular regulation.},
}
@article {pmid41277692,
year = {2025},
author = {Jang, H and Kang, JE and Kim, H and Kim, JR and Park, J and Go, SR and Lee, YH and Kang, H and Park, Y and Kim, S and Jung, Y and Kim, SJ and Lim, EK and Jung, J and Woo, EJ and Park, KH and Kang, T},
title = {CRISPR/Cas12a2 enables ultra-sensitive amplification-free RNA detection.},
journal = {Nucleic acids research},
volume = {53},
number = {21},
pages = {},
pmid = {41277692},
issn = {1362-4962},
support = {//National Research Foundation of Korea/ ; 2021M3H4A1A02051048//Ministry of Science and ICT/ ; 2023R1A2C2005185//Ministry of Science and ICT/ ; RS-2024-00438316//Ministry of Science and ICT/ ; RS-2024-00348576//Ministry of Science and ICT/ ; RS-2024-00459749//Ministry of Science and ICT/ ; RS-2025-00554718//Ministry of Science and ICT/ ; 2021M3A9G802559922//Ministry of Science and ICT/ ; RS-2022-NR071772//Ministry of Science and ICT/ ; RS-2021-NR059435//Ministry of Science and ICT/ ; CRC22024-500//Ministry of Science and ICT/ ; 2020R1A5A1018052//Ministry of Science and ICT/ ; GTL25061-000//Ministry of Science and ICT/ ; //Korea Evaluation Institute of Industrial Technology/ ; RS-2022-00154853//Ministry of Trade, Industry and Energy/ ; RS-2024-00432382//Ministry of Trade, Industry and Energy/ ; RS-2024-00403563//Ministry of Trade, Industry and Energy/ ; //Korea Environmental Industry and Technology Institute/ ; 2021003370003//Ministry of Environment/ ; //Korea Health Industry Development Institute/ ; RS-2025-02213315//Ministry of Health and Welfare/ ; RS-2024-00401639//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture/ ; KGM1322511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM1032511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM1062511//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; KGM5382531//Korea Research Institute of Bioscience and Biotechnology Research Initiative Program/ ; //Research Institute of Bioscience and Biotechnology/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *RNA, Viral/genetics/analysis ; *SARS-CoV-2/genetics/isolation &amp; purification ; *CRISPR-Associated Proteins/genetics/metabolism ; Humans ; *COVID-19/diagnosis/virology ; Limit of Detection ; Endodeoxyribonucleases ; RNA, Guide, CRISPR-Cas Systems/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; Bacterial Proteins ; },
abstract = {Advances in clustered regularly interspaced short palindromic repeat (CRISPR) technologies have led to the use of diverse CRISPR-associated (Cas) proteins in diagnostic applications. Herein, we present a CRISPR/Cas12a2-based amplification-free RNA detection method that exhibits sub-attomolar sensitivity and substrate versatility. Cas12a2, a recently characterized RNA-guided nuclease, uniquely integrates bimolecular recognition through CRISPR RNA (crRNA)-target complementarity and protospacer flanking sequence identification, enabling highly specific trans-cleavage of single-stranded DNA, double-stranded DNA, and RNA. We have optimized key biochemical parameters, including pH, ionic strength, and temperature, to enhance the catalytic efficiency of Cas12a2. Based on the optimal activity conditions of Cas12a2, we have achieved ultra-sensitive viral RNA detection with a limit of detection of 46.7 aM through the strategic design and cooperative activation of crRNAs targeting conserved regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. The diagnostic accuracy of the Cas12a2-based assay has been demonstrated for 26 SARS-CoV-2 variants, and it has further resulted in the definitive diagnosis of 317 clinical samples. This work establishes Cas12a2 as a promising molecular diagnostic tool that provides an amplification-free, rapid, and versatile solution for RNA detection. The adaptability and simplicity of the platform render it particularly well suited for point-of-care applications, paving the way for next-generation CRISPR diagnostics.},
}
@article {pmid41277735,
year = {2025},
author = {Zhang, L and He, X and Hu, J and Bai, H and Yao, Y and Hu, WW},
title = {Recent advances in nanozyme-CRISPR/Cas biosensors.},
journal = {Chemical communications (Cambridge, England)},
volume = {61},
number = {100},
pages = {19735-19749},
doi = {10.1039/d5cc05585g},
pmid = {41277735},
issn = {1364-548X},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Nanostructures/chemistry ; Humans ; },
abstract = {The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system, renowned for precise DNA recognition and potent trans-cleavage activity, has become a promising tool for biosensing. Nanozymes, a new class of artificial enzymes that combine the physicochemical properties of nanomaterials with the catalytic functions of natural enzymes, offer high surface area and versatile functionalization, enabling efficient target binding and catalysis to markedly boost biosensing performance. With advances across disciplines, the integration of nanozymes and CRISPR/Cas has become prominent in biosensing. Nanozyme-enhanced CRISPR/Cas biosensors can substantially improve detection sensitivity and expand sensing modes for bioanalysis, with potential for direct target detection without pre-amplification. In this review, we comprehensively examine recent strategies in nanozyme-enhanced CRISPR/Cas biosensing, with particular emphasis on advances in bioanalytical applications. We further critically assess the challenges and prospects of using nanozymes to improve CRISPR/Cas performance for biosensing and offer insights for the design of next-generation biosensors for precise, rapid, on-site detection of nucleic acids, proteins, and small molecules in clinical samples.},
}
@article {pmid41277755,
year = {2025},
author = {Wang, Z and Zhang, D and Wu, Y and Ye, X and Qian, Y and Chen, F},
title = {A capillary SERS sensor based on CRISPR/Cas13a and DS Au-AgNRs for detecting miRNA-221 in serum of hepatocellular carcinoma patients.},
journal = {Analytical methods : advancing methods and applications},
volume = {17},
number = {47},
pages = {9627-9637},
doi = {10.1039/d5ay01517k},
pmid = {41277755},
issn = {1759-9679},
mesh = {*MicroRNAs/blood/genetics ; Humans ; *Carcinoma, Hepatocellular/blood/diagnosis/genetics ; *Liver Neoplasms/blood/diagnosis/genetics ; *Spectrum Analysis, Raman/methods ; Gold/chemistry ; Silver/chemistry ; CRISPR-Cas Systems ; Nanotubes/chemistry ; Biomarkers, Tumor/blood ; Limit of Detection ; },
abstract = {The low early diagnosis rate of hepatocellular carcinoma (HCC) severely impacts patient prognosis, making the development of highly sensitive and specific early diagnostic technologies crucial. MicroRNA-221 (miR-221), an aberrantly overexpressed biomarker in HCC, holds significant diagnostic potential. This paper constructed a capillary surface-enhanced Raman scattering (SERS) sensing platform utilizing CRISPR/Cas13a trans-cleavage and double-shell gold-silver nanorods (DS Au-AgNRs) to detect serum miR-221 in HCC patients. DS Au-AgNRs were synthesized and assembled onto aminated capillaries, followed by conjugation of Cy5-labeled single-stranded DNA (ssDNA) to the DS Au-AgNR surface via Au-S bonds. In the presence of miR-221, activated CRISPR/Cas13a trans-cleavage cleaves the ssDNA, releasing Cy5 from the sensor surface and diminishing the SERS signal, enabling miR-221 quantification. The synthesized DS Au-AgNRs exhibit uniform morphology and size, are uniformly distributed on the capillary, and form numerous "hotspots", thereby significantly enhancing the SERS signal. According to the characteristic peak of Cy5 at 1074 cm[-1], a linear relationship is established between the log concentration of miR-221 and the measured SERS intensity (y = -3527.97 × -35369.60, R[2] = 0.97767), with a LOD as low as 4.17 × 10[-17] M. The sensor demonstrated high specificity and high sensitivity, and its capacity to detect miR-221 expression aligned with qRT-PCR results when analyzing serum samples, confirming that hepatocellular carcinoma patients exhibited significantly higher miR-221 levels compared to healthy individuals. The capillary SERS sensor thus provides an accurate and convenient approach for early HCC diagnosis.},
}
@article {pmid41279041,
year = {2025},
author = {Vera, AO and Avilés-Vázquez, FJ and Ha, T and Choudhary, A and Raines, RT},
title = {Nuclear Localization Signals Enable the Cellular Delivery of an Anti-CRISPR Protein to Control Genome Editing.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
doi = {10.1101/2025.10.28.685205},
pmid = {41279041},
issn = {2692-8205},
abstract = {Precise regulation of Cas9 activity is essential to minimize off-target effects, mosaicism, chromosomal alterations, immunogenicity, and genotoxicity in genome editing. Although type II anti-CRISPR proteins (Acrs) can inhibit and regulate Cas9, their size and anionic charge generally prevent them from crossing the cell membrane. Existing Acr delivery methods employing vectors or electroporation are either slow and persistent or require external equipment, limiting their therapeutic utility. To address these challenges, we developed a cell-permeable Acr (6×NLS-Acr), which uses nuclear localization signals (NLSs) to cross the cell membrane. We conjugated 6×NLS-Acr to a fluorescent dye to elucidate its cellular entry mechanism and directly visualized its binding to a fluorescent Cas9·gRNA complex to study its inhibitory mechanism. 6×NLS-Acr (IC 50 = 0.47 µM) directly transduces human cells, including immortalized cell lines, embryonic stem cells, and 3D cell cultures, within 5 min, inhibiting up to 99% of Cas9 activity and increasing genome-editing specificity by nearly 100%. We further compared 6×NLS-Acr with our anthrax-derived Acr delivery platform. Our results demonstrate that 6×NLS-Acr is the most efficacious cell-permeable CRISPR-Cas inhibitor, significantly enhancing the precision and therapeutic potential of CRISPR-based genome editing.},
}
@article {pmid41280279,
year = {2026},
author = {Li, X and Ye, C and Liu, T and Li, S and Zhang, M and Zhao, Y and Jin, Y and Cheng, J and Yang, G and Li, P},
title = {Engineering genetic elements for microbial protein expression systems: Advances, challenges, applications, and prospects.},
journal = {Synthetic and systems biotechnology},
volume = {11},
number = {},
pages = {370-384},
pmid = {41280279},
issn = {2405-805X},
abstract = {The rising global demand for sustainable protein sources poses critical challenges across food, pharmaceutical, and industrial biotechnology sectors. Microbial expression systems provide scalable and versatile platforms for producing recombinant proteins, including enzymes, therapeutic molecules, and functional food ingredients. These platforms enable efficient biosynthesis of high-value proteins from renewable substrates often via precision fermentation, surpassing conventional methods in yield, cost-efficiency, and environmental sustainability. This review summarizes the genetic regulatory elements that govern gene expression in microbial hosts, with comparative coverage of prokaryotic and eukaryotic systems at transcriptional and translational levels. Key regulatory components, such as promoters, ribosome binding sites (RBS), untranslated regions (UTRs), signal peptides, and terminators, are discussed in the context of host-specific engineering strategies. We highlight advanced engineering approaches, including artificial intelligence (AI) assisted sequence design, CRISPR-Cas-based genome editing, and modular combinatorial optimization of genetic elements. Particular attention is given to the integration of high-throughput screening and predictive modeling tools that accelerate the rational design and optimization of microbial production systems. The review also discusses practical applications in food, pharmaceutical, and industrial enzyme production, emphasizing how genetic element engineering bridges fundamental research and biomanufacturing. Finally, key challenges and future prospects are analyzed to guide the development of next-generation microbial cell factories for sustainable protein production and industrial innovation.},
}
@article {pmid41280798,
year = {2025},
author = {Fronza, F and Verardo, R and Schneider, C},
title = {RepFluo, a Fast Fluorescent In Vitro Assay of Cas9 Activity Exploiting Melting Curve Analysis.},
journal = {ACS omega},
volume = {10},
number = {45},
pages = {53816-53826},
pmid = {41280798},
issn = {2470-1343},
abstract = {Demand for less labor-intensive in vitro assays of the activity of CRISPR/Cas proteins is rising to extend the potential applications of CRISPR in the field of diagnostics. RNA guided DNA endonucleases of the Cas family generate double-strand breaks in the target DNA, which results in two shorter DNA fragments. We hypothesized that this cleavage event could be studied using melting curve analysis, and using SpyCas9, we demonstrate that it is possible to evaluate the activity of Cas proteins by measuring the melting curves of their products. We present here a novel assay for the in vitro activity of Cas9 that exploits melting curve analysis (MCA) to be fast, inexpensive, and widely accessible. The assay can, in fact, be performed with readily available components(?)in its simplest form a real-time thermal cycler and an intercalating dye (SYBR Green I)(?)and produces reliable results with a run-time of 15 min. It does not require external intervention to stop the reaction, which is done by thermal denaturation of the protein directly in the thermal cycler machine. The described advantages, combined with the provided data analysis package, make the assay robust and amenable to high-throughput applications. To increase the accessibility of our assay, we provided an R package that simplifies the analytical process.},
}
@article {pmid41281724,
year = {2025},
author = {Barraclough, A and Bär, I and van Duijl, T and Fijnvandraat, K and Eikenboom, JCJ and Leebeek, FWG and Bierings, R and Voorberg, J and Trasanidou, D},
title = {Correction: Rewriting the script: gene therapy and genome editing for von Willebrand Disease.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1719330},
doi = {10.3389/fgeed.2025.1719330},
pmid = {41281724},
issn = {2673-3439},
abstract = {[This corrects the article DOI: 10.3389/fgeed.2025.1620438.].},
}
@article {pmid41281758,
year = {2025},
author = {Jia, Z and Wu, J and Zhang, J and Zheng, P and Zhang, H and Lin, Y and Pan, T and Wu, M and Song, Y},
title = {Precision Reprogramming in CAR-T Cell Therapy: Innovations, Challenges, and Future Directions of Advanced Gene Editing.},
journal = {International journal of biological sciences},
volume = {21},
number = {15},
pages = {6884-6906},
pmid = {41281758},
issn = {1449-2288},
mesh = {Humans ; *Gene Editing/methods ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/genetics ; CRISPR-Cas Systems/genetics ; Neoplasms/therapy ; T-Lymphocytes ; Animals ; },
abstract = {Chimeric antigen receptor (CAR)-T cell therapy represents a breakthrough in cancer immunotherapy, demonstrating impressive clinical outcomes, particularly for hematologic malignancies. However, its broader therapeutic application, especially against solid tumors, remains limited. Key challenges include T cell exhaustion, limited persistence, cytokine-mediated toxicities, and logistical hurdles associated with manufacturing autologous products. Emerging gene editing technologies, such as CRISPR/Cas systems, base editing, and prime editing, offer novel approaches to optimize CAR-T cells, aiming to enhance efficacy while managing toxicity and improving accessibility. This review comprehensively examines the current landscape of these gene editing tools in CAR-T cell therapy, highlighting the latest advancements, persisting challenges, and future directions. Leveraging gene editing holds the potential to transform CAR-T therapy into a more potent, safer, and broadly applicable modality for cancer and beyond.},
}
@article {pmid41284414,
year = {2025},
author = {Iyer, KA and Tenchov, R and Lotti Diaz, LM and Jain, P and Thite, T and Deng, Y and Zhou, QA},
title = {CRISPR Technology: Transforming the Future of Medicine and Diagnostics.},
journal = {Biochemistry},
volume = {64},
number = {24},
pages = {4628-4660},
pmid = {41284414},
issn = {1520-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods/trends ; *Genetic Therapy/methods/trends ; Neoplasms/therapy/genetics/diagnosis ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Animals ; },
abstract = {In this report, we examine the extensive research landscape of CRISPR with an emphasis on CRISPR therapeutics and showcase our results from an in-depth analysis of the most up-to-date scientific information consisting of more than 53,000 publications encompassing academic journal articles and patents, spanning nearly three decades, extracted from the CAS Content Collection. Our analysis indicates that cancer and infectious diseases are the most explored in the context of CRISPR. Identified gene targets associated with CRISPR-related publications are led by TP53, c-myc, and hemoglobin beta subunit (HBB). Among the many delivery methods, adeno-associated vectors (AAVs) appear to be highly explored. With >140 CRISPR-based therapeutics in the clinical development pipeline and billions of dollars in investment, the field of CRISPR continues to evolve rapidly. We also briefly discuss the ethical implications of CRISPR technology. While some fundamental challenges persist, the future of CRISPR is undoubtedly bright.},
}
@article {pmid41284608,
year = {2025},
author = {Zhu, W and Qi, T and Wu, Z and Zhong, W and Yan, L and Feng, J and Jin, F and Chen, W and Cai, Z and Rui, Y and Maria Da Costa, E and Liu, Q and Fu, Q and Zheng, L},
title = {One-Pot Photocontrolled CRISPR-Cas12b Coupled with Loop-Mediated Isothermal Amplification Assay for Point-of-Care Test of Mycoplasma pneumonia.},
journal = {ACS sensors},
volume = {10},
number = {12},
pages = {9323-9334},
doi = {10.1021/acssensors.5c01679},
pmid = {41284608},
issn = {2379-3694},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Mycoplasma pneumoniae/isolation &amp; purification/genetics ; *Pneumonia, Mycoplasma/diagnosis/microbiology ; *Point-of-Care Testing ; Humans ; *Molecular Diagnostic Techniques/methods ; Limit of Detection ; DNA, Bacterial/genetics/analysis ; },
abstract = {Mycoplasma pneumoniae (MP) is a highly prevalent respiratory pathogen, making the development of point-of-care testing (POCT) methods for its detection essential. The integration of loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12b systems demonstrates remarkable specificity and offers promising potential for MP POCT application. However, the current one-pot LAMP/CRISPR-Cas12b system (HOLMESv2) faces the challenge of low sensitivity due to the premature cleavage of the template by CRISPR, which limits its practical utility. To address this, this study introduces a photocontrolled HOLMESv2 (pHOLMESv2) assay using gRNA with an NPOM-modified spacer region. This modification prevents full base pairing between the gRNA and MP DNA, thereby keeping the CRISPR-Cas12b system in an inactive condition during the LAMP reaction and avoiding unintended cleavage of the DNA template. After completion of the LAMP reaction, light irradiation eliminates the NPOM group from the gRNA, restoring its activity to cleave the LAMP products, resulting in a fluorescence signal. The pHOLMESv2 assay successfully addresses the issue of premature DNA template cleavage, improving the limit of detection (LoD) by 133-fold (7.5 copies/μL). Additionally, this method enables direct detection of samples treated with nucleic acid release agents, eliminating the need for complex extraction, and features lyophilized reagents for enhanced stability, storage, and transport. The efficacy of pHOLMESv2 was assessed with 160 clinical MP samples, achieving a sensitivity of 99.0% and a specificity of 100.0%. The pHOLMESv2 assay, when combined with the developed smartphone-based amplification reader, provides a highly sensitive, specific, portable, and cost-effective MP detection, indicating its potential for significant diagnostic use.},
}
@article {pmid41284662,
year = {2025},
author = {Abad Santos, JC and Garimella, SS and Khanchandani, AN and Shah, PS},
title = {Rapid Optimization of a Light-Inducible System to Control Mammalian Gene Expression.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {225},
pages = {},
doi = {10.3791/68779},
pmid = {41284662},
issn = {1940-087X},
support = {P30 CA093373/CA/NCI NIH HHS/United States ; S10 OD018223/OD/NIH HHS/United States ; },
mesh = {Humans ; HEK293 Cells ; *Optogenetics/methods ; Light ; *CRISPR-Cas Systems ; Flow Cytometry/methods ; },
abstract = {Inducible gene expression tools can open novel applications in human health and biotechnology, but current options are often expensive, difficult to reverse, and have undesirable off-target effects. Optogenetic systems use light-responsive proteins to control the activity of regulators such that expression is controlled with the "flip of a switch". This study optimizes a simplified light activated CRISPR effector (2pLACE) system, which provides tunable, reversible, and precise control of mammalian gene expression. The OptoPlate-96 enables high-throughput screening via flow cytometry for single-cell analysis and rapid optimization of 2pLACE. This study demonstrates how to use the 2pLACE system with the OptoPlate-96 in HEK293T cells to identify the optimal component ratios for maximizing dynamic range and to find the blue light intensity response curve. Similar workflows can be developed for other mammalian cells and for other optogenetic systems and wavelengths of light. These advancements enhance the precision, scalability, and adaptability of optogenetic tools for biomanufacturing applications.},
}
@article {pmid41284724,
year = {2025},
author = {da Silva, GLA and Damasceno, JD and Black, JA and Lapsley, C and McCulloch, R and Tosi, LRO},
title = {ATR, a DNA damage kinase, modulates DNA replication timing in Leishmania major.},
journal = {PLoS genetics},
volume = {21},
number = {11},
pages = {e1011899},
pmid = {41284724},
issn = {1553-7404},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {*Leishmania major/genetics ; *Ataxia Telangiectasia Mutated Proteins/genetics/metabolism ; DNA Damage/genetics ; *DNA Replication/genetics ; CRISPR-Cas Systems ; *DNA Replication Timing/genetics ; *Protozoan Proteins/genetics/metabolism ; },
abstract = {All cells possess mechanisms to maintain and replicate their genomes, whose integrity and transmission are constantly challenged by DNA damage and replication impediments. In eukaryotes, the protein kinase Ataxia-Telangiectasia and Rad3-related (ATR), a member of the phosphatidylinositol 3-kinase-like family, acts as a master regulator of the eukaryotic response to DNA injuries, ensuring DNA replication completion and genome stability. Here we aimed to investigate the functional relevance of the ATR homolog in the DNA metabolism of Leishmania major, a protozoan parasite with a remarkably plastic genome. CRISPR/cas9 genome editing was used to generate a Myc-tagged ATR cell line (mycATR), and a Myc-tagged C-terminal knockout of ATR (mycATRΔC-/-). We show that the nuclear localisation of ATR depends upon its C-terminus. Moreover, its deletion results in single-stranded DNA accumulation, impaired cell cycle control, increased levels of DNA damage, and delayed DNA replication re-start after replication stress. In addition, we show that ATR plays a key role in maintaining L. major's unusual DNA replication program, where larger chromosomes duplicate later than smaller chromosomes. Our data reveals loss of the ATR C-terminus promotes the accumulation of DNA replication signal around replicative stress fragile sites, which are enriched in larger chromosomes. Finally, we show that these alterations to the DNA replication program promote chromosome instability. In summary, our work shows that ATR acts to modulate DNA replication timing, limiting the plasticity of the Leishmania genome.},
}
@article {pmid41284810,
year = {2025},
author = {Park, S and Kim, MM},
title = {Mutation of the Collagen Type 1α Gene in Human Fibrosarcoma Cells Using the CRISPR/Cas9 System.},
journal = {Biochemistry},
volume = {64},
number = {24},
pages = {4794-4804},
doi = {10.1021/acs.biochem.5c00394},
pmid = {41284810},
issn = {1520-4995},
mesh = {Humans ; Collagen Type I, alpha 1 Chain ; *Fibrosarcoma/genetics/metabolism/pathology ; *Collagen Type I/genetics/metabolism ; *CRISPR-Cas Systems ; *Mutation ; Cell Line, Tumor ; Signal Transduction ; Gene Editing ; },
abstract = {Collagen in the connective tissue plays a key role in the expression the aging phenotypes. While collagen production decreases with aging, collagenase expression increases, resulting in collagen breakdown. The purpose of this study is to investigate the change in the expression of proteins and genes related to the collagen signaling pathway, cell cycle, and aging phenotypes of cells with the collagen type 1 α (COL1A1) gene edited by the CRISPR/Cas9 system. The mutation of the COL1A1 gene was induced by the CRISPR/Cas9 system. Sanger DNA sequencing and Indel analyses, Sanger DNA sequencing analysis and Swiss protein modeling analysis were used to verify the induction of mutation. Aging phenotypes in the mutated cells were evaluated by collagen staining assay, SA-β-galactosidase staining assay, RT-PCR assay, Western blot analysis, gelatin zymography, and immunofluorescent staining assay. Sanger DNA sequencing analysis demonstrated that human fibrosarcoma cells with COL1A1 gene mutations were successfully established in this study. Swiss protein modeling analysis displayed the altered structure of COL1A1 in the edited cells. In addition, while collagen production was decreased, the SA-β-galactosidase staining level was increased in the edited cells. It was also found that the expression levels of CDC2, CDk2, and cyclin D were increased by down-regulating p53 and p21 levels through the increased expression of MDM2 in the edited cells. Moreover, the expression levels of MMP-1, MMP-2, MMP-9, AKT, and p-mTOR were reduced in the edited cells. These findings could provide a crucial clue in elucidating the close relationship between collagen production and senescence.},
}
@article {pmid41285016,
year = {2025},
author = {He, RL and Huang, BC and Wu, J and Sun, D and Li, HH and Liu, JQ and Wu, J and Liu, DF and Li, WW},
title = {One-Step Biological Upcycling of Chitin Wastes into Violacein by Engineered Chromobacterium violaceum.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4733-4743},
doi = {10.1021/acssynbio.5c00472},
pmid = {41285016},
issn = {2161-5063},
mesh = {*Chromobacterium/metabolism/genetics ; *Chitin/metabolism ; *Indoles/metabolism ; *Metabolic Engineering/methods ; Fermentation ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {Existing technologies for the valorization of organic wastes have been focused mainly on degradable wastes, while an efficient, low-carbon approach for the upcycling of shell waste is still lacking. Here, we report a one-step chitin biological fermentation process (CBFP), based on the construction of Chromobacterium violaceum engineered strain, for efficiently converting shell waste-derived chitin into high-value violacein. A high-efficiency CRISPR cytosine-base editor (pRK2-BE, 97% editing efficiency) was developed for C. violaceum, which demonstrated cv_4240, cv_1440, and cv_2935 as the major chitin hydrolysis genes and phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS) as the major N-acetyl-glucosamine uptake pathway. The engineered strain WT/pBAD-4, co-overexpressing of cv_4240, cv_1440, cv_2935, and vioABCDE, efficiently utilized colloidal chitin and crystalline chitin as the sole carbon and nitrogen source, achieving violacein yields of 159.78 and 120.95 mg·L[-1], respectively. This study provided an economically viable and environmentally sustainable solution for green upcycling of shell waste.},
}
@article {pmid41285544,
year = {2026},
author = {Gong, C and Wang, Z and Gao, X and He, S},
title = {A review of CRISPR-Cas as a "molecular catcher" for tracking circulating tumor cells and extracellular vesicles.},
journal = {Analytica chimica acta},
volume = {1381},
number = {},
pages = {344675},
doi = {10.1016/j.aca.2025.344675},
pmid = {41285544},
issn = {1873-4324},
mesh = {*Neoplastic Cells, Circulating/pathology/metabolism ; Humans ; *Extracellular Vesicles/metabolism/chemistry ; *CRISPR-Cas Systems ; Biomarkers, Tumor/genetics ; },
abstract = {BACKGROUND: Liquid biopsy is reshaping the landscape of cancer diagnostics by turning a simple blood sample into a rich source of real-time molecular insights. Among its most promising targets are circulating tumor cells (CTCs) and extracellular vesicles (EVs), which carry valuable clues about tumor progression, metastasis, and treatment response. However, traditional workflows for analyzing CTCs and EVs typically rely on immunoaffinity-based enrichment followed by molecular assays such as quantitative Polymerase Chain Reaction (qPCR) or Enzyme-Linked Immunosorbent Assay (ELISA). These approaches are often limited by low sensitivity, high costs, and complex procedures, hindering their widespread clinical use.<br><br>RESULTS: Recent advances in Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas technologies offer a multifaceted approach to biomarker analysis. Cas9 is primarily used for functional gene interrogation to identify and validate targets, while Cas12a and Cas13a serve as direct diagnostic tools, enabling ultrasensitive signal amplification for DNA, RNA, and protein markers. By integrating these distinct Cas effectors with aptamer-based recognition, nanomaterial-assisted enrichment, and hybrid amplification techniques like Hybridization Chain Reaction (HCR), Rolling Circle Amplification (RCA), researchers have developed highly sensitive and programmable platforms for analyzing CTCs and EVs.<br><br>SIGNIFICANCE: Despite ongoing challenges such as off-target effects, Protospacer Adjacent Motif (PAM) sequence limitations, and clinical variability, the field is rapidly evolving. The convergence of CRISPR diagnostics with artificial intelligence, device miniaturization, and multiplexed sensing is accelerating clinical translation. Collectively, these innovations are paving the way for a new era of precision oncology that is fast, flexible, and achievable with only a drop of blood.},
}
@article {pmid41285661,
year = {2026},
author = {Gu, X and Zhang, T and Yao, H and Guo, F and Yang, C and Xu, H and He, X and Ma, Z and Zhang, X and Yu, S and An, R and Wang, F},
title = {CRISPR-Cas12a-integrated pregnancy test strip biosensors: Visual detection of telomerase and miRNA let-7a in cervical cancer diagnostics.},
journal = {Biosensors &amp; bioelectronics},
volume = {294},
number = {},
pages = {118241},
doi = {10.1016/j.bios.2025.118241},
pmid = {41285661},
issn = {1873-4235},
mesh = {Humans ; Female ; *MicroRNAs/genetics/isolation &amp; purification/analysis ; *Uterine Cervical Neoplasms/diagnosis/genetics ; *Biosensing Techniques ; *Telomerase/isolation &amp; purification/genetics/analysis ; CRISPR-Cas Systems/genetics ; HeLa Cells ; Pregnancy ; Limit of Detection ; Reagent Strips/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Cervical cancer is a leading cause of female cancer-related mortality globally, and early screening based on reliable biomarkers is critical for improving prognosis. Telomerase (a key driver of cellular immortalization) and microRNA let-7a (a tumor suppressor with downregulated expression in cervical cancer) are well-validated diagnostic targets, but existing detection methods are hindered by complex procedures, high instrumentation costs, and reliance on specialized technical expertise-limiting their accessibility in resource-constrained settings. To address these limitations, we developed two novel CRISPR-Cas12a-integrated biosensors using commercially available pregnancy test strips (PTS) for instrument-free, visual readout. Both biosensors leverage a core signal mediator, probe 1 ("MB-ssDNA1-hCG"), which links CRISPR-Cas12a activation to visible color development on the PTS. The first Biosensor CRISPR-PTS-Telo detects telomerase activity in one-step without PCR: telomerase-generated (TTAGGG)n repeats activate Cas12a-crRNA1 complex, cleaving the probe 1 to release hCG, achieving a detection limit of 18 HeLa cells-comparable to sensitive laboratory assays. The second Biosensor CRISPR-PTS-let7a detects miRNA let-7a by first converting miRNA signals to Trigger DNA via Assister DNA and probe 2 ("MB-ssDNA2+Trigger"), activating Cas12a-crRNA2 complex, cleaving the probe 1 and inducing PTS coloration. This achieves a detection limit of 25.1 fM for let-7a. Validation with clinical samples (24 cervical tissues and 26 blood samples) confirmed their concordance with gold-standard methods (ELISA for telomerase, RT-qPCR for let-7a). These versatile tools hold significant potential as point-of-care testing (POCT) solutions to facilitate early, accessible cervical cancer screening.},
}
@article {pmid41285673,
year = {2025},
author = {Munusamy, S and Jahani, R and Zheng, H and Chen, J and Kong, J and Zhao, Y and Guan, L and Zhou, S and Guan, X},
title = {Enhanced CRISPR-Cas12a Fluorescent Assay for Detecting Trace Levels of Procalcitonin.},
journal = {ACS applied bio materials},
volume = {8},
number = {12},
pages = {10818-10826},
doi = {10.1021/acsabm.5c01535},
pmid = {41285673},
issn = {2576-6422},
mesh = {*Procalcitonin/analysis/blood ; Humans ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Biocompatible Materials/chemistry/chemical synthesis ; Gold/chemistry ; Materials Testing ; Metal Nanoparticles/chemistry ; Particle Size ; Fluorescence ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and accurate detection of procalcitonin (PCT), a major biomarker for bacterial infections and sepsis, remains a pressing need in clinical diagnostics because sepsis progresses rapidly and may initially present with nonspecific or even subtle symptoms. Herein, we report a CRISPR-Cas12a-based fluorescence biosensing platform for ultrasensitive detection of PCT. The platform employs antibody-functionalized magnetic beads (MBs) for specific protein enrichment and antibody- and oligonucleotide- dual-functionalized gold nanoparticles (AuNPs) for high-density DNA payload. After sandwich complex formation with the target PCT, a programmed ssDNA strand is released by thermal denaturation, which then activates Cas12a collateral cleavage, thereby generating a fluorescence signal. Thorough physicochemical characterizations, including zeta potential, dynamic light scattering, UV-vis spectroscopy, and TEM, were carried out to confirm the successful functionalization of MBs and AuNPs. The developed PCT sensor was highly sensitive with a limit of detection (LOD) reaching 3 pg/mL. Moreover, the biosensor exhibited an excellent specificity toward PCT against clinically relevant interferents such as C-reactive protein (CRP), interleukin-2β (IL-2β), interleukin-6 (IL-6), human serum albumin (HSA), and bovine serum albumin (BSA), and simulated serum sample analysis was successfully carried out with the recoveries ranging from 108 to 122%. The PCT sensing technique developed in this work offers the potential to be expanded to construct a multiplexing platform for simultaneous detection of multiple biomarker species for early and accurate disease diagnosis.},
}
@article {pmid41285723,
year = {2025},
author = {Morshedi Rad, D and Richards, C and Zhand, S and de Alwis, N and Hannan, NJ and Faiz, A and McClements, L and Ebrahimi Warkiani, M},
title = {CRISPR/Cas9-mediated gene editing in trophoblast cells via mechanoporation for preeclampsia insight.},
journal = {Cell death &amp; disease},
volume = {17},
number = {1},
pages = {61},
pmid = {41285723},
issn = {2041-4889},
support = {106628//National Heart Foundation of Australia (Heart Foundation)/ ; DP200101860//Department of Education and Training | Australian Research Council (ARC)/ ; 2021CDF1148//Cancer Institute NSW (Cancer Institute New South Wales)/ ; },
mesh = {*Trophoblasts/metabolism/pathology ; Female ; *Pre-Eclampsia/genetics/pathology/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Pregnancy ; Cell Movement/genetics ; Cell Line ; },
abstract = {Preeclampsia is a severe pregnancy complication marked by impaired trophoblast function and abnormal placental development, leading to significant maternal and fetal morbidity. FK506-binding protein-like (FKBPL) has been identified as a potential biomarker as it is significantly downregulated in early pregnancy stages of women who progress to develop preeclampsia. However, editing the Fkbpl gene in trophoblast cells to create a model of preeclampsia using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is challenging due to inefficient delivery, leading to low editing efficiency and reduced cell viability. To address these challenges, we developed a cost-effective and minimally invasive mechanoporation system using micro-engineered filters to deliver CRISPR/Cas9 plasmid DNA (pDNA) targeting the Fkbpl gene into trophoblast cells. This approach successfully generated cell lines with a 38% knockout (K/O) of Fkbpl expression, significantly reducing cell migration (wildtype (WT): 28.77% ± 4.7 vs. 38% K/O: 4.95% ± 0.8, wound closure, **p < 0.01) and proliferation (WT: 1.26 ± 0.06 vs. 38% K/O: 0.81 ± 0.01, ****p < 0.0001). Lower Fkbpl-K/O efficiency of 17% showed a similar reduction in cell proliferation as the 38% K/O clone. Although a full Fkbpl-K/O in the ACH-3P first-trimester trophoblast cell line was not achieved, the partial K/O provided valuable insights into Fkbpl's role in trophoblast function relevant to preeclampsia pathogenesis. Moreover, treatment with mesenchymal stem cell (MSC)-derived small extracellular vesicles (sEVs) or MSC-sEVs did not restore migratory capacity in Fkbpl-deficient cells (p = 0.14). MSC-sEVs increased proliferation in WT ACH-3P cells at 1 µg (p < 0.05) and 2 µg (p < 0.01) doses, however, were not effective in either 17% or 38% Fkbpl-K/O clones, suggesting that FKBPL is an important mechanism of MSC-sEV-mediated therapeutic effect in trophoblasts in the context of preeclampsia. This study advances gene-editing techniques in placental biology and proposes new therapeutic strategies and mechanisms for pregnancy-related complications. A Schematic overview of CRISPR/Cas9 plasmid delivery using microfiltroporation compared to gold standard electroporation and lipofection technologies in trophoblast cells. A CRISPR/Cas9 plasmid targeting Fkbpl was delivered to the first trimester trophoblast cell line, ACH-3P. Cells were sorted according to green fluorescence protein (GFP) expression, expanded and assessed for changes in cell function using proliferation and migration assays. B Actual images of the isopore silicon nitride (SiN) microfilters used in this study and diagram of cell membrane dynamics in response to mechanoporation. This figure was created with Biorender.com. CRISPR clustered regularly interspaced short palindromic repeats, EP electroporation, MFP microfiltroporation.},
}
@article {pmid41285753,
year = {2025},
author = {Mameli, E and Samantsidis, GR and Viswanatha, R and Kwon, H and Hall, DR and Butnaru, M and Hu, Y and Mohr, SE and Perrimon, N and Smith, RC},
title = {A genome-wide CRISPR screen in Anopheles mosquito cells identifies fitness and immune cell function-related genes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10323},
pmid = {41285753},
issn = {2041-1723},
support = {R01 AI177540/AI/NIAID NIH HHS/United States ; P41 GM132087/GM/NIGMS NIH HHS/United States ; R01 AI182256/AI/NIAID NIH HHS/United States ; 2336877//National Science Foundation (NSF)/ ; GM132087//U.S. Department of Health &amp; Human Services | NIH | National Institute of General Medical Sciences (NIGMS)/ ; AI166857//U.S. Department of Health &amp; Human Services | NIH | National Institute of Allergy and Infectious Diseases (NIAID)/ ; R21 AI166857/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Anopheles/genetics/immunology/drug effects ; *Mosquito Vectors/genetics/immunology/drug effects ; Liposomes ; Clodronic Acid/pharmacology ; CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Malaria/transmission/prevention &amp; control ; Genome, Insect ; *Genetic Fitness ; Female ; },
abstract = {Anopheles mosquitoes are the sole vector of malaria, the most burdensome vector-borne disease worldwide. At present, strategies for reducing mosquito populations or limiting their ability to transmit disease show the most promise for disease control. Therefore, improving our understanding of mosquito biology and immune function may aid new approaches to limit malaria transmission. Here, we perform genome-wide CRISPR screens in Anopheles mosquito cells to identify genes required for fitness and that confer resistance to clodronate liposomes, which are used to ablate immune cells. The cellular fitness screen identifies 1280 fitness-related genes (393 at highest confidence) that are highly enriched for roles in fundamental cell processes. The clodronate screen identifies resistance factors that impair clodronate liposome function. For the latter, we confirm roles in liposome uptake and processing through in vivo validation in Anopheles gambiae that provide new mechanistic detail of phagolysosome formation and clodronate liposome processing. Altogether, we present a genome-wide CRISPR knockout platform in a major malaria vector and identify genes important for fitness and immune-related processes.},
}
@article {pmid41285767,
year = {2025},
author = {Rahimov, F and Ghosh, S and Petiwala, S and Schmidt, M and Nyamugenda, E and Shi, M and Tam, J and Verduzco, D and Singh, S and Avram, V and Modi, A and Espinoza, CA and Lu, C and Wang, J and Keller, A and Macoritto, M and Mahi, NA and Anton, T and Chung, N and Flister, MJ and Katlinski, KV and Biswas, A and den Hollander, AI and Waring, JF and Stender, JD},
title = {A genome-wide CRISPR screen identifies the TNRC18 gene locus as a regulator of inflammatory signaling.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10346},
pmid = {41285767},
issn = {2041-1723},
mesh = {Humans ; *Signal Transduction/genetics ; *Interleukin-1beta/metabolism/genetics ; *Inflammation/genetics/metabolism ; Lipopolysaccharides/pharmacology ; CRISPR-Cas Systems ; U937 Cells ; Finland ; Gene Knockout Techniques ; Genome-Wide Association Study ; Clustered Regularly Interspaced Short Palindromic Repeats ; Gene Expression Regulation ; },
abstract = {Interleukin-1β (IL-1β) is dysregulated in chronic inflammatory diseases, yet the genetic factors influencing IL-1β production remain largely unknown. Myeloid-derived cells are the primary producers of IL-1β, which prompted a genome-wide CRISPR knockout screen in the human myeloid-derived U937 cells treated with lipopolysaccharide (LPS) to mimic inflammatory conditions and sorted for high and low intracellular IL-1β levels. A total of 295 genes are identified as regulators of IL-1β production, with 57 overlapping loci associated with inflammatory diseases, including the TNRC18 gene locus associated with multiple diseases in the Finnish population. U937 cells engineered with the Finnish-enriched rs748670681 risk allele demonstrate decreased expression of TNRC18 and an adjacent gene WIPI2, reduction in LPS-dependent gene activation and cytokine production, but elevation of interferon-responsive gene programs. Transcriptomic profiles for individual knockouts of TNRC18 and WIPI2 attribute the loss of LPS-dependent signaling primarily to TNRC18, which occurs through the modulation of H3K27 acetylation around inflammatory regulatory regions via TNRC18 and its protein interaction network. In contrast, the loss of WIPI2 is characterized by an exacerbation of interferon signaling. These findings delineate the global regulatory mechanisms of IL-1β production and provide molecular insights to the role of the rs748670681 variant in inflammatory diseases.},
}
@article {pmid41285890,
year = {2025},
author = {Kim, S and Won, H and Bae, J and Kim, J and Choi, J and Richar, H and Kim, YG and Choi, HJ},
title = {Structural and functional insights into internal domain replacement in SpCas9 for protein engineering.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41528},
pmid = {41285890},
issn = {2045-2322},
support = {SRFC-MA1801-09//Samsung Science and Technology Foundation/ ; },
mesh = {*Protein Engineering/methods ; *CRISPR-Associated Protein 9/genetics/chemistry/metabolism ; CRISPR-Cas Systems ; Gene Editing/methods ; *Streptococcus pyogenes/enzymology/genetics ; Escherichia coli/genetics/enzymology ; Protein Domains ; },
abstract = {The CRISPR-Cas9 system has emerged as a powerful tool for precise genome editing, with ongoing research focused on enhancing its reliability and expanding its versatility. One effective strategy involves the integration of foreign functional domains into Cas9 to confer new capabilities. However, successful integration requires identification of insertion sites that preserve the protein's structural integrity and function. In this study, we identified a C-terminal region of Streptococcus pyogenes Cas9 (SpCas9), spanning residues 1242-1263, as a viable site for domain replacement. Structural and biochemical analyses of a SpCas9 variant lacking this region confirmed its dispensability for SpCas9 activity. As a proof of concept, we substituted this segment with the evolved E. coli tRNA adenosine deaminase (TadA), a key component of adenine base editors. Functional evaluation of this engineered SpCas9-TadA variant demonstrated deamination efficiency comparable to that of the ABE8e, with the potential to modulate the editing window through linker design. These results highlight the potential of targeted engineering of this region to develop more precise and versatile genome editing tools.},
}
@article {pmid41286106,
year = {2026},
author = {Angelini Stewart, A and Ahrens-Nicklas, RC and Tsai, SQ and Musunuru, K and Giannikopoulos, P and Clelland, CD},
title = {Measurement and clinical interpretation of CRISPR off-targets.},
journal = {Nature genetics},
volume = {58},
number = {1},
pages = {20-27},
pmid = {41286106},
issn = {1546-1718},
support = {K08 NS112330/NS/NINDS NIH HHS/United States ; U01 NS134062/NS/NINDS NIH HHS/United States ; U19 NS132303/NS/NINDS NIH HHS/United States ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Genetic Therapy/methods/adverse effects ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Animals ; },
abstract = {CRISPR genetic therapies are revolutionizing the landscape of preclinical research and clinical studies, providing new potential routes for curative intervention for a range of previously untreatable diseases. As with any therapy, the therapeutic benefits and risks must be weighed against consideration of the disease threat. Genome-related adverse events are an inherent risk of CRISPR genetic therapies, including off-target edits. The perception that CRISPR therapies ought to have near-zero off-targets belies clinical medicine, therapy development and biology, which demonstrate that 'perfect' therapeutics do not exist. Given that not all genomic off-target events are equal, we provide a practical framework to evaluate and assess off-target safety based on the tools available today and ones that will be developed in the future. With the comprehensive information and assessment gathered using these guidelines, we aim to streamline the transition of CRISPR therapeutics from bench to bedside.},
}
@article {pmid41286485,
year = {2025},
author = {Zhao, Y and Liang, Y and Ni, Z and Sun, Q and Zong, Y and Wang, Y},
title = {Advances and prospects of large DNA fragment editing in plants.},
journal = {Nature plants},
volume = {11},
number = {12},
pages = {2461-2475},
pmid = {41286485},
issn = {2055-0278},
mesh = {*Gene Editing/methods ; *Genome, Plant/genetics ; *Crops, Agricultural/genetics ; *DNA, Plant/genetics ; *Plants/genetics ; CRISPR-Cas Systems ; Plants, Genetically Modified/genetics ; },
abstract = {Structural variations drive plant genome evolution and shape agronomic traits. Manipulating structural variations has great potential to improve complex plant traits and enhance agricultural sustainability. Genome editing technologies have evolved from gene knockouts and base editing to the modification of short DNA fragments, and are now advancing towards the precise manipulation of large DNA fragments. This advancement facilitates targeted, large-scale genomic changes such as deletions, insertions, replacements, inversions, translocations and duplications. In this Review, we summarize recent advances in developing technologies for large DNA fragment editing and highlight their key applications in plants as well as their potential to accelerate crop improvement. Finally, we discuss the current challenges and future prospects for these technologies in plant science.},
}
@article {pmid41287270,
year = {2026},
author = {Yao, W and Xu, X and Zhai, X and Ji, T and Zhang, R and Xu, S and Luo, X},
title = {Autocatalytic Circular DNA Powered Plasmonic CRISPR/Cas12a Platform for Ultrasensitive Non-Nucleic Acid Target Sensing.},
journal = {Angewandte Chemie (International ed. in English)},
volume = {65},
number = {3},
pages = {e16838},
doi = {10.1002/anie.202516838},
pmid = {41287270},
issn = {1521-3773},
support = {21505081//National Natural Science Foundation of China/ ; 22374085//National Natural Science Foundation of China/ ; ZR2023MB110//Natural Science Foundation of Shandong Province/ ; },
mesh = {*CRISPR-Cas Systems ; *DNA, Circular/chemistry/metabolism/genetics ; *Biosensing Techniques/methods ; Nucleic Acid Amplification Techniques ; Catalysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {CRISPR/Cas12a-based detection of non-nucleic acid targets faces two major challenges: 1) limited sensitivity due to the inherent inability to pre-amplify non-nucleic acid targets, and 2) suboptimal performance of traditional reporters caused by photobleaching of fluorescent dyes, rapid degradation, and slow reaction kinetics resulting from random molecular collisions. To overcome these limitations, we developed an innovative plasmonic CRISPR/Cas12a platform featuring positive-feedback autocatalytic circular DNA (cir-DNA) amplification. This system synergistically combines spatial confinement effects with plasmon-enhanced fluorescence (PEF) to achieve ultrasensitive detection of non-nucleic acid targets. The engineered cir-DNA enables continuous Cas12a regeneration for autocatalytic signal amplification, while the designed plasmonic spherical nucleic acids significantly accelerate reaction kinetics while enhancing fluorescence signals. This integrated approach reduced the required reaction time to 15 min while improving the detection limit by approximately 52-fold compared to conventional methods. Furthermore, by leveraging a convolutional neural network (CNN) machine learning model, not only the assessment of the risk level of perfluorooctanoic acid (PFOA) based on threshold-positive and threshold-negative serum concentrations but also highly accurate blind testing were both achieved, highlighting its potential for clinical applications such as pregnancy risk assessment.},
}
@article {pmid41288175,
year = {2025},
author = {Prokhorova, PV and Vlasova, NN and Yuzhakov, AG and Gulyukin, AM},
title = {Modern approaches to the construction and use of recombinant viruses.},
journal = {Voprosy virusologii},
volume = {70},
number = {5},
pages = {417-430},
doi = {10.36233/0507-4088-323},
pmid = {41288175},
issn = {2411-2097},
mesh = {Humans ; *Genetic Vectors/genetics ; *Oncolytic Viruses/genetics ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods ; *Vaccines, Synthetic/genetics/immunology ; Animals ; Oncolytic Virotherapy/methods ; Homologous Recombination ; Plasmids/genetics ; },
abstract = {The review describes certain viral vectors and considers various methods for constructing recombinant viruses with special attention paid to the homologous recombination and CRISPR/Cas9 system, and also describes the capabilities of using various cloning vectors (different plasmids, BAC etc.). The review also presents a comparative analysis of the effectiveness and safety of using various viral vectors, both for creating recombinant vaccines and for obtaining oncolytic viruses, as well as medicines for gene therapy.},
}
@article {pmid41288291,
year = {2025},
author = {Liu, H and Liu, Y and Feng, R and Qian, M and Li, Y and Zhai, S and Song, J and Qiu, X},
title = {Homogeneous Femtomolar Detection of P-tau181 via Proximity Extension and CRISPR/Cas Technique.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26560-26569},
doi = {10.1021/acs.analchem.5c04856},
pmid = {41288291},
issn = {1520-6882},
mesh = {Humans ; *tau Proteins/blood/analysis/metabolism ; *CRISPR-Cas Systems ; Limit of Detection ; Phosphorylation ; Nucleic Acid Amplification Techniques/methods ; Polymerase Chain Reaction ; Alzheimer Disease/diagnosis/blood ; },
abstract = {Accurate quantification of site-specific tau phosphorylation in plasma holds great promise for the noninvasive early diagnosis of Alzheimer's disease (AD). Here, we integrated the proximity extension assay (PEA) with nucleic acid amplification techniques-polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA)-and coupled them with CRISPR/Cas12a-mediated fluorescence detection to enable quantitative and homogeneous measurement of threonine-181-phosphorylated tau (p-tau181), a key biomarker of AD. Binding of two PEA probes to a single p-tau181 molecule induces proximity-mediated probe hybridization and extension, thereby converting the protein signal into an amplifiable nucleic acid signal. The resulting double-stranded DNA is subsequently amplified by PCR or RPA and detected through Cas12a trans-cleavage activity. The limits of detection (LODs) for the PEA-PCR-CRISPR/Cas and PEA-RPA-CRISPR/Cas assays were 149.0 fM (6.8 pg·mL[-1]) and 45.4 fM (2.1 pg·mL[-1]), respectively. In fetal bovine serum, LODs of 231.4 fM (10.6 pg·mL[-1]) and 139.2 fM (6.3 pg·mL[-1]) were achieved, demonstrating excellent antimatrix performance. The accuracy of the PEA-RPA-CRISPR/Cas assay in human serum was further validated using a commercial enzyme-linked immunosorbent assay (ELISA) kit. This homogeneous, wash-free approach combines operational simplicity with ultrahigh sensitivity, showing great potential for routine clinical detection and early stage monitoring of AD biomarkers.},
}
@article {pmid41288355,
year = {2025},
author = {Xie, W and Cai, Z and Bao, Z},
title = {Benchmarking the PAM compatibility of Cas12a variants for high-throughput yeast genetic variant engineering.},
journal = {Applied and environmental microbiology},
volume = {91},
number = {12},
pages = {e0161825},
pmid = {41288355},
issn = {1098-5336},
support = {2023YFF1204500//National Key Research and Development Program of China/ ; 22308316//National Natural Science Foundation of China/ ; 226-2025-00043, 226-2022-00214//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *CRISPR-Associated Proteins/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Genetic Engineering/methods ; Benchmarking ; Genetic Variation ; },
abstract = {Saccharomyces cerevisiae is an important organism for basic research and applied biotechnology. Genome editing techniques, particularly CRISPR/Cas9 from Streptococcus pyogenes, have greatly facilitated saturation genome editing in yeast to understand mutant functions on a large scale. However, Cas9 is restricted by its targeting preference for G-rich protospacer-adjacent motif (PAM) sequences. To broaden the targeting scope, we established an efficient homology-integrated CRISPR/Cas12a system to install genetic variants through homologous recombination by targeting T-rich PAMs. We benchmarked the PAM compatibility of PAM-relaxed Cas12a variants and identified the improved LbCas12a (impLbCas12a) as the most efficient and PAM-relaxed variant in S. cerevisiae, showing high editing purity and an editing window centering the double-strand break. We show that our system can be used to perform targeted saturation mutagenesis to reveal functional variants not captured previously. By using a homology-integrated CRISPR RNA array, we utilized the multiplexing capability of CRISPR/Cas12a to realize multiplex genetic variant installation. Our system enriches the yeast genetic variant engineering toolbox, complementing the commonly used CRISPR/Cas9 system.IMPORTANCECRISPR/Cas9 has facilitated yeast functional genomics by generating a large number of precise genetic variants in a very short period of time. This enabled the interrogation of reconstituted natural genetic variants across different genetic backgrounds or entirely synthetic mutations to discover novel or improved functions. However, Cas9 only targets a limited genomic sequence space due to its preference for G-rich PAM sequences. In this study, we close this gap by developing a CRISPR/Cas12a-based system to engineer user-defined genetic variants targeting T-rich PAM sequences. Our system adopts a homology-integrated design and the most PAM-relaxed Cas12a characterized in yeast to date. These features collectively enabled the creation of genetic variant libraries and multiplex edited strains. This genome editing tool can be used together with Cas9-based tools to interrogate a greater genomic sequence space.},
}
@article {pmid41288387,
year = {2025},
author = {Kim, B and Yuk, M and Park, M and Ryu, H and Park, J and Yu, H and Park, S and Hong, JT and Lim, KH and Han, SB and Song, N and Park, H},
title = {CRISPR editing of miR-33 restores ApoE lipidation and amyloid-β metabolism in ApoE4 sporadic Alzheimer's disease.},
journal = {Brain : a journal of neurology},
volume = {148},
number = {12},
pages = {4400-4415},
doi = {10.1093/brain/awaf244},
pmid = {41288387},
issn = {1460-2156},
support = {//National Research Foundation of Korea/ ; 2021R1C1C1006551//Korean government/ ; //Bio &amp; Medical Technology Development Program/ ; //National Research Foundation/ ; RS-2024-00440787//Korean government/ ; },
mesh = {*MicroRNAs/genetics/metabolism ; *Alzheimer Disease/genetics/metabolism/pathology ; Animals ; Humans ; Mice ; *Gene Editing/methods ; *Apolipoprotein E4/genetics/metabolism ; *Lipid Metabolism/genetics ; *Amyloid beta-Peptides/metabolism ; Male ; CRISPR-Cas Systems ; Female ; Astrocytes/metabolism ; Mice, Transgenic ; *Apolipoproteins E/metabolism/genetics ; Aged ; Disease Models, Animal ; },
abstract = {Sporadic Alzheimer's disease (sAD) is marked by dysregulated lipid metabolism, prominently involving apolipoprotein E (ApoE). MicroRNA-33 (miR-33) has emerged as a key regulator of lipid homeostasis, yet its role in sAD remains unclear. This study investigated miR-33 dysregulation in APOE ε4 allele (ApoE4)-associated sAD and explored its therapeutic potential using clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated gene editing. Elevated miR-33 expression was observed in both AD patients, particularly those with ApoE4-associated sAD, and in the ApoE4 mouse model, implicating its role in AD pathology. Using CRISPR/Cas9, we modulated miR-33 expression in astrocytes to regulate ApoE lipidation and ameliorate AD-related pathology. Our results show that targeted miR-33 regulation in astrocytes via CRISPR/Cas9 restores ApoE lipidation and mitigates AD pathology in both in vitro and in vivo AD mice. Additionally, applying this gene therapy approach in ApoE4 sAD patient cell lines highlights its translational potential for therapeutic intervention. In conclusion, our findings elucidate miR-33's role in AD pathogenesis and underscore the therapeutic promise of CRISPR-mediated miR-33 targeting for restoring lipid homeostasis and ameliorating AD pathology. This study provides valuable insights into developing miRNA-based gene therapy strategies for treating sAD.},
}
@article {pmid41288537,
year = {2026},
author = {He, J and Papa, G and Azizi, F and Kirsche, L and Artola-Boran, M and Ferreira Cassio, R and Hotz, AL and Geiger, G and Francas, B and Weber, A and Tzankov, A and Kontarakis, Z and Leary, P and Müller, A},
title = {Gastric Organoid-Based Ectopic and Orthotopic In Vivo CRISPR Screening for Tumor Suppressors in Gastric Cancer.},
journal = {Gastroenterology},
volume = {170},
number = {3},
pages = {495-510},
doi = {10.1053/j.gastro.2025.09.009},
pmid = {41288537},
issn = {1528-0012},
mesh = {Animals ; *Stomach Neoplasms/genetics/pathology/microbiology/metabolism ; *Organoids/pathology ; Mice ; *CRISPR-Cas Systems ; Helicobacter pylori ; Humans ; *Genes, Tumor Suppressor ; Helicobacter Infections/microbiology ; Signal Transduction/genetics ; PTEN Phosphohydrolase/genetics ; Gene Knockout Techniques ; Disease Models, Animal ; },
abstract = {BACKGROUND &amp; AIMS: CRISPR-Cas9 screening is a powerful tool for the in vivo discovery of cancer dependencies. The aim of this study was to perform in vivo CRISPR knockout screening for gastric tumor suppressors using gastric murine organoids in a subcutaneous as well as a surgical model of orthotopic tumor growth.<br><br>METHODS: In vivo screening was performed using a custom library targeting 49 putative gastric tumor suppressor genes, as well as a "cancer genome-wide" library targeting 5000 genes, in immunocompetent and -deficient mice, and in the presence or absence of the gastric pathogen Helicobacter pylori. The top hits were selected for individual validation and mechanistic follow-up.<br><br>RESULTS: Our custom library knockout screens revealed single-guide RNAs targeting Pten, Fbxw7, and genes encoding several components of the transforming growth factor-&#xdf; signaling pathway (Smad4, Tgfbr1, Tgfbr2, and Acvr2a) to be recurrently enriched both in subcutaneously and orthotopically growing tumors. The same, and several additional genes were identified by cancer genome-wide CRISPR screening. Ten of our top hits could be validated individually in vivo. Pten inactivation resulted in large tumors characterized by increased neo-angiogenesis, neutrophil recruitment, and T-cell exclusion. Inactivation of Smad4, Tgfbr1, or Acvr2a all produced phenotypes that were reminiscent of early gastric cancer precursor lesions such as intestinal Alcian blue-positive metaplasia and compensatory hyperplasia. Helicobacter pylori infection failed to affect the mutational landscape of tumors; rather, we found that H pylori modulates the tumor microenvironment and recruits large numbers of tumor-promoting SiglecF[+] neutrophils.<br><br>CONCLUSIONS: In summary, we describe here a versatile model of gastric carcinogenesis that uncouples the genetics of the tumor and the host, and that faithfully recapitulates key risk factors of the malignancy.},
}
@article {pmid41288772,
year = {2025},
author = {Sun, W and Ren, X and Huang, J and Wang, Y and Liu, S},
title = {B-PER tandem assembly CRISPR/Cas12a cascade amplification strategy based fluorescence/colorimetric dual-mode for detection of MC-LR.},
journal = {Mikrochimica acta},
volume = {192},
number = {12},
pages = {853},
pmid = {41288772},
issn = {1436-5073},
support = {32072330//National Natural Science Foundation of China/ ; ZR2022MB066//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Microcystins/analysis ; Quantum Dots/chemistry ; *Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *Colorimetry/methods ; Limit of Detection ; *Nucleic Acid Amplification Techniques/methods ; Marine Toxins ; Fluorescence ; Spectrometry, Fluorescence/methods ; Cadmium Compounds/chemistry ; },
abstract = {Microcystins are the most common, most powerful and most toxic types of cyanobacteria, which seriously threaten the public health and ecological environment. Biosensor has been widely used in Microcystin-Leucine-Arginine (MC-LR) detection. In this study, we report an efficient single-hairpin with double primers (the trigger chain T hybridizes with the left end a, and the amplified product ab hybridizes with the right end a) for bidirectional PER (B-PER) amplification strategy for uniform visual and fluorescence detection of trace amounts of MC-LR in the environment using quantum dots (QDs) as signal reporters. The biosensor is triggered by a single stranded DNA of MC-LR specificity to initiate the amplification by B-PER. From the long product of the B-PER, the trans-cleavage activity of CRISPR/Cas12a is activated, and the Ag[+] is released from the C-Ag[+]-C. The released Ag[+] undergoes cation exchange reaction (CER) with CdTe QDs (QDs), quenching QDs fluorescence and generating visual and fluorescent dual signals. The biosensor can simultaneously complete the display of dual signals of naked eyes and fluorescence, and can successfully used for the detection of the actual environment sample. In addition, the biosensor has low detection limits, high sensitivity, good accuracy and high selectivity. The detection range under the best conditions was 0.05-500 nM, and the detection limit was 0.705 pM. In summary, this strategy provides a general detection platform for detecting trace pollutants in the environment by using biosensors.},
}
@article {pmid41289157,
year = {2026},
author = {van der Meulen, SA and Roemhild, K and Driessen, M and van den Akker, E and Nethe, M},
title = {In vivo modeling of stress erythropoiesis through targeted gene editing of rat hematopoietic stem cells.},
journal = {Blood advances},
volume = {10},
number = {4},
pages = {1281-1292},
pmid = {41289157},
issn = {2473-9537},
mesh = {Animals ; *Erythropoiesis/genetics ; Rats ; *Gene Editing/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; CRISPR-Cas Systems ; Anemia/etiology/genetics/metabolism ; Cadherins/genetics/metabolism ; Disease Models, Animal ; *Stress, Physiological ; },
abstract = {In response to anemia, the erythroid lineage significantly expands. This growth is driven by extramedullary erythropoiesis in mice, but is additionally regulated within the bone marrow (BM) of rats, a process likely conserved in humans due to similar BM architecture. This process is, however, mostly elusive. We identified E-cadherin to mark the expansion of the erythroid lineage in BM from anemic rats. To explore the regulation of erythropoiesis in the BM in response to anemia, we studied the role of E-cadherin in the erythroid lineage of rats. As genetic methods to model erythropoiesis in rats are limited, we established a rat BM transplant model that, combined with CRISPR/Cas9 genome editing, enabled us to examine the control of E-cadherin in BM in response to anemia. We identified CD90+CD44+CD45R- cells to contain hematopoietic stem and progenitor cells (HSPCs) in rats. CD90+CD44+CD45R--enriched HSPCs can be efficiently edited using CRISPR/Cas9, which, upon transplant, induce high BM chimerism. Importantly, we identified that recovery from irradiation-induced anemia involves 2 phases. Phase 1 is marked by expansion of erythroid precursors in the BM, supported by extramedullary erythropoiesis in the spleen. This phase is followed by a second phase, characterized by accelerated terminal differentiation, which is selectively controlled in the BM. Finally, we discovered that genetic inactivation of hematopoietic-expressed E-cadherin delays recovery from radiation-induced anemia. Our work provides novel means to expand our knowledge on hematology, and the opportunity to dissect the molecular regulation underlying the erythroid response(s) to anemia in BM, using rat models.},
}
@article {pmid41289351,
year = {2025},
author = {Su, T and Wei, T and Wang, Z and Wu, H and Fan, Y and Su, S and Zhu, D and Wang, L},
title = {A Pre-Amplification-Free Modular Dual-CRISPR System for Enhanced Pathogen Detection Sensitivity.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26640-26648},
doi = {10.1021/acs.analchem.5c05145},
pmid = {41289351},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems/genetics ; Nucleic Acid Hybridization ; *African Swine Fever Virus/genetics/isolation &amp; purification ; Limit of Detection ; Humans ; *DNA, Viral/analysis/genetics ; Animals ; Nucleic Acid Amplification Techniques ; },
abstract = {CRISPR/Cas12a is extensively utilized for pathogen detection owing to its high specificity and efficiency. However, traditional single-CRISPR/Cas12a encounters challenges due to its limited sensitivity, requiring pre-amplification of nucleic acids. This increases the complexity of the procedure and the potential for cross-contamination and false positives. Herein, a modular dual-CRISPR approach was developed coupled with hybridization chain reaction (HCR) for the universal and sensitive detection of pathogen nucleic acids without the need for pre-amplification. The system comprises two core modules: the first CRISPR/Cas12a recognition module specifically identifies pathogen targets and releases the activating agent, while the second CRISPR/Cas12a signal module is activated by this agent to initiate the HCR reaction for generating a strong fluorescent signal through DNA nanostructure self-assembly. Through rational design, we demonstrate the ability of this dual-CRISPR system to achieve attomolar (aM) level sensitivity for pathogen nucleic acid detection without pre-amplification, showing over six-order-of-magnitude higher sensitivity than a traditional single-CRISPR/Cas12a system. Additionally, the flexibility and versatility of the modular dual-CRISPR system have been confirmed for diverse pathogen targets, such as African swine fever virus (ASFV), severe fever with thrombocytopenia syndrome virus (SFTSV), and human papillomavirus type 16 (HPV-16) DNA. The system's practicality was demonstrated by examining ASFV quality control samples in complex environments. The exploration of the pre-amplification-free dual-CRISPR system offers a new perspective on enhancing pathogen nucleic acid detection systems.},
}
@article {pmid41290964,
year = {2025},
author = {El Menofy, NG and Payoumi, AN and Eissa, MA and El-Sharif, A},
title = {Association of the existence of CRISPR-Cas system and antimicrobial resistance in multi-drug resistant Klebsiella pneumoniae in Egypt.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {41814},
pmid = {41290964},
issn = {2045-2322},
mesh = {*Klebsiella pneumoniae/genetics/drug effects/isolation &amp; purification ; Egypt ; *Drug Resistance, Multiple, Bacterial/genetics ; *CRISPR-Cas Systems/genetics ; *Anti-Bacterial Agents/pharmacology ; Humans ; Microbial Sensitivity Tests ; *Klebsiella Infections/microbiology/drug therapy/epidemiology ; beta-Lactamases/genetics ; Colistin/pharmacology ; Bacterial Proteins/genetics ; },
abstract = {The CRISPR-Cas systems are supposed to be associated with antibiotic susceptibility. Klebsiella pneumoniae (K. pneumoniae) is a major multidrug-resistant (MDR) pathogen that may cause severe infections. This study aimed to detect the antimicrobial resistance (AMR) of K. pneumoniae isolates in addition to determine the association between the existence of CRISPR-Cas systems and the presence of AMR in Egypt. The antibiotic susceptibility patterns of 100 K. pneumoniae isolates were determined using the Kirby Bauer disc diffusion and broth microdilution methods. The frequency of carbapenem resistance encoding genes (blaKPC, blaOXA, blaIMP, blaNDM, and blaVIM), ESBLs encoding genes (blaTEM), aminoglycoside resistance encoding genes (aac(3)-Ia, aac(3)-IIa, colistin resistance encoding genes (mcr-1, mcr-2) and tetracycline resistance encoding genes (tetB) were determined using polymerase chain reaction (PCR). The presence or absence of CRISPR-Cas systems was determined by detection of Cas genes (Cas1 or Cas3) in conjunction with one of CRISPR arrays 1, 2 or 3. Kirby Bauer disc diffusion revealed that 95% of isolates were MDR. The resistance rates of K. pneumoniae isolates to amikacin, meropenem, and colistin were 76%, 67%, and 41% respectively by broth microdilution assay. Among selected 41 K. pneumoniae, the frequency of ESBLs; blaTEM was 92.7%, while the frequency of blaNDM blaOXA blaVIM blaIMP and blaKPC was 95.1%, 95.1%, 39%, 19.5% and 14.6% respectively. The frequency of mcr-1 and mcr-2 was 70.7% and 65.9%. Additionally, the frequency of aac(3)-Ia was 12.2%, and aac(3)-IIa was 87.8%, while the frequency of tetB was 100%. Our isolates exhibited varied profiles for CRISPR-Cas systems, where 65.9% were positive for CRISPR-Cas system. No general significant positive correlation between AMR and the presence ofCRISPR-Cas system was detected; however, a significant difference is present for imipenem, colistin and chloramphenicol phenotypic resistance andfor aac(3)-IIa and mcr-1 genes (P value > 0.1). A significant positive correlation was detected between AMR for imipenem, colistin and chloramphenicol and for aac(3)-IIa and mcr-1 genes and thepresence of CRISPR-Cas system.},
}
@article {pmid41291135,
year = {2025},
author = {Hann, E and Majumdar, D and Layton, D and Fareh, M and Cahill, DM and Ziemann, M and Ujvari, B and Schat, KA and Challagulla, A},
title = {Systematic evaluation of CrRNA design parameters for optimized Cas13d-mediated RNA targeting in chicken cells.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {256},
pmid = {41291135},
issn = {1438-7948},
mesh = {Animals ; Chickens/genetics ; *CRISPR-Cas Systems ; Cell Line ; RNA, Messenger/genetics ; },
abstract = {The CRISPR-Cas13 system has emerged as a powerful platform for programmable RNA targeting, offering efficient and sequence-specific silencing of coding and non-coding transcripts. The RNA-targeting capabilities of CRISPR-Cas13 have been harnessed to silence transcripts harbouring pathogenic mutations and combat infectious diseases. However, the molecular basis of on-target and collateral activity are not completely understood, limiting the utility of Cas13 systems. In this study, we delineate the principles for the development of effective crRNAs by targeting DsRed fluorescence reporter and synthetic influenza mRNA in chicken fibroblast DF1 cells. To systematically determine the optimal design for RfxCas13d crRNA, we investigated the minimum length of the crRNA, importance of protospacer flanking sequence, degree of mismatch tolerance, and off target effects. Our data reveal variable knockdown levels between crRNAs, in which several crRNAs achieved over 95% target knockdown. We show that crRNAs exhibit a high degree of tolerance to single-nucleotide mismatches, regardless of their position in the spacer sequence. However, 4-nt mismatches between the spacer and the target significantly reduces targeting efficacy, whereas eight nucleotide mismatches completely abolish the activity of RfxCas13d. Finally, we compared targeting efficiency and collateral activity of two widely used RfxCas13d and HfCas13d variants. Our data extend current understanding of Cas13d-mediated RNA targeting and offer a framework for rational crRNA design to enhance effectiveness in diverse applications, including antiviral strategies.},
}
@article {pmid41291345,
year = {2025},
author = {Wang, Z and Zhang, Y and Xu, N and Liu, L and Zhang, M and Huang, S and Su, C and Liu, T and Duan, K},
title = {Improving soybean fatty acid profiles by CRISPR/Cas12a-mediated gene editing of GmFAD2 and GmFAD3.},
journal = {Plant cell reports},
volume = {44},
number = {12},
pages = {282},
pmid = {41291345},
issn = {1432-203X},
support = {32172499//National Natural Science Foundation of China/ ; JSSCTD202342//Shuangchuang Program of Jiangsu Province/ ; },
mesh = {*Glycine max/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Fatty Acids/metabolism ; Soybean Oil/metabolism ; Plants, Genetically Modified ; Linoleic Acid/metabolism ; *Plant Proteins/genetics/metabolism ; Oleic Acid/metabolism ; Fatty Acid Desaturases/genetics/metabolism ; },
abstract = {Using CRISPR/Cas12a, we engineered novel soybean germplasms by knocking out GmFAD2 (GmFAD2-1A, GmFAD2-1B) and GmFAD3 (GmFAD3A, GmFAD3B) genes, yielding elevated oleic or linoleic acid content. Soybean oil contains high levels of polyunsaturated fatty acids (PUFAs), which are known to reduce cholesterol levels and help prevent hypertension, thereby contributing significantly to human health. However, the chemical instability of PUFAs makes them susceptible to oxidation, a process that generates harmful trans-fatty acids. To address this issue, precise modulation of fatty acid composition in soybeans becomes critically important for health applications. In this study, we employed CRISPR/Cas12a gene editing technology to selectively knock out the GmFAD2 (GmFAD2-1A, GmFAD2-1B) and GmFAD3 (GmFAD3A, GmFAD3B) genes in soybean. This approach successfully created novel soybean germplasms with distinct fatty acid profiles: one with elevated oleic acid content and another with increased linoleic acid levels. These engineered variants provide valuable options for utilizing soybean oil with optimized fatty acid compositions tailored for specific health and nutritional purposes.},
}
@article {pmid41292010,
year = {2026},
author = {Chen, C and Kassim, Y and Xu, X and He, X and Lin, H and Dang, Y and Shi, Y and Wang, H and Wang, S and Zhang, K},
title = {Gene editing and multi-omics approaches to study early embryogenesis in cattle.},
journal = {Reproduction, fertility, and development},
volume = {38},
number = {1},
pages = {},
doi = {10.1071/RD25167},
pmid = {41292010},
issn = {1448-5990},
mesh = {Animals ; Cattle/embryology ; *Gene Editing/veterinary ; *Embryonic Development/genetics ; Proteomics ; *Metabolomics/methods ; Genomics/methods ; CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; Single-Cell Analysis ; Multiomics ; },
abstract = {Advancements in multi-omics profiling and targeted molecular functional tools have led to significant progress in our understanding of early embryonic development in mammals. This is very relevant in the beef and dairy industries for exploring the etiology of early embryo loss. This review highlights how state-of-the-art single-cell and integrative low-input omics technologies, including single-cell RNA-seq, ATAC-seq, metabolomics, and proteomics, have uncovered complex developmental dynamics during the first week of bovine embryogenesis. This review also provides a concurrent overview of the application of functional tools, including CRISPR-Cas9, RNA editing, base editing, and Trim-Away, in the analysis of critical genes/proteins during the various stages of early embryo development in cattle. The integration of high-dimensional molecular profiling with targeted manipulations enables researchers to analyze key developmental events with unparalleled resolution. It is evident that these approaches provide a more comprehensive mechanistic depiction of early embryogenesis, thereby informing translational efforts in improving bovine fertility.},
}
@article {pmid41292075,
year = {2025},
author = {Du, J and Hu, J and An, J and Li, H and Chen, B and Luo, J and Li, S and Teng, Y and Yuan, T and Zhu, X and Jiang, L and Xiong, E and Yang, R},
title = {Guanine-Quadruplex-Engineered crRNA Enables Light-Activated CRISPR/Cas12a System for Robust One-Pot Viral Assay.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26580-26589},
doi = {10.1021/acs.analchem.5c04848},
pmid = {41292075},
issn = {1520-6882},
mesh = {*G-Quadruplexes ; *CRISPR-Cas Systems/genetics ; Nucleic Acid Amplification Techniques/methods ; Humans ; *Guanine/chemistry ; *Light ; CRISPR-Associated Proteins/metabolism ; Bacterial Proteins ; Endodeoxyribonucleases ; },
abstract = {Conventional one-pot detection platforms integrating CRISPR/Cas12a with isothermal amplification significantly streamline the nucleic acid detection workflow, while minimizing the risk of aerosol contamination. However, the intrinsic cleavage activity of the CRISPR/Cas12a system can substantially interfere with the nucleic acid amplification efficiency, ultimately compromising detection sensitivity. Herein, we develop a light-activated CRISPR/Cas12a system by engineering the crRNA with a guanine-quadruplex (G4) motif at its 3'-terminal, achieving precise regulation of Cas12a activity via photoswitching G4 structure formation. Through coupling with a recombinase polymerase amplification (RPA) reaction, we establish a one-pot detection platform that demonstrates superior detection performance compared to traditional Cas12a-based one-pot systems. The detection sensitivity has been improved by 2 orders of magnitude, reaching a level of 1 copy/μL. Notably, the platform demonstrated comparable sensitivity and specificity to PCR, the gold standard method, in detecting clinical samples, such as Epstein-Barr virus (EBV) and Influenza A virus (IAV), making it a promising technology for clinical diagnostics.},
}
@article {pmid41292433,
year = {2025},
author = {Lan, F and Chen, A and Ding, Y and Yang, C and Zhang, P and Fang, X},
title = {Sensitive and Specific Analysis of miRNAs in Single Tumor-Derived Extracellular Vesicles Using CRISPR-Based Nanoflow Cytometry.},
journal = {Analytical chemistry},
volume = {97},
number = {48},
pages = {26521-26531},
doi = {10.1021/acs.analchem.5c04700},
pmid = {41292433},
issn = {1520-6882},
mesh = {*MicroRNAs/analysis/genetics ; Humans ; *Extracellular Vesicles/chemistry/genetics/metabolism ; *CRISPR-Cas Systems ; *Prostatic Neoplasms/genetics/diagnosis ; *Flow Cytometry/methods ; Male ; Limit of Detection ; Biomarkers, Tumor/genetics ; *Nanotechnology ; },
abstract = {Tumor-derived extracellular vesicle (TEV) microRNAs (miRNAs) are promising cancer biomarkers but pose detection challenges due to their low abundance and sequence homology. Here, we present a CRISPR/Cas13a-based nanoflow cytometry (nFCM) platform integrated with a DNA-guided orthogonal membrane fusion strategy for ultrasensitive miRNA detection of TEVs at the single particle level. TEVs were identified with aptamers against CD63 and EpCAM markers to create an orthogonal barcode-anchored TEV (Orth-TEV). Meanwhile, liposomes preloaded with CRISPR/Cas13a molecular sensing components were modified with cholesterol-tagged DNA probes to produce Tags-CRISPR/Cas13a@Lipo. The complementary DNA sequences on the Orth-TEV and Tags-CRISPR/Cas13a@Lipo vesicles facilitated zipper-like hybridization, thereby achieving specific membrane fusion to effectively eliminate the interference of nontarget vesicles or free molecules. The resulting TEV-CRISPR/Cas13a@Lipo vesicles allow in situ detection of three prostate cancer (PCa)-associated miRNAs in a single TEV via nFCM with a low detection limit (LOD) of 14.7 (miR-153), 16.0 (miR-183), and 23.7 (miR-940) particles/mL, respectively. The approach was further applied to plasma samples from PCa patients and healthy donors, showing significantly elevated miRNA signals in PCa-derived TEV. ROC analysis yielded AUC values of 0.931, 0.923, and 0.869 for the three target miRNAs, confirming excellent diagnostic performance. To enhance classification accuracy, we conducted a statistical multivariate analysis based on the PCA-LDA model, which achieved perfect group separation and a diagnostic accuracy of 91.3%. Overall, this CRISPR/Cas13a-based nFCM platform offers a robust, accurate, and clinically applicable platform for single-vesicle miRNA profiling with broad potential in liquid biopsy-based cancer diagnosis.},
}
@article {pmid41292787,
year = {2025},
author = {Majumder, P and Cahir, CW and Roberts, CG and Patel, DJ and Marraffini, LA},
title = {Cap1 forms a cyclic tetra-adenylate-induced membrane pore during the type III-A CRISPR-Cas immune response.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41292787},
issn = {2692-8205},
support = {R01 GM129430/GM/NIGMS NIH HHS/United States ; P30 CA008748/CA/NCI NIH HHS/United States ; R01 AI141507/AI/NIAID NIH HHS/United States ; R01 GM149834/GM/NIGMS NIH HHS/United States ; R01 GM145888/GM/NIGMS NIH HHS/United States ; },
abstract = {During type III CRISPR-Cas immunity in prokaryotes, RNA-guided recognition of viral (phage) transcripts stimulates the Cas10 complex to convert ATP into cyclic oligoadenylates. These act as signaling molecules that bind to CARF proteins and activate their effector domains. Here, we report the structure and function of the Cap1 effector, composed of a pair of transmembrane helices (TM1/2), a CARF-like (CARFL) domain and a domain of unknown function (DUF4579). Cryo-EM studies on apo- and ligand-bound states of Cap1 in glyco-diosgenin detergent revealed the formation of tetrameric complexes in both states, with one cyclic tetra-adenylate molecule bound in a pocket composed by the four CARFL domains. Binding of cA4 triggers conformational changes that widen an otherwise narrow pore formed by the four TM1/2 domains. In vivo, Cap1 activation results in membrane depolarization, a growth arrest of the bacterial host and the abrogation of the viral lytic cycle. Our findings reveal the mechanistic basis of membrane depolarizarion mediated by cyclic nucleotide signaling during the type III CRISPR-Cas response.},
}
@article {pmid41293956,
year = {2025},
author = {Zang, J and Niklaus, S and Neuhauss, SCF},
title = {An EAAT2b/SLC1A2b-mediated chloride leak current enables rapid cone photoreceptor signalling.},
journal = {Open biology},
volume = {15},
number = {11},
pages = {250347},
pmid = {41293956},
issn = {2046-2441},
support = {/SNSF_/Swiss National Science Foundation/Switzerland ; },
mesh = {Animals ; *Retinal Cone Photoreceptor Cells/metabolism ; Zebrafish/genetics/metabolism ; *Excitatory Amino Acid Transporter 2/metabolism/genetics ; *Signal Transduction ; Electroretinography ; *Chlorides/metabolism ; *Zebrafish Proteins/metabolism/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; Gene Editing ; },
abstract = {Excitatory amino acid transporters not only mediate high-affinity glutamate uptake but also conduct an uncoupled chloride current. In zebrafish, a whole-genome duplication gave rise to two eaat2 paralogues with distinct roles. Excitatory amino acid transporter 2a (SLC1A2b, GLT-1) functions primarily in Müller glia as a glutamate transporter, whereas excitatory amino acid transporter 2b is expressed in cone photoreceptors and exhibits a prominent glutamate-independent chloride current. We hypothesized that this leak current stabilizes the cone resting membrane potential, thereby supporting rapid visual signalling. In order to test this hypothesis, we generated eaat2b knockout zebrafish using CRISPR-Cas9-mediated genome editing. While eaat2b mutants showed no gross morphological abnormalities, they exhibited reduced electroretinogram b-wave amplitudes. Consistent with our hypothesis, eaat2b-deficient larvae displayed a significant reduction in flicker fusion electroretinogram power at each stimulus frequency, indicating impaired temporal processing likely due to delayed repolarization of cone photoreceptors. Our findings reveal a critical role for an excitatory amino acid transporter 2b-mediated chloride anion leak current in regulating the kinetics of photoreceptor responses. This functional innovation, enabled by a whole-genome duplication in the teleost lineage, highlights how gene duplications can lead to the acquisition of physiologically relevant new functions.},
}
@article {pmid41294335,
year = {2026},
author = {Paulin, OKA and Tsavou, A and Priest, EL and Griffiths, JS and Lortal, L and Kempf, A and Chow, EWL and Pang, LM and Wickramasinghe, DN and Lyon, CM and Hernday, AD and Wang, Y and Richardson, JP and Naglik, JR},
title = {The combinatorial action of hyphal growth and candidalysin is critical for promoting Candida albicans oropharyngeal infection.},
journal = {mBio},
volume = {17},
number = {1},
pages = {e0330425},
pmid = {41294335},
issn = {2150-7511},
support = {R15AI185747//National Institute of Allergy and Infectious Diseases/ ; R15 AI185747/AI/NIAID NIH HHS/United States ; 214229_Z_18_Z/WT_/Wellcome Trust/United Kingdom ; /WT_/Wellcome Trust/United Kingdom ; BBSRC: UKRI717/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; },
mesh = {*Candida albicans/genetics/growth &amp; development/pathogenicity ; *Hyphae/growth &amp; development/genetics ; Animals ; *Fungal Proteins/genetics/metabolism ; Mice ; *Virulence Factors/genetics/metabolism ; Humans ; Gene Deletion ; Disease Models, Animal ; Aspartic Acid Endopeptidases/genetics/metabolism ; Epithelial Cells/microbiology ; Female ; *Candidiasis, Oral/microbiology ; Virulence ; Oropharynx/microbiology ; Candidiasis/microbiology ; CRISPR-Cas Systems ; },
abstract = {Candida albicans is one of the most common fungal pathogens, yet much remains unknown about how its virulence factors cooperate to promote pathogenicity. To investigate this, CRISPR-Cas9 technology was used to create a panel of 19 single, double, triple, and quadruple deletion mutant strains targeting four established virulence factors: ALS3 (adhesin/invasin), ECE1 (candidalysin toxin), HGC1 (hypha formation regulator), and SAP2 (protease). In vitro, the deletion of each gene had differing impacts across multiple characterization assays. The hgc1∆/∆ mutant was unable to form hyphae under inducing conditions, leading to downstream impairment of epithelial invasion. The als3∆/∆ mutant exhibited significantly reduced adhesion and invasion into epithelial cells, resulting in attenuated cellular damage. The ece1∆/∆ mutant displayed significantly reduced epithelial damage, cell signaling, and immune activation. The phenotype of the sap2∆/∆ mutant resembled that of wild type but was unable to degrade protein. In an immunocompromised murine model of oropharyngeal infection, hyphal growth and candidalysin production were the dominant drivers of elevated fungal burden, innate immune responses, and mortality. Following a 5-day infection with hgc1∆/∆ and ece1∆/∆ single gene deletion strains, mice had survival rates of 100% and 80%, respectively, compared to 15% in wild-type infected mice. Notably, 100% survival was also observed following challenge with all hgc1∆/∆ and ece1∆/∆ combination mutants. This study demonstrates that specific C. albicans virulence attributes act in combination to promote mucosal infection, with hyphal growth and candidalysin production being a critical driver of oropharyngeal infection.IMPORTANCECandida albicans has been classified by the WHO as a "critical priority" pathogen, highlighting the urgent need for a greater understanding of the mechanisms that enable it to cause disease. C. albicans possesses numerous virulence attributes, but how they synergize during infection is not well understood. Here, using reverse genetics, we dissect the individual and combinatorial roles of four C. albicans virulence factors (Als3p, candidalysin, hyphal growth, and Sap2p) in vitro and in an in vivo murine model of oropharyngeal candidiasis. Increasing the number of C. albicans gene deletions correlated with reduced oral fungal burden, with hyphal growth and candidalysin together being critical for infection, inflammation, and mortality during oropharyngeal infection. These findings demonstrate that virulence attributes act cooperatively as a collective network to promote pathogenicity, a finding also observed in plant fungal pathogens. Our approach has identified specific fungal virulence factors that can be targeted for new treatment strategies against C. albicans infections.},
}
@article {pmid41294729,
year = {2025},
author = {Guo, W and Jiang, M and Xie, Y and Xu, H and Sun, Z},
title = {Recognition Element-Based Strategies for Rapid Detection of Foodborne Pathogens: Recent Progress and Perspectives.},
journal = {Biosensors},
volume = {15},
number = {11},
pages = {},
pmid = {41294729},
issn = {2079-6374},
support = {BK20241924//Natural Science Foundation of Jiangsu Province/ ; SH2024010//Key Research and Development Program of Zhenjiang City/ ; SH2024112//Key Research and Development Program of Zhenjiang City/ ; NY2023002//Key Research and Development Program of Zhenjiang City/ ; },
mesh = {*Biosensing Techniques/methods ; *Food Microbiology ; Humans ; *Foodborne Diseases/microbiology/diagnosis ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; Molecular Imprinting ; },
abstract = {The detection of foodborne pathogens is of great significance for safeguarding food safety and public health. In recent years, rapid detection technologies based on diverse recognition elements have advanced considerably, driven by progress in molecular biology, materials science, and information technology. This review takes recognition elements as the central theme and systematically outlines the mechanisms and research progress of antibodies, nucleic acid aptamers, nucleic acid amplification techniques, CRISPR/Cas systems, molecular imprinting technology, peptides, and small-molecule receptors in foodborne pathogen detection, while comparing their performance in terms of specificity, sensitivity, stability, and applicability. In addition, this review further elaborates on the developmental trends of detection platforms, including multi-target and multimodal integration, microfluidics combined with portable point-of-care testing (POCT) systems, and intelligent terminals empowered by artificial intelligence algorithms. These trends provide new perspectives for improving detection systems in terms of throughput, portability, and intelligence. Overall, this review aims to serve as a comprehensive reference for the development of rapid, accurate, and intelligent detection systems for foodborne pathogens.},
}
@article {pmid41294822,
year = {2025},
author = {Bucheeri, S and Alcibahy, Y and Bucheeri, Y and Bucheeri, S and Alhermi, A and Butler, AE},
title = {CRISPR as a Tool to Uncover Gene Function in Polycystic Ovary Syndrome: A Literature Review of Experimental Models Targeting Ovarian and Metabolic Genes.},
journal = {Cells},
volume = {14},
number = {22},
pages = {},
pmid = {41294822},
issn = {2073-4409},
mesh = {*Polycystic Ovary Syndrome/genetics/metabolism ; Animals ; Female ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Disease Models, Animal ; *Ovary/metabolism/pathology ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Polycystic ovary syndrome (PCOS) is a complex disorder characterized by reproductive abnormalities such as hyperandrogenism, ovulatory dysfunction, and polycystic ovarian morphology, and is frequently accompanied by metabolic disturbances such as insulin resistance, obesity and dyslipidemia. Genome-wide association studies (GWASs) have identified several susceptibility loci, yet little is known about their functional implications. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a powerful gene editing tool in bridging this gap by allowing researchers to directly target candidate genes in ovarian and metabolic pathways. For instance, experimental models have highlighted the role of CYP17A1 and DENND1A.V2 in androgen excess, anti-Müllerian hormone (AMH) in follicular arrest, and insulin receptor substrate 1 (IRS1) and PPARγ in insulin signaling and adipogenesis. To highlight the multifactorial nature of PCOS, animal models, including zebrafish and rodents, have been used to reveal interactions between reproductive and metabolic phenotypes. Nevertheless, most studies remain restricted to single-gene models, and dual-gene models or combined gene editing and hormonal induction models remain underexplored. Future research integrating precision editing, multi-omic platforms, and patient-derived organoids may provide more accurate disease models and novel therapeutic strategies.},
}
@article {pmid41294842,
year = {2025},
author = {Nagaoka, K and Kobayashi, Y and Kakimi, K},
title = {NeoPAIR-T: Functional Mapping of Neoantigen-TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System.},
journal = {Cells},
volume = {14},
number = {22},
pages = {},
pmid = {41294842},
issn = {2073-4409},
support = {24ama221321h0002//Japan Agency for Medical Research and Development (AMED)/ ; 23H02762//JSPS KAKENHI/ ; },
mesh = {Humans ; Jurkat Cells ; *Receptors, Antigen, T-Cell/metabolism/genetics ; *Antigens, Neoplasm/immunology/genetics/metabolism ; Genes, Reporter ; *CRISPR-Cas Systems/genetics ; Coculture Techniques ; Antigen-Presenting Cells/immunology/metabolism ; },
abstract = {Targeting mutation-derived neoantigens is a promising strategy for personalized immunotherapies. However, identifying true neoantigens and cognate T cell receptors (TCRs) remains challenging because computational prediction of neoantigen peptides is uncertain and most tumor-infiltrating lymphocytes are bystanders rather than tumor-reactive, necessitating functional validation. Here, we developed NeoPAIR-T (Neoantigen-TCR Pairing Assay using reporter T cells), a functional assay based on co-culture of TCR-T reporter cells and autologous antigen-presenting cells (APCs) to screen neoantigen-TCR pairs. Reporter T cells are Jurkat-derived cells engineered to express a luciferase/eGFP dual reporter, providing quantitative readouts of TCR activation, while APCs are immortalized autologous cells transfected with tandem minigenes (TMGs) encoding predicted neoantigens, bypassing peptide synthesis. NeoPAIR-T also includes TCRα-knockout with targeted knock-in of candidate TCRs at the TCRβ locus to prevent mispairing and enables parallel testing of multiple reporter T cell clones co-cultured with the same APCs for efficient identification of functional pairs. Using lung cancer samples, whole-exome and RNA sequencing predicted 63 candidate peptides assembled into three TMGs. Single-cell RNA/TCR sequencing identified eight TCR clonotypes, introduced into reporter T cells and tested in parallel. Co-culture with TMG-expressing APCs revealed two functional neoantigen-TCR pairs validated by peptide assays (EC50: 10[-9.2]-10[-6.7] M). Collectively, NeoPAIR-T streamlines neoantigen-TCR identification for vaccine and TCR-T applications.},
}
@article {pmid41296247,
year = {2026},
author = {Aksoy, MO and Rozynek, J and Stachowiak, M and Szczerbal, I},
title = {The role of the CEBPB gene in porcine adipogenesis: a study using CRISPR/Cas9-edited mesenchymal stem cells.},
journal = {Journal of applied genetics},
volume = {67},
number = {2},
pages = {477-486},
pmid = {41296247},
issn = {2190-3883},
support = {2018/29/B/NZ2/00956//Narodowe Centrum Nauki/ ; },
mesh = {Animals ; *Adipogenesis/genetics ; *Mesenchymal Stem Cells/cytology/metabolism ; *CRISPR-Cas Systems/genetics ; Swine/genetics ; *CCAAT-Enhancer-Binding Protein-beta/genetics ; *Gene Editing ; Cell Differentiation/genetics ; Promoter Regions, Genetic ; Adipocytes/cytology/metabolism ; },
abstract = {Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 is a powerful tool for gene editing and the regulation of gene expression. It enables the introduction of targeted mutations, thereby facilitating functional studies of specific genes in various cellular processes. In this study, we aimed to generate a deletion in the promoter region of the CEBPB gene, which encodes a transcription factor involved in adipogenesis, and to evaluate the impact of this modification on the adipogenic differentiation potential of porcine mesenchymal stem cells (MSCs). A 575-bp deletion was introduced in the target region, resulting in the generation of both homozygous and heterozygous mutant cells. Adipogenic differentiation was assessed by quantifying transcript levels of adipocyte marker genes (GATA2, CEBPA, PPARG, and FABP4) at days 0, 4, 6, 8, and 10 of the differentiation process. Disruption of CEBPB expression led to the downregulation of these adipogenic markers, indicating impaired adipocyte differentiation. Additionally, to assess the proliferative capacity of the edited cells, the expression levels of proliferation-associated genes (CCND1, MCM2, and PCNA) were measured. A reduction in their transcript levels was observed in both homozygous and heterozygous mutant cells. These findings indicate that both homozygous and heterozygous deletions in the CEBPB promoter completely block adipogenesis and alter MSC proliferation, highlighting the pivotal role of CEBPB not only in adipogenic differentiation but also in the regulation of cell proliferation in porcine mesenchymal stem cells. These results provide new insights into the molecular mechanisms underlying adipose tissue development and have implications for pig breeding strategies aimed at optimizing carcass composition, as well as for biomedical research focused on adipose tissue biology.},
}
@article {pmid41296365,
year = {2025},
author = {Chen, W and Fan, L and Dong, M and Gao, L and Du, X and Wang, L and Su, S and Liu, LE and Wu, Y and Ding, L},
title = {Emerging CRISPR/Cas-Based Strategies for Extracellular Vesicle Detection: A Comprehensive Review.},
journal = {ACS sensors},
volume = {10},
number = {12},
pages = {9135-9152},
doi = {10.1021/acssensors.5c02441},
pmid = {41296365},
issn = {2379-3694},
mesh = {*Extracellular Vesicles/metabolism/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Biosensing Techniques/methods ; },
abstract = {Extracellular vesicles (EVs), which carry a variety of molecules such as proteins and nucleic acids, have great potential for broad application in liquid biopsy. However, achieving highly sensitive detection of biomarkers within EVs remains a significant challenge. The emergence of CRISPR/Cas systems─adaptive immune mechanisms found in bacteria and archaea that defend against foreign genetic elements─offers new opportunities to address this issue through powerful nucleic acid recognition and cleavage capabilities. Compared to other EV detection techniques, CRISPR/Cas-based biosensors exhibit superior sensitivity, specificity, and operational efficiency, making them a compelling platform for clinical translation. Thus, to promote the application of EVs in disease diagnosis, disease monitoring, and therapeutic evaluation, this review focuses on the state-of-the-art CRISPR/Cas systems (specifically CRISPR/Cas9, CRISPR/Cas12, CRISPR/Cas13, and CRISPR/Cas14) as well as the latest applications of CRISPR/Cas-based EV detection techniques.},
}
@article {pmid41298443,
year = {2025},
author = {Wei, T and Yang, X and Jiang, C and Liao, K and Ye, H and Luo, HY and Liu, YR and Wang, PY and Meng, F and Dou, SX and Rong, Z and Li, H},
title = {Osmotic pressure regulates DNA labelling and transcription with dCas9-SunTag system in live cells.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {773},
pmid = {41298443},
issn = {2041-1723},
support = {12122402, 12074043//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Osmotic Pressure/physiology ; *DNA/metabolism/genetics ; Humans ; *Transcription, Genetic ; Cell Nucleus/metabolism/genetics ; CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/metabolism/genetics ; Telomere/metabolism/genetics ; HeLa Cells ; },
abstract = {Extracellular osmotic pressure is a key modulator of intracellular biophysical properties and cellular functions. However, its impact on the cell nucleus remains elusive, largely due to the challenges in real-time measurement of local environmental properties and reaction kinetics at specific loci within the nucleus. Here, we employ the dCas9-SunTag system to investigate the biophysical response at target DNA loci to osmotic pressure alterations. We reveal that variations in extracellular osmotic pressure modulate the efficiency of dCas9-SunTag-mediated fluorescent labelling rapidly and reversibly, with hypoosmotic condition increasing and hyperosmotic condition decreasing the number and fluorescence intensity of foci for telomeres and genes. Strikingly, osmotic pressure also regulates gene transcription with the dCas9-SunTag system, mirroring its effects on fluorescent labelling, as evidenced by changes in mRNA burst frequency. The underlying mechanism is that osmotic pressure shifts the binding-unbinding equilibrium of specific proteins to dCas9-SunTag complex by altering intranuclear crowding. These findings not only highlight the role of mechanical cues in modulating DNA-related processes within the nucleus, but also establish the dCas9-SunTag system as a sensitive probe for intranuclear crowding in response to extracellular cues, notably osmotic pressure.},
}
@article {pmid41298482,
year = {2025},
author = {Aguado-Alvaro, LP and Garitano, N and Esser-Skala, W and Sayers, J and Del Valle, C and Alameda, D and Mendieta-Esteban, J and Calleja-Cervantes, ME and Goñi-Salaverri, A and Zazpe, J and de Vito, AR and Marchese, F and Alignani, D and Cudini, J and Gross, T and Rábago, G and Narayan, N and Martinez, L and Martinez, S and Huntly, B and Riley, P and Gonzalez, A and Taylor-King, JP and Fortelny, N and Pelacho, B and Lara-Astiaso, D},
title = {Identification of epigenetic regulators of fibrotic transformation in cardiac fibroblasts through bulk and single-cell CRISPR screens.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11660},
pmid = {41298482},
issn = {2041-1723},
mesh = {Humans ; Fibrosis/genetics ; *Epigenesis, Genetic ; Single-Cell Analysis/methods ; Myofibroblasts/metabolism/pathology ; *Myocardium/pathology/metabolism/cytology ; *Fibroblasts/metabolism/pathology ; Animals ; Chromatin/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Cas Systems ; Cell Differentiation/genetics ; Cells, Cultured ; Mice ; Transcription Factors/metabolism/genetics ; },
abstract = {Cardiac fibrosis is mediated by the persistent activity of myofibroblasts, which differentiates from resident cardiac fibroblasts in response to tissue damage and stress signals. The signaling pathways and transcription factors regulating fibrotic transformation have been thoroughly studied. In contrast, the roles of chromastin factors in myofibroblast differentiation and their contribution to pathogenic cardiac fibrosis remain poorly understood. Here, we combined bulk and single-cell CRISPR screens to characterize the roles of chromatin factors in the fibrotic transformation of primary cardiac fibroblasts. We uncover strong regulators of fibrotic states including Srcap and Kat5 chromatin remodelers. We confirm that these factors are required for functional processes underlying fibrosis including collagen synthesis and cell contractility. Using chromatin profiling in perturbed cardiac fibroblasts, we demonstrate that pro-fibrotic chromatin complexes facilitate the activity of well-characterized pro-fibrotic transcription factors. Finally, we show that KAT5 inhibition alleviates fibrotic responses in patient-derived human fibroblasts.},
}
@article {pmid41298570,
year = {2025},
author = {Kiseleva, AA and Timonova, EM and Berezhnaya, AA and Kolozhvari, AE and Kochetov, AV and Salina, EA},
title = {Fine tuning wheat heading time through genome editing of transcription factor binding sites in Ppd-1 gene promoter.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42034},
pmid = {41298570},
issn = {2045-2322},
support = {FWNR&#x2011;2024&#x2011;0009//The Ministry of Science and Higher Education of the Russian Federation/ ; 075&#x2011;15&#x2011;2025&#x2011;51//The Kurchatov Genomic Center of ICG SB RAS/ ; },
mesh = {*Triticum/genetics/growth &amp; development ; *Promoter Regions, Genetic ; *Gene Editing/methods ; *Transcription Factors/metabolism/genetics ; Binding Sites ; Gene Expression Regulation, Plant ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Mutation ; Photoperiod ; },
abstract = {Increasing the productivity and adaptability of agricultural plants often depends on optimizing heading time. To develop common wheat lines with accelerated heading and investigate its regulation, we targeted the PPD-1 genes, which control photoperiod sensitivity. Large deletions in the promoter regions of these genes are known to disrupt their expression, resulting in early heading. Using CRISPR/Cas9 genome editing, we generated wheat plants with mutations in the promoter regions of the Ppd-D1 and Ppd-B1 genes. These mutations included nucleotide substitutions, deletions, and insertions ranging from several to hundreds of base pairs, occurring within probable transcription factor binding sites, that may influence gene expression. Under short-day conditions, we assessed PPD-1 gene expression in T0 plants and T2 lines with different mutations. Our analysis revealed that deletions spanning the CHE transcription repressor binding sites altered gene expression patterns, supporting the hypothesis regarding the role of these cis-elements in regulating PPD-1 expression. Furthermore, plants with different mutations displayed distinct diurnal expression patterns, suggesting the involvement of additional transcription factors in the regulation of this gene. Evaluation of heading time in T1 and T2 families with different mutations demonstrated that plants with mutations affecting the "core region", including the CHE binding sites, initiated heading significantly earlier than those without mutations.},
}
@article {pmid41298934,
year = {2025},
author = {Lee, SY and Birkholz, N and Lee, JH and Fineran, PC and Park, HH},
title = {Regulation of anti-CRISPR operons by structurally distinct families of Aca proteins.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1698},
pmid = {41298934},
issn = {2399-3642},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; PE25150//Korea Polar Research Institute (KOPRI)/ ; },
mesh = {*Operon ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation, Bacterial ; *Viral Proteins/genetics/metabolism/chemistry ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Binding ; Bacteriophages/genetics ; Crystallography, X-Ray ; Models, Molecular ; },
abstract = {CRISPR-Cas systems provide bacteria with adaptive immunity against bacteriophages and mobile genetic elements, driving an evolutionary arms race in which phages deploy anti-CRISPR (Acr) proteins. Acr proteins are often co-encoded in operons with anti-CRISPR-associated (Aca) proteins, which coordinate the regulation of acr gene expression. Here, we reveal the molecular basis of DNA binding that mediates transcriptional repression by two distinct Aca family members: Aca7 and Aca11. Crystal structures of Aca7 and Aca11 highlight conserved helix-turn-helix (HTH) motifs within α-helix bundles, providing a universal DNA-binding platform. Aca7 forms a symmetrical dimer to recognize a 19-bp inverted repeat (IR) within the acrIF11-aca7 operon. Strikingly, Aca11 binds 22-bp IRs in two distinct promoters, suggesting that Aca proteins can control multiple target operons. Mutagenesis and electrophoretic mobility shift assays (EMSAs) confirm that dimerization and sequence-specific IR recognition are essential for DNA binding. Despite mechanistic similarities, these and other Aca proteins exhibit notable differences. Structural comparisons across Aca families reveal that while monomer structures are generally similar with conserved HTH motifs, the structures of their dimeric functional units vary significantly. These structural differences might be essential for Aca proteins to bind to various promoters and regulate the expression of different Acr proteins.},
}
@article {pmid41298993,
year = {2025},
author = {Heu, CC and Benowitz, KM and Matzkin, LM and Allan, CW and LeRoy, DM and Li, X and Tabashnik, BE and Carrière, Y and Fabrick, JA},
title = {Editing the kinesin-12 gene affects responses to Bt toxin Cry1Ac in Helicoverpa zea.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {45378},
pmid = {41298993},
issn = {2045-2322},
support = {2020-33522-32268//USDA, National Institute of Food and Agriculture, Biotechnology Risk Assessment Research Grants Program/ ; 2020-67013-31924//USDA, National Institute of Food and Agriculture, Agriculture and Food Research Initiative/ ; 2020-22620-023-000D//USDA, Agricultural Research Service/ ; },
mesh = {Animals ; Bacillus thuringiensis Toxins ; *Endotoxins/pharmacology ; *Gene Editing ; *Hemolysin Proteins/pharmacology ; *Kinesins/genetics ; *Moths/genetics/drug effects ; *Bacterial Proteins/pharmacology ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; Bacillus thuringiensis/genetics ; *Insect Proteins/genetics ; },
abstract = {Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) are used globally to manage key insect pests. However, the evolution of resistance to Bt proteins in at least 11 pest species has reduced the effectiveness of Bt crops. Resistance to crystalline (Cry) Bt proteins including Cry1Ac produced by Bt cotton is a major problem in Helicoverpa zea (also known as bollworm and corn earworm), one of the most economically damaging pests in the United States. A previous genome-wide association study identified a nonsense point mutation in a kinesin-12 gene that was associated with resistance to Cry1Ac in a lab-selected strain of H. zea. Here, we used CRISPR/Cas9 gene editing to knock out the kinesin-12 gene in a Cry1Ac-susceptible laboratory strain, which caused a 4.0-fold increase in resistance to Cry1Ac. Conversely, gene editing that repaired the natural kinesin-12 nonsense mutation in a lab-selected resistant strain increased susceptibility to Cry1Ac by 3.8-fold. These complementary results provide compelling evidence that kinesin-12 plays a role in the mode of action of Cry1Ac against H. zea.},
}
@article {pmid41299842,
year = {2025},
author = {Shi, C and Yu, Z and Tan, H and Li, W and Wang, Y and Wang, Y and Zhang, Q and Man, Y},
title = {One-Pot CRISPR-Based Isothermal Amplification for Nucleic Acid Detection: A Comparative Review of Different Strategies.},
journal = {ACS sensors},
volume = {10},
number = {12},
pages = {9108-9134},
doi = {10.1021/acssensors.5c00806},
pmid = {41299842},
issn = {2379-3694},
mesh = {*Nucleic Acid Amplification Techniques/methods ; *CRISPR-Cas Systems/genetics ; *Nucleic Acids/analysis/genetics ; Humans ; Molecular Diagnostic Techniques ; },
abstract = {Nucleic acid detection plays an important role in pathogen monitoring and disease diagnosis. CRISPR one-pot assays combined with isothermal amplification are emerging as promising point-of-care technologies that simplify workflows while increasing sensitivity and specificity. However, the incompatibility inherent in the one-pot reaction of isothermal amplification and CRISPR detection limits their practical application. This review comprehensively analyzes diverse advanced one-pot CRISPR-based isothermal amplification strategies developed to overcome this fundamental challenge. These strategies primarily encompass physical separation strategies (utilizing lid-bottom, internal ledge, nested tube, and membrane approaches), phase separation strategies (employing glycerol, sucrose, and gel matrices), reaction system optimization strategies (fine-tuning reaction parameters and incorporating specialized additives), non-PAM and suboptimal PAM strategies, improved Cas enzyme strategies (enhanced Cas12 and Cas13 variants), light-controlled approaches (PC-oligonucleotides, NPOM-dt modification, and acylation modification), and microfluidic chip integration strategies (centrifugal microfluidic chips, droplet microfluidic chips, and microarray chips). These methodological approaches have achieved important advances in simplifying operational processes, enhancing sensitivity, shortening detection cycles, and minimizing cross-contamination risks. The review further synthesizes critical insights regarding current opportunities, technical challenges, and future directions for one-pot CRISPR-based isothermal amplification technologies in nucleic acid detection, providing valuable guidance for researchers and practitioners in this evolving field.},
}
@article {pmid41300710,
year = {2025},
author = {Iksat, N and Madirov, A and Zhanassova, K and Masalimov, Z},
title = {Artificial Intelligence-Assisted CRISPR/Cas Systems for Targeting Plant Viruses.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
pmid = {41300710},
issn = {2073-4425},
support = {grant No. BR21882269//the Science Committee of the Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Artificial Intelligence ; *Plant Viruses/genetics ; *Gene Editing/methods ; *Plant Diseases/virology/genetics ; Crops, Agricultural/genetics/virology ; },
abstract = {Plant viral infections continue to pose a significant and ongoing threat to global food security, especially in the context of climatic instability and intensive agricultural practices. The CRISPR/Cas system has emerged as a powerful tool for developing virus-resistant crops by enabling precise modifications to viral genomes or plant susceptibility factors. Nonetheless, the efficacy and dependability of CRISPR-based antiviral approaches are limited by challenges in guide RNA design, off-target effects, insufficiently annotated datasets, and the intricate biological dynamics of plant-virus interactions. This paper summarizes the latest advancements in the incorporation of artificial intelligence (AI) methodologies, including machine learning and deep learning algorithms, into the CRISPR design and optimization framework. It examines how convolutional and recurrent neural networks, transformer architectures, and generative models like AlphaFold2, RoseTTAFold, and ESMFold can be used to predict protein structures, score sgRNAs, and model host-virus interactions. AI-enhanced methods have been proven to improve target specificity, Cas protein performance, and in silico validation. This paper aims to establish a foundation for next-generation genome editing strategies against plant viruses and promote the adoption of AI-powered CRISPR technologies in sustainable agriculture.},
}
@article {pmid41300715,
year = {2025},
author = {Sapakhova, Z and Kanat, R and Daurov, D and Daurova, A and Shamekova, M and Zhambakin, K},
title = {The Enhancement of Fungal Disease Resistance in Major Staple Crops Using CRISPR-Cas Technology.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
pmid = {41300715},
issn = {2073-4425},
support = {BR21882269//Ministry of Science and Higher Education of the Republic of Kazakhstan/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Disease Resistance/genetics ; *Plant Diseases/microbiology/genetics ; *Crops, Agricultural/genetics/microbiology ; Gene Editing/methods ; Fungi/pathogenicity/genetics ; Plants, Genetically Modified/genetics ; },
abstract = {Fungal pathogens represent a major constraint to global agricultural productivity, causing a wide range of plant diseases that severely affect staple crops such as cereals, legumes, and vegetables. These infections result in substantial yield losses, deterioration of grain and produce quality, and significant economic impacts across the entire agri-food sector. Among phytopathogens, fungi are considered the most destructive, causing a wide range of diseases such as powdery mildew, rusts, fusarium head blight, smut, leaf spot, rots, late blight, and other fungal pathogens. Traditional plant protection methods do not always provide long-term effectiveness and environmental safety, which requires the introduction of innovative approaches to creating sustainable varieties. CRISPR-Cas technology opens up new opportunities for targeted genome editing, allowing the modification or silencing of susceptibility genes and thus increasing plant resistance to fungal infections. This review presents current achievements and prospects for the application of CRISPR-Cas technology to increase the resistance of major agricultural crops to fungal diseases. The implementation of these approaches contributes to the creation of highly productive and resistant varieties, which is crucial for ensuring food security in the context of climate change.},
}
@article {pmid41300819,
year = {2025},
author = {Kansal, R},
title = {Curing Sickle Cell Disease by Allogeneic Hematopoietic Stem Cell (HSC) Transplantation Toward In Vivo HSC Gene Therapy.},
journal = {Genes},
volume = {16},
number = {11},
pages = {},
pmid = {41300819},
issn = {2073-4425},
mesh = {*Anemia, Sickle Cell/therapy/genetics ; Humans ; *Hematopoietic Stem Cell Transplantation/methods ; *Genetic Therapy/methods ; Gene Editing/methods ; CRISPR-Cas Systems ; Transplantation, Homologous ; Animals ; Hematopoietic Stem Cells/metabolism ; },
abstract = {Sickle cell disease comprises a group of prevalent inherited disorders defined by an underlying sickle cell allele that forms sickle hemoglobin. The incidence of this disease is rising, with more than 500,000 children born with it globally. The disease carries significant morbidity and mortality. Its only curative treatment was an allogeneic hematopoietic stem cell (HSC) transplant (HSCT) until late 2023, when two one-time gene therapies were approved for treating patients aged 12 years or older with severe sickle cell disease. This work aims to inform readers about these two gene therapies: one lentiviral-based and the other nonviral. The latter is based on the Nobel Prize-winning discovery of clustered, regularly interspaced, short, palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 proteins and single-guide RNA (sgRNA)-based genome editing. Both approved gene therapies require an autologous HSCT with ex vivo genetically edited autologous hematopoietic stem and progenitor cells. Therefore, access to these gene therapies is limited to specialized centers with expertise in HSCTs. This review is meant for students, researchers, and clinical practitioners. It explains the basis for both approved gene therapies, their mechanisms of action, differences, risks, and other lentiviral-based and CRISPR-Cas9-based ex vivo gene therapies for sickle cell disease in clinical development. Additionally, it discusses the current state of preclinical studies for in vivo HSC gene therapy for sickle cell disease, which utilize advanced genome editing technologies developed after CRISPR-Cas9-sgRNA-based genome editing. In vivo HSC gene therapy, after it is clinically developed, would eliminate the need for an HSCT in receiving gene therapy and vastly increase access for numerous patients worldwide, even in low-income countries with the most significant disease burden.},
}
@article {pmid41301473,
year = {2025},
author = {Gibril, BAA and Chai, X and Xu, J},
title = {From Correlation to Causation: Defining Gene and RNA Function in Poultry Muscle Biology Using In Vivo Genetic Tools.},
journal = {Biomolecules},
volume = {15},
number = {11},
pages = {},
pmid = {41301473},
issn = {2218-273X},
support = {20242BCE50051//Science and Technology Research Project of the Education Department of Jiangxi Province/ ; },
mesh = {Animals ; *Muscle, Skeletal/metabolism ; Muscle Development/genetics ; *Poultry/genetics ; Muscular Diseases/genetics ; Transcriptome ; CRISPR-Cas Systems ; *RNA/genetics/metabolism ; },
abstract = {A central challenge in functional genomics is understanding the difference between correlative transcriptomic observations and definitive causal understanding of gene function in vivo. Poultry skeletal muscle, a system of significant agricultural and biological importance, demonstrates this challenge. While transcriptomic studies have cataloged extensive RNA expression dynamics during muscle development and in growth-related myopathies like wooden breast, establishing causative roles for these molecules is lacking. This review synthesizes how advanced genetic tools are now enabling a shift from correlation to causation in avian muscle biology. We detail how viral vectors (e.g., adenovirus, lentivirus, and RCAS) and CRISPR/Cas9 systems have provided direct in vivo validation of the functional roles of specific mRNAs, miRNAs, lncRNAs, and circRNAs in regulating myogenesis, hypertrophy, and atrophy. We contrast this success in fundamental biology with the study of myopathies, which remains largely descriptive. Here, a wealth of transcriptomic data has identified dysregulated pathways, including ECM remodeling, metabolism, and inflammation, but functional validation for most candidates is absent. We argue that the critical next step is to apply this established functional genomics toolkit to disease models. By defining causal mechanisms, this research will not only address a major agricultural issue but also provide a model for using genetic tools to dissect complex traits in a post-genomic era.},
}
@article {pmid41301547,
year = {2025},
author = {Mohammed, A and Ibrahim, NA and Basher, NS},
title = {Protein Engineering and Drug Discovery: Importance, Methodologies, Challenges, and Prospects.},
journal = {Biomolecules},
volume = {15},
number = {11},
pages = {},
pmid = {41301547},
issn = {2218-273X},
support = {IMSIU-DDRSP2501//Imam Mohammad ibn Saud Islamic University/ ; },
mesh = {*Drug Discovery/methods ; *Protein Engineering/methods ; Humans ; Animals ; Recombinant Proteins/therapeutic use/chemistry/genetics ; },
abstract = {Protein engineering is a rapidly evolving field that plays a critical role in transforming drug discovery and development. This innovative field harnesses the unique structural and functional properties of engineered proteins, such as monoclonal antibodies, nanobodies, therapeutic enzymes, and cytokines, to address complex diseases more effectively than traditional small-molecule drugs. These biologics not only enhance therapeutic specificity but also minimize adverse effects, marking a significant advancement in patient care. However, the journey of protein engineering is not without challenges. Issues related to protein folding, stability, and potential immunogenicity pose significant complications. Additionally, navigating the complex regulatory landscape can delay the transition from laboratory to clinical application. Addressing these hurdles requires the integration of cutting-edge technologies, including phage and yeast display technology, CRISPR, and advanced computational modeling, which enhance the predictability and efficiency of protein design. In this review, we explore the multifaceted impact of protein engineering on modern medicine, highlighting its potential to transform treatment paradigms, methodologies, challenges, and the successful development and approval of recombinant protein-based therapies. By navigating the complexities and leveraging technological advancements, the field is poised to unlock new therapeutic possibilities, ultimately improving patient outcomes and transforming healthcare.},
}
@article {pmid41301629,
year = {2025},
author = {Wang, Y and Li, L and Liang, Y and Xu, K and Ye, Y and He, M},
title = {Phage Therapy for Acinetobacter baumannii Infections: A Review on Advances in Classification, Applications, and Translational Roadblocks.},
journal = {Antibiotics (Basel, Switzerland)},
volume = {14},
number = {11},
pages = {},
pmid = {41301629},
issn = {2079-6382},
support = {82302568//National Natural Science Foundation of China/ ; 2308085QH283//Anhui Provincial National Science Foundation/ ; 2022xkjT012//Basic and Clinical Collaboration Enhancement Program Foundation of Anhui Medical University/ ; },
abstract = {The global spread of carbapenem-resistant Acinetobacter baumannii (CRAB) poses a severe public health threat, driving growing interest in phage-based precision antibacterial strategies. This systematic review synthesizes recent advances in the field of A. baumannii phage. Modern taxonomy, based on whole-genome phylogeny, has reclassified the majority of A. baumannii phages into the class Caudoviricetes, revealing distinct evolutionary clades that correlate with host tropism and biological properties, superseding the traditional morphological families (Myoviridae, Siphoviridae, Podoviridae). To overcome limitations of natural phage therapy, such as narrow host range, cocktail therapies (ex vivo resistance mutation rates < 5%) and phage-antibiotic synergism (enabling antibiotic efficacy at 1/4 minimum inhibitory concentration) have significantly enhanced antibacterial efficacy. Preclinical models demonstrate that phage therapy efficiently clears pathogens in pneumonia models and promotes the healing of burn wounds and diabetic ulcers via immunomodulatory mechanisms. Technical optimizations include nebulized inhalation delivery achieving 42% alveolar deposition, and thermosensitive hydrogels enabling sustained release over 72 h. Genetic engineering approaches, such as host range expansion through tail fiber recombination and CRISPR/Cas-mediated elimination of lysogeny, show promise. However, the genetic stability of engineered phages requires further validation. Current challenges remain, including limited host spectrum, the absence of clinical translation standards, and lagging regulatory frameworks. Future efforts must integrate metagenomic mining and synthetic biology strategies to establish a precision medicine framework encompassing resistance monitoring and personalized phage formulation, offering innovative solutions against CRAB infections.},
}
@article {pmid41301838,
year = {2025},
author = {Dziedzic, A and Kubina, R and Skonieczna, M and Madej, M and Fiegler-Rudol, J and Abid, M and Nadhim, D and Tanasiewicz, M},
title = {CRISPR Genome Editing in Personalized Therapy for Oral and Maxillofacial Diseases: A Scoping Review.},
journal = {Biomedicines},
volume = {13},
number = {11},
pages = {},
pmid = {41301838},
issn = {2227-9059},
abstract = {Background: CRISPR/Cas genome editing is emerging as a powerful tool in oral and maxillofacial medicine, with potential applications in personalized therapies for conditions that currently lack durable treatments. Objectives: This scoping review aimed to map existing evidence on CRISPR-based applications in oral and maxillofacial fields, rather than to assess treatment effectiveness. Methods: A systematic search of PubMed, Scopus, Web of Science, and ClinicalTrials.gov (2012-2024) identified studies and registered trials involving CRISPR with oral health relevance. Eligible articles included peer-reviewed experimental reports and clinical trials. Results: From 1437 records, 121 studies met inclusion criteria: 106 preclinical reports and 15 clinical or translational studies. Investigated domains included oral cancer therapy, hereditary craniofacial syndromes, regenerative strategies, infectious disease models, and pathogen detection. Early clinical efforts focus mainly on CRISPR-edited T-cell immunotherapies in oncology. Major barriers include off-target effects, delivery challenges, regulatory complexity, and ethical concerns. Conclusions: CRISPR-based bioengineering shows strong promise for precision care in oral and maxillofacial medicine. However, current evidence remains largely preclinical and heterogeneous. No clinical recommendations can yet be made, and translation will depend on rigorous late-phase trials, ethical oversight, and health-economic evaluation.},
}
@article {pmid41302913,
year = {2025},
author = {Ayaz, S and Kong, WW and Wang, J and Liu, SH and Xu, JP},
title = {Host Immunity Mechanisms Against Bacterial and Viral Infections in Bombyx mori.},
journal = {Insects},
volume = {16},
number = {11},
pages = {},
pmid = {41302913},
issn = {2075-4450},
abstract = {The domesticated silkworm, Bombyx mori, is a highly valued biodiversity and economic asset, acclaimed for its silk production, besides making important contributions to various scientific disciplines. However, the sericulture industry faces ongoing threats from bacterial and viral infections, which severely impact silkworm health and silk yield. This review provides a comprehensive overview of the innate immune response of B. mori against bacterial and viral pathogens, emphasizing the fundamental molecular and cellular defense mechanisms. We explore the humoral and cellular immune response using antimicrobial peptides (AMPs), pattern recognition receptors (PRRs) like peptidoglycan recognition protein (PGRP), and glucan recognition protein (GRP), which activate canonical signaling pathways. The review further highlights the molecular mechanisms underlying the silkworm's defense against viruses, incorporating RNA interference (RNAi), apoptosis, and distinct signaling pathways such as Toll and Imd, JAK/STAT, and STING. We also discussed the viral suppression strategies and modulation of host metabolism during infection. Furthermore, the review explores the recent use of CRISPR-Cas gene editing to enhance disease resistance, presenting a promising avenue for mitigating pathogen-induced losses in sericulture. By elucidating these mechanisms, the work provides a synthesis that is critical in terms of developing particular interventions and developing more resistant silkworm strains to ensure that the industry of sericulture becomes viable and productive.},
}
@article {pmid41303344,
year = {2025},
author = {Zhang, X and Che, J and Li, Z and Bao, B and Fan, C},
title = {The Mutation of piezo1 Weakens the Intermuscular Bones in Zebrafish and Crucian Carp.},
journal = {International journal of molecular sciences},
volume = {26},
number = {22},
pages = {},
pmid = {41303344},
issn = {1422-0067},
support = {2023YFD2400300//National Key Research and Development Program of China/ ; 32170514//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Carps/genetics ; *Ion Channels/genetics/metabolism ; *Mutation ; *Zebrafish Proteins/genetics/metabolism ; *Bone and Bones/metabolism ; CRISPR-Cas Systems ; *Fish Proteins/genetics/metabolism ; },
abstract = {Intermuscular bones (IBs), unique skeletal features found only in teleost fishes, pose significant challenges to food processing and consumption. While recent studies have identified several key genetic regulators of IB development, the role of mechanosensory mechanisms remains largely unexplored. This study investigated the role of Piezo1, a critical mechanosensitive ion channel, in IB formation using zebrafish and crucian carp models. Our findings demonstrated that piezo1 was expressed in the myoseptum of zebrafish, and CRISPR/Cas9-mediated knockout of this gene resulted in shorter and smaller IBs. Similar knockout experiments in crucian carp confirmed the conserved role of Piezo1 across cyprinid species. These results established Piezo1 as a key regulator of IB development, providing new insights into the molecular mechanisms underlying this process and suggesting potential strategies for breeding IB-free fish strains through modulation of mechanosensory pathways.},
}
@article {pmid41304743,
year = {2025},
author = {Zhang, H and Li, Y and Li, J and Li, X and Li, T},
title = {Base and Prime Editing for Inherited Retinal Diseases: Delivery Platforms, Safety, Efficacy, and Translational Perspectives.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
pmid = {41304743},
issn = {1999-4923},
support = {U22A20311//he National Natural Science Foundation of China/ ; 82571246//the National Natural Science Foundation of China/ ; 82388101//the National Natural Science Foundation of China/ ; 23J41900200//Science and Technology Commission of Shanghai Municipality/ ; SHWSRS(2025)_071//Shanghai "Rising Stars of Medical Talents" Youth Development Program/ ; },
abstract = {Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous spectrum of disorders that lead to progressive and irreversible vision loss. Gene therapy is the most promising emerging treatment for IRDs. While gene augmentation strategies have demonstrated clinical benefit and results within the first approved ocular gene therapy, their application is restricted by adeno-associated virus (AAV) packaging capacity and limited efficacy for dominant mutations. Recent breakthroughs in precision genome editing, particularly base editing (BE) and prime editing (PE), have provided alternatives capable of directly correcting pathogenic variants. BE enables targeted single-nucleotide conversions, whereas PE further allows for precise insertions and deletions, both circumventing the double-strand DNA cleavage or repair processes typically induced by conventional CRISPR-Cas editing systems, thereby offering advantages in post-mitotic retinal cells. Preclinical investigations across murine and non-human primate models have demonstrated the feasibility, molecular accuracy, and preliminary safety profiles of these platforms in targeting IRD-associated mutations. However, critical challenges remain before clinical application can be realized, including limited editing efficiency in photoreceptors, interspecies variability in therapeutic response, potential risks of off-target effects, and barriers in large-scale vector manufacturing. Moreover, the delivery of genome editors to the outer retina remains suboptimal, prompting intensive efforts in capsid engineering and the development of non-viral delivery systems. This review synthesizes the current progress in BE and PE optimization, highlights innovations in delivery platforms that encompass viral and emerging non-viral systems and summarizes the major barriers to clinical translation. We further discuss AI-driven strategies for the rational design of BE/PE systems, thereby outlining their future potential and perspectives in the treatment of IRDs.},
}
@article {pmid41304764,
year = {2025},
author = {Alidriss, OM and AlSudais, H and Alhumaidan, OS and Altwaijry, HD and Bakhsh, A and Almuhanna, Y and Alkudmani, ZS and Alqarni, IA and Alenazi, D and Aljasham, AT and Jamous, YF},
title = {Targeted Drug Delivery Strategies in Overcoming Antimicrobial Resistance: Advances and Future Directions.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
pmid = {41304764},
issn = {1999-4923},
abstract = {Antimicrobial resistance (AMR) is a present, pressing global public health crisis associated with rising morbidity and mortality rates due to previously curable infectious disease. Targeted drug delivery is an important approach to address AMR due to its ability to improve the therapeutic performance of antibiotics without leading to any adverse effects or organ toxicities. In this review we explore molecular mechanisms of AMR and drawbacks of conventional antibiotic therapies and discuss unique drug delivery approaches to compensate these. Nanoparticulate carrier systems, stimuli-responsive systems, antibody-drug conjugates, and CRISPR-Cas systems are some of the carrier method designs that are promising for tackling hard to treat infections related to pathogenic strains and biofilms due to their features. Many of these are among the most significant advances in the field. However, there are many challenges to be overcome, with biological limitations, scaling and regulatory challenges, etc., before they can be employed in commercial applications. Materials are being developed, and an approach standardized and applicable to future work is in development to improve the efficiency of targeted delivery systems. Controlled drug delivery, which could be the answer to an increasing AMR problem, will not only help in alerting awareness among individuals but will also help in prolonging the activity of antibiotics by providing synergistic interdisciplinary solutions. This review emphasizes the complementary role of targeted drug delivery in transitioning from laboratory investigations to clinical therapy. It addresses underrepresented aspects, including new materials, scalability, regulatory considerations, and ethical implications, while offering a roadmap for translating innovations into next-generation antimicrobials.},
}
@article {pmid41304797,
year = {2025},
author = {Sharma, A and Sharma, V and Sharma, S and Sharma, S and Sharma, M and Sivanesan, I},
title = {Advanced Nanosystems and Emerging Therapies: Innovations in Tuberculosis Treatment and Drug Resistance.},
journal = {Pharmaceutics},
volume = {17},
number = {11},
pages = {},
pmid = {41304797},
issn = {1999-4923},
abstract = {Tuberculosis (TB) remains a significant worldwide health challenge due to the limitations of conventional treatments and the rising incidence of drug-resistant Mycobacterium tuberculosis strains. This review consolidates the advancements in nanotechnology-based therapeutics, inhalable formulations, CRISPR-Cas tools, host-directed therapies (HDTs), and nanoparticle-based vaccine development aimed at enhancing TB management. Novel nanocarriers such as liposomes, solid-lipid nanoparticles (SLNs), dendrimers, and polymeric nanoparticles (NPs) offer enhanced bioavailability of drugs, sustained release, as well as targeted delivery to infected macrophages, thereby reducing systemic toxicity and dosing frequency. Inhalable nanomedicines provide localized delivery to the pulmonary site, enhancing the concentration of the drug at the primary site of infection. CRISPR-Cas technology is emerging as a transformative approach to disabling drug-resistant genes and enhancing diagnostic precision. HDTs, including agents like vitamin D and metformin, show potential in modulating host immune responses and enhancing pathogen clearance. Nanoparticle-based vaccines, including mRNA and antigen-conjugated platforms, aim to overcome the limitations of the BCG vaccine by enhancing antigen presentation and eliciting stronger, longer-lasting immunity. Collectively, these modalities mark a shift toward more personalized, effective, and less toxic TB therapies. However, challenges such as regulatory approval, safety, scalability, and accessibility remain. This review highlights the integrated potential of nanomedicine, gene editing, and immunomodulation to transform TB care and combat drug resistance, paving the way for more robust and durable treatment strategies.},
}
@article {pmid41304933,
year = {2025},
author = {Yue, Y and Xu, Z and Soteyome, T and Premarathna, M and Yin, X and Liu, J},
title = {Phage Encapsulation and Delivery Technology: A Strategy for Treating Drug-Resistant Pathogenic Microorganisms.},
journal = {Pharmaceuticals (Basel, Switzerland)},
volume = {18},
number = {11},
pages = {},
pmid = {41304933},
issn = {1424-8247},
abstract = {Antimicrobial resistance (AMR) is one of the most critical challenges to global public health in the 21st century, posing a significant threat to healthcare systems and human health due to treatment failure and high mortality. The World Health Organization (WHO) estimates that, without effective interventions, AMR-associated infections could cause 10 million deaths annually and economic losses of up to 100 trillion US dollars by 2050. The rapid spread of drug-resistant strains, especially in hospital and community settings, has significantly reduced the efficacy of traditional antibiotics. With the continuous advancements in relevant research, bacteriophage (Phage) therapy is constantly innovating in the antimicrobial field. The application of frontier technologies, such as phage cocktails and engineered phages, has significantly enhanced the broad spectrum and high efficiency of phage therapy, which is gradually becoming a new generation of tools to replace antibiotics and effectively combat pathogenic bacteria. However, phage therapy is facing several challenges, including phage inactivation by gastric acid, enzymes, ultraviolet light, and mechanical stress, as well as the potential risk of bacterial phage resistance. Advanced encapsulation technologies such as electrospun fibers, liposomes, chitosan nanoparticles, and electrospray provide solutions to these problems by protecting phage activity and enabling controlled release and targeted delivery. This review addresses phage therapeutic studies of Salmonella, Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes, summarizes the recent advances in phage research, and details the current development and applications of encapsulated phage technologies across various delivery modes.},
}
@article {pmid41305485,
year = {2025},
author = {Iftehimul, M and Hasan, NA and Bass, D and Bashar, A and Haque, MM and Santi, M},
title = {Combating White Spot Syndrome Virus (WSSV) in Global Shrimp Farming: Unraveling Its Biology, Pathology, and Control Strategies.},
journal = {Viruses},
volume = {17},
number = {11},
pages = {},
pmid = {41305485},
issn = {1999-4915},
support = {2022/21/Other//Ocean Country Partnership Programme (OCPP), Blue Planet Fund/ ; },
mesh = {*White spot syndrome virus 1/pathogenicity/immunology/physiology/genetics ; Animals ; *Aquaculture/methods ; *Penaeidae/virology/immunology ; Immunity, Innate ; Viral Vaccines/immunology ; },
abstract = {White Spot Syndrome Virus (WSSV) is one of the most devastating viral pathogens affecting shrimp, causing severe economic losses to the global farmed shrimp trade. The globalization of live shrimp trade and waterborne transmission have facilitated the rapid spread of WSSV across major shrimp-producing countries since its initial emergence. The present review gives an updated account of WSSV biology, pathology, transmission dynamics, and recent developments in control measures. The virus, a double-stranded DNA virus of the Nimaviridae family, utilizes advanced immune evasion strategies, resulting in severe mortality. Shrimp lack adaptive immunity and hence rely predominantly on innate immunity, which is insufficient to mount an effective response against severe infections. Traditional disease control measures such as augmented biosecurity, selective breeding, and immunostimulants have, despite extensive research, achieved only limited success. New biotechnological tools such as RNA interference, CRISPR-Cas gene editing, and nanotechnology offer tremendous potential for disease mitigation. In parallel, the development of DNA and RNA vaccines targeting WSSV structural proteins, such as VP28, holds significant promise for stimulating the shrimp immune system. This review highlights the urgent need for a convergent approach to sustainable disease management in global shrimp aquaculture, with interdisciplinarity playing a pivotal role in shaping the future of WSSV control.},
}
@article {pmid41305525,
year = {2025},
author = {Verma, N and O'Mahony, A and Mohammad, R and Keiser, D and Mosman, CW and Holden, D and Starr, K and Bauer, J and Bauer, B and Suntisukwattana, R and Atthaapa, W and Tantituvanont, A and Nilubol, D and Gladue, DP},
title = {The First CRISPR-Based Therapeutic (SL_1.52) for African Swine Fever Is Effective in Swine.},
journal = {Viruses},
volume = {17},
number = {11},
pages = {},
pmid = {41305525},
issn = {1999-4915},
mesh = {Animals ; *African Swine Fever/therapy/virology ; Swine ; *African Swine Fever Virus/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Genome, Viral ; },
abstract = {African swine fever virus (ASFV) is a high-consequence pathogen that causes African swine fever (ASF), for which mortality rates can reach 90-100%, with death typically occurring within 14 days. ASF is currently a highly contagious pandemic disease responsible for extensive losses in pig production in multiple affected countries suffering from extended outbreaks. While a limited number of vaccines to prevent ASF are in use in south-east Asia, vaccines are not widely available, are only effective against highly homologous strains of ASFV, and must be used prior to an outbreak on a farm. Currently, there is no treatment for ASF and culling affected farms is the only response to outbreaks on farms to try and prevent spreading. CRISPR/Cas systems evolved as an adaptive immune response in bacteria and archaea that function by cleaving and disrupting the genomes of invading bacteriophage pathogens. CRISPR technology has since been leveraged into an array of endonuclease-based systems used for nucleic acid detection, targeting, genomic cleavage, and gene editing, making them particularly well-suited for development as sequence-specific therapeutic modalities. The programmability of CRISPR-based therapeutics offers a compelling new way to rapidly and specifically target pathogenic viral genomes simply by using different targeting guide RNAs (gRNA) as an adaptable antiviral modality. Here, we demonstrate for the first time a specific CRISPR/Cas9 multiplexed gRNA system that targets the African swine fever viral genome, resulting in sequence-specific cleavage, leading to the reduction in the viral load in infected animals, and subsequent recovery from an otherwise lethal dose of ASFV. Moreover, animals that recovered had protective immunity to subsequent homologous ASFV infection.},
}
@article {pmid41305866,
year = {2026},
author = {Naderi, S and Williamson, J and Sun, H and Joshi, S and Spera, RJ and Zaib, S and Sharma, S and Sun, C and Brodovskiy, A and Zawar, I and Kapur, J},
title = {Hydroxycarboxylic Acid Receptor 2 Mediates β-hydroxybutyrate's Antiseizure Effect in Mice.},
journal = {Annals of neurology},
volume = {99},
number = {3},
pages = {809-824},
pmid = {41305866},
issn = {1531-8249},
support = {K23 AG084893/AG/NIA NIH HHS/United States ; R01 NS120945/NS/NINDS NIH HHS/United States ; R37 NS119012/NS/NINDS NIH HHS/United States ; R37N119012//United States National Institute of Health (NINDS)/ ; R01NS120945//United States National Institute of Health (NINDS)/ ; },
mesh = {Animals ; Mice ; *3-Hydroxybutyric Acid/pharmacology/therapeutic use ; *Seizures/drug therapy/metabolism ; *Receptors, G-Protein-Coupled/metabolism/genetics ; Mice, Knockout ; Male ; Hippocampus/drug effects/metabolism ; Mice, Inbred C57BL ; *Anticonvulsants/pharmacology/therapeutic use ; Diet, Ketogenic ; },
abstract = {OBJECTIVE: The ketogenic diet, a high-fat, low-carbohydrate regimen, is often used to treat drug-resistant seizures and is being studied for Alzheimer's disease and other neuropsychiatric disorders. However, its mechanism of action remains unclear. β-hydroxybutyrate, a primary circulating ketone body produced by the ketogenic diet, may mediate its effects on seizures by binding to a recently identified Gi-coupled receptor: hydrocarboxylic acid receptor 2 (HCAR2).<br><br>METHODS: RNAscope in situ hybridization assay and real-time quantitative polymerase chain reaction were used to assess HCAR2 expression in the mouse brain. We generated HCAR2[-]/[-] using the CRISPR-Cas technique on an S129 mouse background. Whole-cell current-clamp was performed to measure the passive and active membrane properties of hippocampal dentate granule cells. The voltage-clamp was performed to record synaptic currents. Two complementary in vivo mouse models-continuous hippocampal stimulation to induce status epilepticus (SE) and kindling-were used to induce seizures.<br><br>RESULTS: HCAR2 was localized in dentate granule cells and microglia. In mice with HCAR2, &#x3b2;-hydroxybutyrate reduced neuronal excitability by hyperpolarizing the resting membrane potential, raising the action potential threshold, and reducing the firing frequency of dentate granule cells. &#x3b2;-hydroxybutyrate suppressed excitatory synaptic transmission. These effects were nullified in HCAR2[-]/[-] mice. HCAR2[-]/[-] mice showed no cognitive impairment. Moreover, &#x3b2;-hydroxybutyrate did not affect seizures in HCAR2[-]/[-] mice. However, it diminished both the duration and severity of seizures in HCAR2[+]/[+] mice.<br><br>INTERPRETATION: These findings demonstrate that HCAR2 mediates &#x3b2;-hydroxybutyrate's antiseizure effects by regulating neuronal excitability and synaptic transmission. These studies propose a new mechanism for the antiseizure action of the ketogenic diet. ANN NEUROL 2026;99:809-824.},
}
@article {pmid41306592,
year = {2025},
author = {Shi, L and Chen, H and Zhang, Z and Wang, Y and Ren, W and Huang, J},
title = {Evolving HPV diagnostics: current practice and future frontiers.},
journal = {Frontiers in cellular and infection microbiology},
volume = {15},
number = {},
pages = {1681779},
pmid = {41306592},
issn = {2235-2988},
mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; Female ; *Molecular Diagnostic Techniques/methods/trends ; *Papillomaviridae/genetics/isolation &amp; purification ; Uterine Cervical Neoplasms/virology/diagnosis ; Early Detection of Cancer/methods ; High-Throughput Nucleotide Sequencing ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Polymerase Chain Reaction/methods ; Mass Screening/methods ; },
abstract = {Human papillomavirus (HPV) infection serves as a primary causative agent of cervical cancer, highlighting the importance of early screening and detection in mitigating the incidence and mortality rates of HPV-related diseases. Over the past decades, HPV detection technologies have evolved considerably, transitioning from traditional methods to more advanced, patient-centered approaches. This review provides a comprehensive overview of both established and emerging HPV detection strategies, with a particular focus on their clinical applicability, technical advantages, and limitations. Conventional methods such as hybrid capture and PCR-based assays remain the backbone of clinical screening, offering robust sensitivity and specificity. However, their reliance on invasive sampling and centralized laboratory infrastructure limits accessibility and patient compliance, particularly in low-resource settings. To address these limitations, emerging technologies-including CRISPR/Cas systems, droplet digital PCR (ddPCR), next-generation sequencing (NGS), isothermal amplification techniques (IAT) and artificial intelligence (AI) combined with hpv screening offer enhanced accuracy, rapid turnaround, and the potential for point-of-care deployment. In parallel, innovations in sampling such as self-collected vaginal swabs and liquid biopsy using urine, blood, or extracellular vesicles are improving test acceptability and broadening screening coverage. By summarizing current progress and highlighting ongoing challenges, this review aims to guide the development of more precise, non-invasive, and scalable HPV detection strategies to reduce the global burden of HPV-related disease, support global prevention efforts, and guide public health policies.},
}
@article {pmid41307501,
year = {2025},
author = {Sahu, S and Boukherroub, R and Ritzenthaler, C and Szunerits, S},
title = {Emerging technologies for in-field plant virus detection: innovations and future directions.},
journal = {The Journal of general virology},
volume = {106},
number = {11},
pages = {},
pmid = {41307501},
issn = {1465-2099},
mesh = {*Plant Viruses/isolation &amp; purification/genetics ; *Plant Diseases/virology ; Biosensing Techniques/methods ; High-Throughput Nucleotide Sequencing ; CRISPR-Cas Systems ; Nanotechnology/methods ; },
abstract = {Plant virus infections pose a substantial threat to crop quality and productivity, contributing to considerable economic losses in global agriculture annually. Traditionally, laboratories have widely adopted serological techniques, such as ELISA, and molecular methods, including quantitative PCR, for virus diagnostics. More recently, sophisticated next-generation sequencing approaches have been introduced to improve the efficiency and reliability of virus detection and identification. However, the development of sensitive, rapid and low-cost methods for the on-site detection, quantification and identification of plant viruses remains an ongoing challenge and is still in its early days. Point-of-care technologies have not fully realized their potential in agriculture due to numerous challenges, such as the elevated cost of development, lack of standardized validation and insufficient field testing. Therefore, future success depends on addressing these technical, economic and regulatory hurdles, as well as considering the specific user needs within the agricultural context. In this mini-review, recent advancements in biosensing for on-site plant virus monitoring, involving nanotechnology-based sensors, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, electrochemical and modern field-effect transistor-based sensors offering high sensitivity, speed and portability, are discussed. These technologies, when integrated with smartphone applications and/or machine learning modules, could enable real-time, field-deployable diagnostics for early disease management and sustainable agriculture. The aim is to raise awareness among plant virologists about this panel of emerging diagnostic concepts that could help improve current methods, ultimately facilitating the management of plant viral diseases.},
}
@article {pmid41307588,
year = {2025},
author = {Kumar, V and Verma, P},
title = {Advances in microbial biotechnology for sustainable wastewater reclamation: recent trends and future prospects.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {12},
pages = {478},
pmid = {41307588},
issn = {1573-0972},
mesh = {*Wastewater/microbiology/chemistry ; *Biotechnology/methods/trends ; *Water Purification/methods ; Biodegradation, Environmental ; Bacteria/metabolism/genetics ; Microalgae/metabolism ; Bioelectric Energy Sources ; Waste Disposal, Fluid/methods ; Microbial Consortia ; },
abstract = {The growing demand for freshwater, coupled with the increasing volume of industrial and municipal wastewater, has intensified the need for sustainable and eco-friendly reclamation strategies. Recent advancements in microbial biotechnology have emerged as promising tools for developing cost-effective, efficient, and environmentally sustainable wastewater treatment (WWT) strategies for reuse and safe disposal. This mini-review explores current innovations, such as microbial consortia, bioaugmentation, and the microalgae-bacteria nexus, which have shown promising results in nutrient removal, enhanced degradation of complex pollutants (including emerging contaminants), and biomass valorization. Moreover, bioelectrochemical systems, such as microbial fuel cells (MFC) and microbial electrolysis cells (MEC), have revolutionized WWT by facilitating pollutant degradation while simultaneously generating bioelectricity or biohydrogen. This article also critically examines the role of CRISPR-based tools and 'omics' approaches, which have enabled the development of novel microbial strains and degradative pathways, enhancing wastewater reclamation in challenging environments. Furthermore, advancements through the integration of multi-omics and artificial intelligence, digital twins, and Internet of Things (IoT) for microbial optimization and real-time process control are discussed. The review highlights the role of microbial systems in resource recovery, supporting a circular economy by transforming wastewater into valuable bioresources. Additionally, this review addresses the major challenges and proposes future research directions for effective wastewater treatment. The novelty of this manuscript is that no single review explores the cutting-edge microbial biotechnologies for wastewater reclamation, uniquely integrating CRISPR-Cas genome editing, multi-omics analyses, and artificial intelligence-driven optimization to advance pollutant degradation and real-time process control in one place. This study concludes that by implementing multi-omics and artificial intelligence (AI)-driven optimization process for wastewater treatment can be effective towards wastewater treatment while simultaneously minimizing the environmental pollution.},
}
@article {pmid41308487,
year = {2026},
author = {Rodríguez-Estévez, D and Gil-Durán, C and Silva, R and Palma, D and Vaca, I and Chávez, R},
title = {CRISPR/Cas9-mediated development of Penicillium roqueforti strains deficient in roquefortine C and mycophenolic acid enables toxin-free blue cheese production.},
journal = {International journal of food microbiology},
volume = {446},
number = {},
pages = {111535},
doi = {10.1016/j.ijfoodmicro.2025.111535},
pmid = {41308487},
issn = {1879-3460},
mesh = {*Penicillium/genetics/metabolism ; *Cheese/microbiology/analysis ; *Mycophenolic Acid/metabolism ; *CRISPR-Cas Systems ; *Mycotoxins/biosynthesis ; Food Microbiology ; *Indoles/metabolism ; Heterocyclic Compounds, 4 or More Rings ; Piperazines ; },
abstract = {Penicillium roqueforti, a key fungus in the manufacture of blue-veined cheeses, can produce mycotoxins such as roquefortine C and mycophenolic acid. The production of these metabolites is highly strain- and condition-dependent. In industrial manufacture, hypotoxigenic P. roqueforti strains are typically used as controlled adjunct starters under standardized conditions, resulting in minimal mycotoxin accumulation, whereas naturally matured or artisan cheeses display more variable strain composition and ripening environments, which can elevate risk. In this context, the development of strains incapable of mycotoxin biosynthesis represents an important step toward safer cheese products. Here, we report the generation of P. roqueforti strains lacking the ability to synthesize roquefortine C and mycophenolic acid using CRISPR/Cas9. Single and double mutants deficient in one or both mycotoxins were obtained. Laboratory-scale cheeses produced under artisan-like conditions with these engineered strains contained no detectable levels of the target mycotoxins, in contrast to cheeses made with the wild-type strain. All mutants retained the ability to colonize cheese but displayed altered fungal biomass production compared to the native strain. These differences were consistent in curd and laboratory media and were not associated with changes in lipolytic or proteolytic activities. Further analyses revealed that while the absence of mycophenolic acid did not affect NaCl sensitivity, the lack of roquefortine C increased sensitivity to salt. Collectively, these results demonstrate the feasibility of producing mycotoxin-free blue cheeses using strains deficient in roquefortine C and mycophenolic acid biosynthesis, thereby laying the foundation for developing mycotoxin-free cheeses with engineered atoxigenic P. roqueforti strains.},
}
@article {pmid41308567,
year = {2025},
author = {Downton, P and Bates, N and Woods, S and Adamson, A and Sergouniotis, PI},
title = {Genome editing of a low-penetrance albinism-associated variant in TYR in patient-derived pluripotent stem cells.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103855},
doi = {10.1016/j.scr.2025.103855},
pmid = {41308567},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; *Monophenol Monooxygenase/genetics/metabolism ; *Gene Editing/methods ; *Albinism/genetics/pathology ; CRISPR-Cas Systems ; Cell Differentiation ; Male ; },
abstract = {TYR encodes tyrosinase, the enzyme catalysing the initial steps of melanin biosynthesis in melanocytes and retinal pigment epithelia (RPE). TYR c.1205G>A (p.Arg402Gln) is a common genetic variant associated with several pigmentation traits. Notably, when this variant is encountered in specific haplotypic backgrounds in the homozygous state, it predisposes to albinism. We generated an induced pluripotent stem cell (iPSC) line from an affected individual carrying such a homozygous genotype (UMANi255-A), and then used CRISPR-Cas9 to correct the TYR c.1205G>A variant (UMANi255-A-1). The resulting iPSC lines demonstrate capacity for multi-lineage differentiation, providing a useful in vitro model for studying pigmentation biology.},
}
@article {pmid41309229,
year = {2025},
author = {Song, BS and Baek, YH and Kim, EH and Kwon, HI and Kim, AH and Lee, SH and Son, YB and Kim, SH and Song, MS and Choi, YK and Park, SJ},
title = {Development of an RT-LAMP-CRISPR/Cas12a assay for rapid and specific detection of Bandavirus dabieense.},
journal = {Journal of microbiology (Seoul, Korea)},
volume = {63},
number = {11},
pages = {e2506013},
doi = {10.71150/jm.2506013},
pmid = {41309229},
issn = {1976-3794},
support = {2022R1C1C1004704//National Research Foundation of Korea/ ; RS-2023-00301974//Ministry of Education/ ; //Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry/ ; RS-2022-IP322088//Ministry of Agriculture, Food and Rural Affairs/ ; },
mesh = {*Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Humans ; *CRISPR-Cas Systems ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; RNA, Viral/genetics ; *RNA Viruses/genetics/isolation &amp; purification ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Bandavirus dabieense, a single-stranded RNA virus, is the causative agent of severe fever with thrombocytopenia syndrome (SFTS), a disease associated with high fatality rates. Early and accurate diagnosis is essential for improving clinical outcomes, particularly given the limited therapeutic options and high mortality rates associated with SFTS. However, while highly sensitive, conventional diagnostic methods such as PCR and qRT-PCR require specialized laboratory facilities and trained personnel, making them impractical for rapid detection in resource-limited settings. To address these challenges, we developed a rapid and highly sensitive assay for Bandavirus dabieense detection by integrating reverse transcription loop-mediated isothermal amplification (RT-LAMP) with CRISPR/Cas12a technology. LAMP primers and guide RNA sequences were designed to target the L gene, ensuring broad detection across viral genotypes. The optimized assay demonstrated a detection limit of 5 RNA copies per reaction, showing more sensitivity than qRT-PCR, and exhibited 100% concordance with qRT-PCR results in clinical samples. Given its speed, accuracy, and field applicability, this LAMP-CRISPR/Cas12a-based assay represents a promising diagnostic tool for early SFTSV detection, particularly in resource-constrained environments where conventional molecular diagnostics are not readily available.},
}
@article {pmid41309382,
year = {2025},
author = {Dao, TO and Park, HE and Lee, JH and Kim, KM and Trinh, MP and Kang, HL and Yoo, HS and Shin, MK},
title = {Advances and Challenges in Mycobacterial Genetic Engineering: Techniques for Knockout, Knockdown and Overexpression.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2507051},
pmid = {41309382},
issn = {1738-8872},
mesh = {*Genetic Engineering/methods ; *Gene Knockout Techniques/methods ; *Mycobacterium/genetics ; CRISPR-Cas Systems ; *Gene Knockdown Techniques/methods ; DNA Transposable Elements ; Homologous Recombination ; Gene Transfer Techniques ; },
abstract = {Genetic engineering of mycobacteria is challenging due to their hydrophobic cell wall structure and slow growth rates. Despite these obstacles, significant progress has been made to develop genetic engineering tools to study gene function and pathogenesis in these organisms. This review comprehensively explores the current methodologies employed in the genetic modification of mycobacteria, focusing on gene knockout, knockdown, and overexpression systems. Techniques covered include homologous recombination, recombineering, transposon mutagenesis, CRISPR-Cas systems, conditional expression strategies, and phage-mediated gene delivery. The mechanism, advantages, and limitations of those methods are critically analyzed, with particular emphasis on the adaptability of these tools to various mycobacterial species. By providing a detailed comparative analysis of available genetic tools, this review is a practical guide for researchers aiming to develop targeted and efficient genetic modifications in Mycobacterium species, accelerating discoveries in pathogenesis, drug resistance, and vaccine development.},
}
@article {pmid41309578,
year = {2025},
author = {Arana, S and Du, PP and Vaughan-Jackson, A and Enright, N and Spees, K and Valbuena, R and Garcia, CA and Nguyen, T and Venida, A and Seczynska, M and Bintu, L and Lehner, PJ and Prolo, LM and Bassik, MC},
title = {Reduced Cas9 transgene silencing by incorporation of intron sequences.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10656},
pmid = {41309578},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; R01 HG011866/HG/NHGRI NIH HHS/United States ; U54 CA261719/CA/NCI NIH HHS/United States ; R01HG011866//U.S. Department of Health &amp; Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; },
mesh = {*Introns/genetics ; *Transgenes/genetics ; *Gene Silencing ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *CRISPR-Associated Protein 9/genetics/metabolism ; HEK293 Cells ; Chromatin/metabolism/genetics ; Animals ; Cell Line ; },
abstract = {Silencing remains a significant challenge for exogenous gene expression, limiting both the penetrance and expressivity of transgenes. In particular, silencing of Cas9 expression is a major technical limitation for many gene editing and CRISPR screening applications. Here, we demonstrate that including introns in Cas9 expression cassettes significantly reduces silencing across multiple cell lines. Notably, the incorporation of an intron into a CRISPRa construct results in reduced silencing, increased expression levels, and markedly enhanced activation of target genes. We investigate diverse intron sequences and discover that T-rich introns over 2 kb confer the greatest protection against silencing. In addition, we find that introns can work synergistically with chromatin opening elements to further mitigate silencing, suggesting regulatory mechanisms are acting at both the DNA and RNA level to silence exogenous genes. Our work highlights the potential of introns to optimize genetic constructs for enhanced expression and improved cellular engineering requiring constitutive expression of large transgenes.},
}
@article {pmid41309688,
year = {2025},
author = {Zhang, F and Peng, Y and Fan, D and Song, G and Gao, X and Tian, Y},
title = {Engineering a CRISPR-associated IscB system for developing miniature genome-editing tools in human cells and mouse embryos.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10693},
pmid = {41309688},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Embryo, Mammalian/metabolism ; HEK293 Cells ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR-Associated Proteins/genetics/metabolism ; },
abstract = {IscB, as the putative ancestor of Cas9, possesses a compact size, making it suitable for in vivo delivery. OgeuIscB is the first IscB protein known to function in eukaryotic cells but requires a complex TAM (NWRRNA). Here, we characterize a CRISPR-associated IscB system, named DelIscB, which recognizes a flexible TAM (NAC). Through systematically engineering its protein and sgRNA, we obtain enDelIscB with an average 48.9-fold increase in activity. By fusing enDelIscB with T5 exonuclease (T5E), we find that enDelIscB-T5E displays robust efficiency comparable to that of enIscB-T5E in human cells. Moreover, by fusing cytosine or adenosine deaminase with enDelIscB nickase, we establish efficient miniature base editors (ICBE and IABE). Finally, we efficiently generate mouse models by microinjecting mRNA/sgRNA of enDelIscB and enDelIscB-T5E into mouse embryos. Collectively, our work presents a set of enDelIscB-based miniature genome-editing tools with great potential for diverse applications in vivo.},
}
@article {pmid41309901,
year = {2025},
author = {Moroi, K and Yamamoto, T and Kurita, T},
title = {Double-strand break-free and transgene-free genome editing in the microalga Nannochloropsis oceanica using removable vectors containing the CRISPR base editing system.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42431},
pmid = {41309901},
issn = {2045-2322},
mesh = {*Gene Editing/methods ; *Microalgae/genetics ; *CRISPR-Cas Systems ; *Genetic Vectors/genetics ; Plasmids/genetics ; DNA Breaks, Double-Stranded ; Transgenes ; *Stramenopiles/genetics ; },
abstract = {The accumulation of lipids by algae makes them attractive for carbon-neutral fuel production; however, the industrial-scale production of algal lipids has yet to be achieved. Currently, researchers are trying to improve the lipid productivity of algal strains using genome editing for molecular breeding with CRISPR-Cas9, which allows the efficient alteration of genomic information. However, CRISPR-based gene modification via double-strand breaks sometimes induces unintended large deletions that are toxic to host cells. Here, we applied the cytidine base editor combined with an episomal vector backbone containing a centromere and autonomous replication sequence to the microalga Nannochloropsis oceanica. The cytosine base editor introduces cytidine-to-thymidine base substitutions using deaminase without double-strand breaks, and an episomal vector enables plasmid removal after base substitution. We succeeded in inducing cytidine-to-thymidine substitution at the six target sites of five endogenous genes. The base substitution activity ranged from 29.2% to 47.6% on cytidine bases at the 16th to 19th positions from the protospacer adjacent motifs. The removal of base editor plasmids was also detected, which is essential for constructing transgene-free strains. Our results provide insights into the applicability of further technologies in the genetic modification of microalgae.},
}
@article {pmid41310261,
year = {2025},
author = {Spaans, GW and van der Berg, JP and Bouwman, LMS and Kleter, GA},
title = {Advancements in genomic crop techniques and considerations for regulation and food safety.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {49},
pmid = {41310261},
issn = {1573-9368},
mesh = {*Crops, Agricultural/genetics/growth &amp; development ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Food Safety ; Gene Editing ; Plant Breeding ; *Genomics/methods ; Food, Genetically Modified ; Mutagenesis ; Humans ; Genome, Plant ; },
abstract = {Advancements in genomic crop techniques have led to the development of new genetic technologies, such as base- and prime editing, but improvements have been made to existing conventional techniques as well. Fields in which these advancements occur include targeted mutagenesis, conventional random mutagenesis, and developments with null segregants, e.g., crops from which transgenic elements have been crossed out. In this review, we describe the developments in these three fields and provide considerations concerning regulatory and safety aspects. Because of differences in legislation of modern biotechnology between countries or regions, regulatory challenges are to be expected given the ongoing developments in genomic crop techniques. Moreover, the nature of the mutations induced with these newly developed techniques is not different from those induced with conventional techniques, making the modified crop plants indistinguishable from non-modified counterparts of the same crop species. Thus, enforcement of regulations cannot solely rely on technical analytical methods. Also, potential off-target or unintended effects in the primary mutants remain underexplored. Yet, these do not raise safety concerns owing to the experience with the crop breeding practice of iterative cycles for desirable traits selection, as well as the segregation and discard of unwanted phenotypes. Given that regulation will always change after innovation and developments within the sector advance rapidly, we advocate that both authorities and the breeding sector pro-actively implement a food safety culture. Such a safety culture will help developers of genomic technologies in crops to identify potential food safety issues at an early stage of development of future products.},
}
@article {pmid41310509,
year = {2025},
author = {Ekrami, A and Taheri, B and Daneshfar, S and Moradi, M and Ghorbani, A and Akhash, N and Jafarzadeh, Z and Farshadzadeh, Z and Saki, M},
title = {Occurrence of CRISPR-Cas genes and lack of association with antibiotic resistance in Shigella isolates collected from patients with diarrhea in Ahvaz, southwest Iran.},
journal = {BMC infectious diseases},
volume = {25},
number = {1},
pages = {1666},
pmid = {41310509},
issn = {1471-2334},
abstract = {BACKGROUND: So far, few studies have examined the association between CRISPR-Cas and antibiotic resistance in Shigella isolates. Hence, this study sought to address this issue in Shigella species isolated from stool samples of patients with diarrhea in Ahvaz, southwest Iran.<br><br>METHODS: In this cross-sectional study, stool samples were collected from 103 children (3&#x2013;14 years) with diarrhea admitted to Abuzar Hospital affiliated to the Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. All samples were cultured on the xylose lysine desoxycholate agar and hektoen enteric agar and incubated at 37&#xa0;&#xb0;C for 24&#xa0;h. Primary identification of the Shigella species was performed by biochemical tests and polymerase chain reaction (PCR). Antibiotic resistance rates were evaluated by the Kirby-Bauer disc diffusion. The prevalence of 12 CRISPR-Cas genes was investigated by PCR.<br><br>RESULTS: Overall, 83 Shigella isolates were identified by standard biochemical tests. Finally, 72 Shigella isolates including 45 S. flexneri (46.8%) and 27 (53.2%) S. sonnei were confirmed by PCR. The most effective antibiotics were trimethoprim/sulfamethoxazole (n&#x2009;=&#x2009;48, 66.7%), imipenem (n&#x2009;=&#x2009;46, 63.9%), and ceftazidime (n&#x2009;=&#x2009;43, 59.7%), respectively. All isolates harbored at least one of the CRISPR-Cas genes. Occurrence of CRISPR-Cas genes was as follows: CRa (100.0%), CRb (100.0%), CRc (100.0%), CSe2 (100.0%), CRf (95.8%), CSe1_Cas3 (87.5%), Cas2_Cas (84.7%), Cas (81.9%), CRd (80.6%), Cas7 (76.4%), Cas6e_Cas5 (72.2%), and CRe (48.6%). There was no significant association between the occurrence of CRISPR-Cas elements and resistance to any antibiotic in Shigella isolates (P-value&#x2009;&#x2265;&#x2009;0.9999), except for Cas gene with ceftriaxone and cefepime.<br><br>CONCLUSION: This study revealed high resistance rates of various antibiotics in Shigella isolates. However, there was no significant association between the existences of CRISPR-Cas genes with antibiotic resistance. Further investigation with higher sample size is needed to confirm this observation.<br><br>SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12879-025-12100-0.},
}
@article {pmid41313207,
year = {2026},
author = {Wang, Y and Qin, Z and Wang, Q and Yang, Y and Gu, C and Yu, F and Wu, Y and Zhang, Lx},
title = {An RPA-CRISPR/Cas12a-based rapid and sensitive nucleic acid method for detection of Toxoplasma gondii in tissue and blood samples.},
journal = {Microbiology spectrum},
volume = {14},
number = {1},
pages = {e0155025},
pmid = {41313207},
issn = {2165-0497},
support = {231111111500//Key Research and Development Special Project of Henan Province of China/ ; 2022YFD1800200, 2023YFD1801200//National Basic Research Program of China/ ; },
mesh = {*Toxoplasma/genetics/isolation &amp; purification ; Animals ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; DNA, Protozoan/genetics ; *Toxoplasmosis/diagnosis/parasitology/blood ; Limit of Detection ; *Toxoplasmosis, Animal/diagnosis/parasitology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Toxoplasma gondii is a zoonotic pathogen that can infect humans and a wide range of warm-blooded animals, posing a significant threat to human health and the livestock industry. The development of a time-saving, highly sensitive, and specific method for the detection of T. gondii in tissue and blood samples is crucial to the monitoring, prevention, and control of toxoplasmosis. In this study, we evaluated the efficiency of a previously described method, termed REPORT, that integrates recombinase polymerase amplification with CRISPR/Cas12a for the detection of T. gondii nucleic acids. We evaluated the limit of detection (LOD) and specificity of the extended REPORT method using prepared target DNA in addition to tissue and blood samples. Furthermore, we validated the accuracy of T. gondii detection in clinical samples using the REPORT-based method in comparison with nested PCR based on the B1 gene. Sensitivity tests showed that the LOD of the REPORT-based fluorescence method and the lateral flow strip method were 3.7 copies /μL for target DNA, 3.1 tachyzoites/g for tissue samples, and five tachyzoites/mL for blood samples. Specificity tests suggested that the REPORT method had good specificity and did not cross-react with several common parasites. The method performed well for clinical DNA samples, demonstrating its ability for use in on-site detection.IMPORTANCEToxoplasma gondii can infect over 200 species of warm-blooded animals, including humans, posing not only a significant threat to public health systems but also causing substantial economic losses to the global livestock industry. Current diagnostic methods are slow, equipment-dependent, and impractical for field use. This study addresses these limitations by developing REPORT, a rapid, ultrasensitive nucleic acid test combining recombinase polymerase amplification and CRISPR/Cas12a. The REPORT detects T. gondii in tissue and blood samples within 1 h at low cost, requiring only a portable heater. Its visual results (fluorescence or test strips) enable on-site use without specialized training, achieving 100% accuracy versus nested PCR. With a sensitivity of 3.1 parasites per gram of tissue and five parasites per milliliter of blood, this method revolutionizes toxoplasmosis screening in resource-limited clinics, farms, and food safety inspections, empowering timely interventions to curb transmission and improve public health outcomes.},
}
@article {pmid41313840,
year = {2026},
author = {Mohammad, SI and Kareem, AK and Vasudevan, A and Rekha, MM and Jabir, MS and Nayak, P and AlKhafaje, Z and Arora, V and Kadhum, W and Chennakesavulu, K},
title = {Genome editing of immune checkpoints: CRISPR-mediated PD-1 inhibition in cancer.},
journal = {Seminars in oncology},
volume = {53},
number = {1},
pages = {152438},
doi = {10.1016/j.seminoncol.2025.152438},
pmid = {41313840},
issn = {1532-8708},
mesh = {Humans ; *Gene Editing/methods ; *Neoplasms/genetics/therapy/immunology ; *Programmed Cell Death 1 Receptor/antagonists &amp; inhibitors/genetics ; Animals ; *CRISPR-Cas Systems ; *Immune Checkpoint Inhibitors/therapeutic use/pharmacology ; B7-H1 Antigen/antagonists &amp; inhibitors/genetics ; Immunotherapy, Adoptive/methods ; },
abstract = {The programmed cell death protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) immune checkpoint is a primary mechanism by which tumors evade immune surveillance, limiting the efficacy of cytotoxic T lymphocytes (CTLs) and tumor-infiltrating lymphocytes (TILs). Although immune checkpoint blockade therapies have revolutionized cancer treatment, their efficacy is restricted by acquired resistance, T-cell exhaustion, and tumor heterogeneity. The advent of CRISPR-Cas9 genome editing provides a precise and versatile approach to disrupt PD-1 or PD-L1, directly enhancing anti-tumor immune responses. Preclinical studies demonstrate that ex vivo PD-1 knockout in primary human T cells or TILs enhances proliferation, cytokine production, and cytotoxicity, resulting in improved tumor clearance in xenograft and humanized mouse models. In chimeric antigen receptor (CAR) T cell therapy, CRISPR-mediated disruption of PD-1 improves effector function, persistence, and resistance to exhaustion, with universal and allogeneic CAR-T platforms benefiting from multiplex genome editing. Direct PD-L1 knockout in tumor cells, often facilitated via nanoparticle- or biomaterial-assisted delivery, reshapes the immunosuppressive tumor microenvironment, promotes T cell infiltration, and enhances the efficacy of adoptive cellular therapy. Combination approaches integrating PD-1 editing with viral antigen targeting, long noncoding RNA (lncRNA) modulation, or conventional checkpoint blockade demonstrate synergistic anti-tumor effects. Clinically, early-phase trials in non-small cell lung cancer, mesothelin-positive solid tumors, and hematological malignancies establish the feasibility, safety, and preliminary efficacy of PD-1-deficient T cells. Despite these promising outcomes, challenges such as off-target effects, delivery efficiency, immunogenicity, long-term persistence, and regulatory considerations remain. This review aims to comprehensively evaluate preclinical and clinical studies investigating CRISPR-mediated PD-1/PD-L1 inhibition across various cancers, summarize mechanistic insights, and highlight translational opportunities and challenges for clinical implementation.},
}
@article {pmid41314751,
year = {2025},
author = {Fatima, M and Tariq, I and Tariq, A and Talib, S and Fatima, M and Shehzadi, M and Aqib, AI},
title = {Pharmacogenomics and CRISPR-based therapies.},
journal = {Progress in brain research},
volume = {297},
number = {},
pages = {319-343},
doi = {10.1016/bs.pbr.2025.08.009},
pmid = {41314751},
issn = {1875-7855},
mesh = {Humans ; *Pharmacogenetics/methods ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Precision Medicine/methods ; *Genetic Therapy/methods ; Animals ; },
abstract = {Pharmacogenomics and CRISPR-based treatments are two areas of precision medicine that are advancing together. Pharmacogenomics involves studying how differences in someone's genes can change the effect of medications on them. Pharmacogenomics helps reduce adverse reactions to drugs and improve healing by choosing and measuring drugs according to a patient's genetic information. Additionally, CRISPR-Cas systems now serve as leading genome editing tools that allow precise alterations at given points of the genome. CRISPR technology's use in pharmacogenomics creates new opportunities for modifying gene expression, fixing harmful mutations, and creating innovative treatment approaches. A more proactive approach to illness treatment is supported by this synergy, in which genetic factors serve as both direct targets for intervention and a basis for medication selection. This chapter examines the theoretical and practical frameworks that link CRISPR-based treatments with pharmacogenomics, emphasizing recent uses in pharmacoresistance, cancer, and monogenic diseases. To guarantee safe and fair deployment, it also covers the ethical, legal, and technical issues that need to be resolved. When combined, these technologies hold the potential to revolutionize medicine by facilitating individualized and curative drugs.},
}
@article {pmid41314912,
year = {2026},
author = {Chen, L and Ouyang, W and Hu, Y and Peng, L and Chen, P and Guo, W and Yang, H and Xu, J and Pan, M and Xu, D and Wang, X and Zhang, C and Chen, S and Hao, Q and Yuan, S and Huang, Y and Shan, Z and Yang, Z and Xia, R and Hewezi, T and Chen, H and Tran, LP and Zhou, X and Cao, D},
title = {Creating artificial miR2118a/b to boost yield and broad-spectrum resistance in soybean via CRISPR/Cas9-targeted mutation.},
journal = {Trends in biotechnology},
volume = {44},
number = {4},
pages = {1149-1166},
doi = {10.1016/j.tibtech.2025.10.022},
pmid = {41314912},
issn = {1879-3096},
mesh = {*Glycine max/genetics/growth &amp; development/microbiology ; *CRISPR-Cas Systems/genetics ; *MicroRNAs/genetics ; Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Disease Resistance/genetics ; Mutation ; Plant Diseases/genetics/microbiology ; Pseudomonas syringae ; Gene Expression Regulation, Plant ; },
abstract = {While regulatory functions of mature miRNAs are well established, the functions of miRNAs* and their potential for genetic engineering in crop improvement remain underexplored. Here, we used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) to generate artificial miR2118a/b (amiR2118a/b) by editing miR2118a/b-5p and obtained several amir2118a/b mutants in soybean (Glycine max). miR2118a/b-5p modifications altered the secondary structure of precursor amiR2118a/b (pre-amiR2118a/b) and reduced mature miR2118a/b levels. These amir2118a/b mutants retained the ability to initiate biogenesis of phased small interfering RNAs (phasiRNAs), albeit with a reduced abundance compared with wild-type (WT) plants. Furthermore, these mutants upregulated the expression of genes related to growth and defense under normal and Pseudomonas syringae pv. glycinea (Psg)-infected conditions, respectively. Notably, two transgene-free amir2118 mutants exhibited enhanced resistance to Psg, soybean cyst nematode (SCN), and root-knot nematode (RKN), and achieved increased yield under pathogen-free field conditions. This study provides a strategy to generate artificial miRNAs (amiRNAs) for crop improvement through the CRISPR/Cas system by mutating miRNAs* in crops.},
}
@article {pmid41315077,
year = {2025},
author = {Loedige, KW and White, AL and McMurrough, TA and Stead, BE and Edgell, DR},
title = {A buffer-tuning strategy to profile domain-specific activity of chimeric I-TevI/CRISPR gene editors in vitro.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {42742},
pmid = {41315077},
issn = {2045-2322},
support = {RGPIN-2022-05459//Natural Sciences and Engineering Research Council of Canada/ ; RGPIN-2022-05459//Natural Sciences and Engineering Research Council of Canada/ ; ALLRP 571374 - 21//Mitacs/ ; ALLRP 571374 - 21//Mitacs/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Buffers ; RNA, Guide, CRISPR-Cas Systems/genetics ; Protein Domains ; DNA Cleavage ; *Endodeoxyribonucleases/metabolism/genetics ; Humans ; DNA/metabolism ; },
abstract = {Protein-DNA interactions can be manipulated in vitro by changing buffer conditions. Here, we develop a methodology to map the cleavage preferences of chimeric gene editors that are fusions of the I-TevI nuclease domain to CRIPSR nucleases by manipulating in vitro salt concentrations. We found that DNA cleavage by the I-TevI (Tev) nuclease domain at CNNNG sites was de-coupled from the gRNA-targeted site in low salt buffers. For TevCas12a, this non-targeted cleavage activity was enriched at Tev CNNNG cleavage motifs optimally positioned within a 30-bp window upstream of a Cas12a TTTV PAM site. Non-targeted cleavage did not require Cas12a nuclease activity or specific Cas12a gRNA targeting. Similar non-targeted products were observed in low salt buffer conditions for TevSaCas9, Tev-meganuclease and Tev-zinc finger editors. Cas12a and SaCas9 activity at gRNA-directed sites and sites with multiple mismatches were also sensitive to buffer salt concentration. Oxford Nanopore sequencing revealed a remarkably similar Tev CNNNG cleavage preference at different salt concentrations and in different fusion contexts, emphasizing the robustness and specificity of Tev activity. More generally, our work highlights the sensitivity of gene editors to in vitro reaction conditions and how these conditions can be leveraged to functionally dissect the activity of individual domains of chimeric gene editors.},
}
@article {pmid41315225,
year = {2025},
author = {Wen, HP and Yu, C and Bi, S and Jiang, LH and Wang, ZG and Yao, Z and Pang, DW and Liu, SL},
title = {Programmable targeted RNA degradation via dCas13d-directed chaperone-mediated autophagy (dCasCMA).},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10738},
pmid = {41315225},
issn = {2041-1723},
support = {22293032//National Natural Science Foundation of China (National Science Foundation of China)/ ; 22374138//National Natural Science Foundation of China (National Science Foundation of China)/ ; 21977054//National Natural Science Foundation of China (National Science Foundation of China)/ ; 24JCZDJC01240//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; 23JCYBJC01880//Natural Science Foundation of Tianjin City (Natural Science Foundation of Tianjin)/ ; },
mesh = {Humans ; Animals ; *Chaperone-Mediated Autophagy/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA Stability/genetics ; *CRISPR-Cas Systems/genetics ; Mice ; HEK293 Cells ; Autophagy ; },
abstract = {RNA-targeted degradation technologies offer significant promise for treating diseases by selectively disrupting gene expression. However, a robust method to specifically, efficiently, and programmability degrade targeted RNAs in mammalian cells is still in demand. Here, we present a versatile platform, dCas13d-directed chaperone-mediated autophagy (dCasCMA), which integrates the precise targeting capabilities of dCas13/CRISPR with the degradation efficiency of chaperone-mediated autophagy (CMA) to achieve efficient degradation of specific RNAs. By combining dCas13d with a CMA-targeting motif and customizable guide RNA (gRNA), the platform allows for accurate targeting of both exogenous and endogenous RNAs in cells. Moreover, the incorporation of multiplexed gRNA expression arrays enables the simultaneous degradation of multiple RNA targets during viral pathogenesis in live cells and in vivo. Our findings emphasize the platform's modular design, which enables flexible combinations of dCCTM components with user-defined gRNA sequences. This versatility positions it as a promising tool for developing innovative therapies for various diseases.},
}
@article {pmid41315365,
year = {2025},
author = {Wang, Y and Liao, Y and Sun, Y and Mitra, B and Guo, R and Piedras, BI and White, S and Tang, HY and Asara, JM and Tempera, I and Lieberman, PM and Gewurz, BE},
title = {The CTLH ubiquitin ligase substrates ZMYND19 and MKLN1 negatively regulate mTORC1 at the lysosomal membrane.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10731},
pmid = {41315365},
issn = {2041-1723},
support = {R01 AI164709/AI/NIAID NIH HHS/United States ; PF-24-1250090-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; P01CA269043//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; P01 CA269043/CA/NCI NIH HHS/United States ; R01AI164709//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; PF-23-1144614-01-IBCD//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1194768-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; PF-24-1308318-01-TBE//American Cancer Society (American Cancer Society, Inc.)/ ; R01 DE033907/DE/NIDCR NIH HHS/United States ; R00 DE031016/DE/NIDCR NIH HHS/United States ; },
mesh = {Humans ; *Lysosomes/metabolism ; *Mechanistic Target of Rapamycin Complex 1/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; HEK293 Cells ; Cell Line, Tumor ; CRISPR-Cas Systems ; *Intracellular Membranes/metabolism ; Signal Transduction ; Phosphatidylinositol 3-Kinases/metabolism ; Cell Proliferation ; },
abstract = {Most Epstein-Barr virus-associated gastric carcinoma (EBVaGC) harbor non-silent mutations that activate phosphoinositide 3 kinase (PI3K) to drive downstream metabolic signaling. To gain insights into PI3K/mTOR pathway dysregulation in this context, we perform a human genome-wide CRISPR/Cas9 screen for hits that synergistically blocked EBVaGC proliferation together with the PI3K antagonist alpelisib. Multiple subunits of carboxy terminal to LisH (CTLH) E3 ligase, including the catalytic MAEA subunit, are among top screen hits. CTLH negatively regulates gluconeogenesis in yeast, but not in higher organisms. The CTLH substrates MKLN1 and ZMYND19, which highly accumulated upon MAEA knockout, associate with one another and with lysosome outer membranes to inhibit mTORC1. Rather than perturbing mTORC1 lysosomal recruitment, ZMYND19 and MKLN1 block the interaction between mTORC1 and Rheb and also with mTORC1 substrates S6 and 4E-BP1. Thus, CTLH enables cells to rapidly tune mTORC1 activity at the lysosomal membrane via the ubiquitin/proteasome pathway.},
}
@article {pmid41316351,
year = {2025},
author = {Qin, Z and Wang, Y and Sun, M and Wang, Q and Duan, J and Gu, C and Zhang, X and Yu, F and Wu, Y and Xu, H and Li, J and Zhang, L},
title = {Development of a field-deployable RPA-CRISPR/Cas12a assay for the detection of Cyclospora cayetanensis in human feces.},
journal = {Parasites &amp; vectors},
volume = {19},
number = {1},
pages = {4},
pmid = {41316351},
issn = {1756-3305},
support = {232102110088//Henan Province Scientific and Technological Project/ ; 2023YFD1801200//National Key Research and Development Program of China/ ; 231111111500//Key Research and Development Project of Henan Province/ ; },
mesh = {*Cyclospora/isolation &amp; purification/genetics ; Humans ; *Feces/parasitology ; *Cyclosporiasis/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; Biosensing Techniques/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Cyclospora is an emerging intestinal pathogenic protozoan transmitted through foodborne and waterborne routes. At least 19 countries in the world have recorded outbreaks of cyclosporiasis, mainly associated with the consumption of contaminated fresh agricultural products. The lack of a sensitive immediate test is one of the major obstacles to the rapid diagnosis of cyclosporiasis. The target interference mechanisms of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been adapted into versatile and efficient genome manipulation and disease-curing technologies, while also being promising for point-of-care testing (POCT) applications. It can serve as an excellent rapid and specific detection tool.<br><br>METHODS: The recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system were combined to develop a detection method for C. cayetanensis (termed RECCT-Cay) via visual observation of fluorescent readings under blue light and field diagnosis using lateral flow strip (LFS) biosensors.<br><br>RESULTS: The detection limit of the established RECCT-Cay was 7 copies/&#x3bc;L. Under simulated clinical conditions, the detection limit was 30 oocysts per gram of stool. At the same time, the established detection platform can distinguish C. cayetanensis from the closely related Eimeria spp. The results of our constructed assay were compared with nested PCR, and the detection results of 30 clinical stool samples were consistent, with three samples positive for C. cayetanensis. Based on the RECCT-Cay detection principle, a portable suitcase-sized device has been designed, which can conduct rapid on-site detection of clinical samples.<br><br>CONCLUSIONS: The RECCT-Cay platform features rapid speed, high sensitivity, and the capability for field detection, making it a promising tool for use in remote areas.},
}
@article {pmid41316685,
year = {2026},
author = {Megarani, DV and Yang, L and Siler, HJ and Quijano Cardé, EM and Martyniuk, CJ and Hick, PM and Becker, JA and Soto, E and Surachetpong, W and Yanong, RPE and Subramaniam, K},
title = {One-Pot RT-LAMP CRISPR/Cas12b Platform for Rapid Detection of Tilapia Lake Virus.},
journal = {Journal of fish diseases},
volume = {49},
number = {5},
pages = {e70087},
doi = {10.1111/jfd.70087},
pmid = {41316685},
issn = {1365-2761},
support = {2023-67015-39481//National Institute of Food and Agriculture/ ; },
mesh = {Animals ; *Fish Diseases/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Sensitivity and Specificity ; *RNA Virus Infections/veterinary/diagnosis/virology ; *Molecular Diagnostic Techniques/veterinary/methods ; Aquaculture ; *RNA Viruses/isolation &amp; purification ; *Tilapia ; },
abstract = {Tilapia Lake Virus (TiLV) is a significant threat to global tilapia aquaculture, highlighting the need for rapid and accurate diagnostic methods to manage outbreaks and minimise economic losses. This study presents the development and partial validation of a one-pot assay integrating RT-LAMP with the CRISPR/Cas12b system for sensitive and specific TiLV detection. This assay amplifies viral RNA using RT-LAMP, while CRISPR/Cas12b enables a real-time detectable signal. Targeting a conserved region in TiLV segment four, the assay achieves results within 75 min at 62°C, with easy visualisation using a portable fluorescence viewer. It demonstrated high sensitivity, with a 95% limit of detection of 79.6 copies (95% CI: 48-132 copies), and high specificity, with no cross-reaction to other fish RNA or DNA viruses. Based on a validation panel of 261 samples from 9 source populations, the assay exhibited 92% diagnostic sensitivity (95% CI: 87%-96%) and 100% diagnostic specificity (95% CI: 97%-100%). When assessed as a non-lethal sample, gills provided a reliable and less invasive alternative despite lower viral loads compared to internal organs. Therefore, this partially validated one-pot assay is potentially practical for enhancing TiLV detection and disease management in aquaculture systems, especially in field settings and resource-limited laboratories.},
}
@article {pmid41317788,
year = {2026},
author = {Brogan, DJ and Lin, CP and Benetta, ED and Wang, T and Chen, F and Li, H and Lin, C and Komives, EA and Akbari, OS},
title = {Synthetic Type III-E CRISPR-Cas Effectors for Programmable RNA-targeting.},
journal = {Journal of molecular biology},
volume = {438},
number = {2},
pages = {169566},
doi = {10.1016/j.jmb.2025.169566},
pmid = {41317788},
issn = {1089-8638},
mesh = {*CRISPR-Cas Systems/genetics ; *RNA/genetics/metabolism ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Protein Domains ; *Gene Editing/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The recent discovery of the type III-E class of CRISPR-Cas effectors has reshaped our fundamental understanding of CRISPR-Cas evolution and classification. Type III-E effectors are composed of several Cas7-like domains and a single Cas11-like domain naturally fused together to create a single polypeptide capable of targeting and degrading RNA. Here we identified a novel type III-E-like effector composed of three Cas7 domains and a Cas1 domain which was not active but could be engineered into an active chimeric RNA-targeting Cas effector by domain additions and swaps from other type III-E effectors. The results reveal that various domains in type III-E effectors can be swapped for the equivalent domain from a different type III-E effector. Remarkably, the Cas1 domain located at the C-terminus of Cas7-1 could be swapped in place of the Cas11 domain located between the Cas7.1 and the Cas7.2 domains of DiCas7-11. The results reveal a new modality for engineering type III-E effectors from the blueprints found in nature.},
}
@article {pmid41317988,
year = {2026},
author = {Arya, SK and Goodman, CL and Palli, SR},
title = {The expanding toolkit of insect cell culture: a new era in biotechnology.},
journal = {Current opinion in insect science},
volume = {74},
number = {},
pages = {101465},
doi = {10.1016/j.cois.2025.101465},
pmid = {41317988},
issn = {2214-5753},
support = {R01 GM070559/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; *Insecta/genetics/cytology ; *Biotechnology/methods ; *Cell Culture Techniques/methods ; Cell Line ; Baculoviridae/genetics ; CRISPR-Cas Systems ; },
abstract = {Insect cell culture has become an essential platform in modern biotechnology, valued for its safety, scalability, and ability to perform complex post-translational modifications. This review highlights the latest and most important advances in the field. We focus on efforts at developing and engineering new insect cell lines, innovations in expression systems, especially the baculovirus expression vector system and the transformative impact of CRISPR/Cas9-based genome editing. Additionally, we explore breakthroughs that improve the efficiency of recombinant protein production and discuss key challenges such as viral contamination and expression instability. Collectively, these developments mark an important step forward in insect cell biotechnology and are expected to enhance the efficiency and scalability of producing vaccines and biopharmaceuticals. Together, these innovations illustrate a transition from cataloging cell line development to understanding the mechanisms and engineering principles driving these advances. This review not only summarizes recent progress but also provides perspective on how foundational lepidopteran models have guided innovations now extending into dipteran, hemipteran, and hymenopteran systems, shaping the future of insect biotechnology.},
}
@article {pmid41318543,
year = {2025},
author = {Pernaci, C and Johnson, A and Gillette, S and Warden, AS and McCormick, C and Weiser-Novak, S and Ramirez, G and Broersma, EH and Mishra, P and Sivakumar, A and Cherqui, S and Coufal, NG},
title = {Microgliopathy as a primary mediator of neuronal death in models of Friedreich's Ataxia.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {81},
pmid = {41318543},
issn = {2041-1723},
support = {R01NS135162//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; TRAN1-13983//California Institute for Regenerative Medicine (CIRM)/ ; EDUC2-08388//California Institute for Regenerative Medicine (CIRM)/ ; R01 AG086443/AG/NIA NIH HHS/United States ; S10 OD025060/OD/NIH HHS/United States ; R01 NS135162/NS/NINDS NIH HHS/United States ; },
mesh = {*Friedreich Ataxia/pathology/genetics/metabolism ; Animals ; *Microglia/pathology/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mice ; Frataxin ; Disease Models, Animal ; Iron-Binding Proteins/genetics/metabolism ; Cell Death ; Mitochondria/metabolism/pathology ; *Neurons/pathology/metabolism ; Purkinje Cells/pathology/metabolism ; Trinucleotide Repeat Expansion ; Iron/metabolism ; Male ; Female ; White Matter/pathology ; CRISPR-Cas Systems ; },
abstract = {Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by a GAA repeat expansion in the frataxin (FXN) gene, leading to a severe reduction of the mitochondrial FXN protein, crucial for iron metabolism. While microglial inflammation is observed in FRDA, it remains unclear whether immune dysfunction is a primary disease mediator or a secondary reactionary phenotype. Utilizing patient-derived induced pluripotent stem cells (iPSCs), we report an intrinsic microglial phenotype of stark mitochondrial defects, iron overload, lipid peroxidation, and lysosomal abnormalities. These factors drive a pro-inflammatory state that contributes to neuronal death in co-culture systems. In a murine xenograft model, transplanted human FRDA microglia accumulate in white matter and the Purkinje cell layer, resulting in Purkinje neuron loss in otherwise healthy brains. Notably, CRISPR/Cas9-mediated correction of the GAA repeat reverses microglial defects and mitigates neurodegeneration. Here, we suggest that microglial dysfunction serve as a disease driver and a promising therapeutic target in FRDA.},
}
@article {pmid41319833,
year = {2026},
author = {Hu, H and Ke, X and Xiao, H and Shang, X and Yin, Q and Li, J and Li, X and Hu, Z and Qian, P and Wang, M},
title = {Genomic assembly, rescue, and characterization of a functional pseudorabies virus.},
journal = {Virologica Sinica},
volume = {41},
number = {1},
pages = {97-106},
pmid = {41319833},
issn = {1995-820X},
mesh = {Animals ; *Herpesvirus 1, Suid/genetics ; *Genome, Viral ; Chlorocebus aethiops ; Vero Cells ; Swine ; Mice ; Pseudorabies/virology ; Gene Editing ; Mice, Inbred BALB C ; CRISPR-Cas Systems ; Swine Diseases/virology ; },
abstract = {With recent advances in synthetic biology methods, the genomes of several large DNA viruses have been de novo synthesized and assembled, leading to the functional rescue of the respective viruses. Pseudorabies virus (PRV), a large DNA virus belonging to the family Herpesviridae, causes severe diseases in swine, resulting in significant economic losses to the global pig farming industry. Genome editing is crucial for attenuating virulence and developing safer vaccines for PRV. However, its complex repetitive sequences and extremely high GC-rich genome pose significant challenges for genetic manipulation. In this study, we developed a PRV genome assembly platform using yeast-based transformation-associated recombination (TAR) technology. The genome of a prevalent genotype II variant strain, PRV-GX-2011 (GenBank number PV405324.1), was divided into nine A-level fragments and cloned into vectors via TAR. Subsequently, three B-level fragments were generated by recombining three A-level fragments each. In vitro CRISPR/Cas9-mediated editing was introduced to insert an egfp gene into the non-coding intergenic region between UL23 and UL22 genes. Infectious viruses were rescued by co-transfection of linearized B-level fragments in Vero cells, and an isolated virus, PRV-GX-Syn1, was purified via plaque assay. While PRV-GX-Syn1 exhibited reduced viral titer and smaller plaque size compared to the parental strain, its morphological characteristics remained indistinguishable from the parental virus. In BALB/c mice, PRV-GX-Syn1 caused lethal infection, producing lung pathology comparable to the parental strain. This TAR-based platform offers faster and more flexible genomic modification of PRV, facilitating both basic research and PRV-based vaccine vectors.},
}
@article {pmid41319963,
year = {2026},
author = {Kong, H and Wang, S and Zhuo, C and Zhong, Q and Xu, Y and Lao, YH and Lv, S and Xie, X and Yuan, Q and Li, K and Tao, Y and Li, M},
title = {Nanovesicles integrating PD-1-mediated targeting and CRISPR/Cas9-based CD47 editing for dual immune checkpoint blockade.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114480},
doi = {10.1016/j.jconrel.2025.114480},
pmid = {41319963},
issn = {1873-4995},
mesh = {*CD47 Antigen/genetics ; CRISPR-Cas Systems ; Animals ; Gene Editing ; *Immune Checkpoint Inhibitors/administration &amp; dosage ; *Programmed Cell Death 1 Receptor/genetics/immunology ; Humans ; B7-H1 Antigen/immunology ; Mice ; Cell Line, Tumor ; *Neoplasms/therapy/immunology/genetics ; Mice, Inbred C57BL ; Female ; Immunotherapy/methods ; },
abstract = {Immunotherapy with immune checkpoint inhibitors has revolutionized cancer treatment, yet many tumors evade immune surveillance through multiple suppressive mechanisms. In particular, the adaptive immune checkpoint programmed death 1 (PD-1)/programmed death-ligand 1 (PD-L1) and the innate "don't eat me" signal CD47/signal-regulatory protein alpha (SIRPα) represent two distinct pathways that cancers exploit to avoid T-cell attack and macrophage phagocytosis, respectively. Herein, we present BITE (Biomimetic Immune Targeting and Editing), a genetically engineered biomimetic nanoplatform designed to concurrently blockade both pathways by combining PD-1-mediated tumor targeting with CRISPR/Cas9 gene editing of CD47. BITE nanovesicles display PD-1 on their surface, enabling selective binding to PD-L1-expressing tumor cells and local disruption of PD-1/PD-L1 signaling. Simultaneously, they deliver a CRISPR/Cas9 payload that knocks out the CD47 gene in tumor cells, abolishing the anti-phagocytic signal and thus activating innate immune clearance. We demonstrate that BITE efficiently homes to PD-L1-positive tumors in vitro and in vivo, achieves significant CD47 gene disruption in tumor cells, and triggers robust phagocytosis by macrophages. In a mouse tumor model, dual checkpoint blockade by BITE reshapes the tumor microenvironment, yielding increased infiltration of CD4[+] T cells, CD8[+] T cells, and M1 macrophages; treatment with BITE induces pronounced tumor regression and extended survival, outperforming single-target controls. Our results establish a proof-of-concept for this dual-function nanovesicle approach, highlighting its potential to engage both adaptive and innate immunity synergistically. The BITE platform offers a versatile and targeted strategy to overcome immune resistance in cancer, representing a promising therapeutic avenue in biomedical engineering and nanomedicine.},
}
@article {pmid41321825,
year = {2025},
author = {Senthilraja, G and Sandhya, M and Priyadharshini, E and Anand, T and Kavitha, M and Tharmalingam, N},
title = {Targeting effector proteins of plant pathogens as a strategy for durable plant disease resistance.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1681047},
pmid = {41321825},
issn = {1664-302X},
}
@article {pmid41323291,
year = {2025},
author = {Li, Y and Hall-Ponselè, AM},
title = {Plant Cell Strain Improvement Through Engineering Biology for Industrial Plant Cell Culture.},
journal = {Engineering biology},
volume = {9},
number = {1},
pages = {e70002},
pmid = {41323291},
issn = {2398-6182},
abstract = {Plant cell culture (PCC) presents a promising and sustainable alternative to traditional agricultural methods for producing specialty bioactive compounds. However, its widespread industrial application has been hindered by challenges such as low yields, cell line instability and inconsistent product quality. engineering biology (EB) offers a powerful toolkit to overcome these limitations by systematically improving plant cell lines. This review focuses on the application of EB principles to enhance PCC for the production of high-value bioactives from an industry-oriented perspective. We explore three core pillars of the EB toolkit: (1) Multiomics and in silico design, which leverage comprehensive data integration and predictive modelling for rational target identification; (2) gene manipulation and pathway bioengineering, encompassing precise genome editing (e.g., CRISPR/Cas), synthetic gene circuits and directed evolution for targeted metabolic reprogramming and (3) biosensors for high-throughput screening and real-time monitoring, enabling rapid testing and optimisation of engineered cell lines. The synergistic integration of these tools within the iterative design-build-test-learn (DBTL) cycle is highlighted as a key strategy for accelerating strain improvement. Ultimately, the convergence of these EB approaches is transforming PCC into a robust platform for producing pharmaceuticals, functional foods and green chemicals, contributing to a biobased economy with a minimal ecological footprint.},
}
@article {pmid41324601,
year = {2025},
author = {Bhattacharya, S and Goyal, K and Satpati, P},
title = {Thermodynamics of PAM Recognition by Cas9 of Streptococcus pyogenes.},
journal = {Journal of chemical information and modeling},
volume = {65},
number = {24},
pages = {13328-13337},
doi = {10.1021/acs.jcim.5c01934},
pmid = {41324601},
issn = {1549-960X},
mesh = {*Streptococcus pyogenes/enzymology ; *Thermodynamics ; *CRISPR-Associated Protein 9/metabolism/chemistry ; *CRISPR-Cas Systems ; DNA/chemistry/metabolism/genetics ; Gene Editing ; Base Sequence ; },
abstract = {The CRISPR/Cas9 system from Streptococcus pyogenes (SpCas9) requires a canonical 5'-NGG-3' PAM sequence in target DNA for effective genome editing. Base-specific interactions between the guanines (second and third position) and arginine dyad (R1333 and R1335) ensured specificity. We evaluated the PAM recognition strength of SpCas9 by using alchemical free energy calculations, revealing the energetics that influence genome editing accuracy. SpCas9 does not discriminate at the first position of the NGG sequence, but it penalizes mutations in the second and third positions. SpCas9 imposes a higher penalty for guanine mutation in the third PAM position compared to the second due to the greater conformational rigidity of R1335 in relation to R1333. Conformational rigidity of R1335 prevents side-chain readjustment for new protein-DNA interactions in noncanonical PAMs. A guanine-to-cytosine substitution in either the second or third position of canonical PAM disrupts direct protein-PAM interactions and leads to solvent exposure. This happens due to strong electrostatic repulsion between the arginine dyad's guanidinium groups and the amine group of cytosine. Interestingly, the strength of SpCas9 in disfavoring a single cytosine substitution (by >10 kcal/mol) is comparable to that of disfavoring double base substitutions in the NGG sequence. The ability of SpCas9 to differentiate between noncanonical and canonical PAMs (ΔΔG) is directly related to the number of direct interactions between SpCas9 and the PAM sequence, as well as the degree of solvent exposure. Loss of direct interactions and increased solvent exposure enhance ΔΔG. The calculated ΔΔG adequately explains the observed differences in DNA cleavage activity of SpCas9 across various DNA substrates with different PAM sequences. This study connects thermodynamics, structures, and activity to elucidate PAM selectivity in SpCas9 and may also apply to other CRISPR/Cas systems, offering valuable insights for the rational design of Cas9 variants with modified PAM specificities.},
}
@article {pmid41324747,
year = {2025},
author = {Priyanka, SS and Iqbal, G and Nidarshan, NC and Kumari, K and Vanjre, S and Rasal, K and Sonwane, A and Brahmane, M and Goswami, M},
title = {Avenues of genome editing for color trait improvement in ornamental fishes: current status and future perspectives.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {51},
pmid = {41324747},
issn = {1573-9368},
mesh = {Animals ; *Gene Editing/methods ; *Fishes/genetics/growth &amp; development ; *Pigmentation/genetics ; CRISPR-Cas Systems/genetics ; Animals, Genetically Modified/genetics/growth &amp; development ; },
abstract = {Ornamental fish industries are growing sectors contributing significantly to livelihood, trade and export, driven by the worldwide demand for colourful and unique species. Pigmentation is the focal point of the visual appeal of ornamental fish, market value, and species-specific interaction. The pigment cells are called chromatophores, derived from neural crest cells and controlled by sophisticated genetic mechanisms, conferring these fish with distinctive colours and patterns. Historically, selective breeding and dietary pigment supplementation have been applied to enhance colouration. Such traditional practices, however, are prone to disadvantages such as slow development, genetic ambiguity, and unforeseen consequences. With the invention of genome editing, such as CRISPR-Cas9, researchers now have a sensitive and powerful tool to control pigmentation traits at the genetic level. Central pigmentation gene manipulation, such as Tyr, Mc1r, and Slc45a2, can allow researchers to introduce stable and uniform color changes. Such enhancements confer unparalleled control of fish colour, a promising avenue in the ornamental fish industry. This review discusses the genetic nature of fish pigmentation. It reports recent advances in CRISPR-based modifications and describes their possible applications and implications for future ornamental fish breeding.},
}
@article {pmid41325351,
year = {2025},
author = {Jansen van Vuuren, J and Matthews, MC and Robène, I and Rozsasi, S and Campa, M and Burger, J and Viljoen, A and Mostert, D},
title = {Combined Recombinase Polymerase Amplification CRISPR/Cas12a Assay for Detecting Fusarium oxysporum f. sp. cubense Tropical Race 4.},
journal = {Journal of visualized experiments : JoVE},
volume = {},
number = {225},
pages = {},
doi = {10.3791/68841},
pmid = {41325351},
issn = {1940-087X},
mesh = {*Fusarium/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Recombinases/genetics/chemistry/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Plant Diseases/microbiology ; DNA, Fungal/genetics/analysis ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Regular and accurate surveillance stands central to the efficient management of plant diseases. It can indicate which course of action is most appropriate, and whether prevention, eradication, or no action is required. Surveillance based on symptomology in host plants alone is often not reliable due to similarities in the symptoms caused by biotic and abiotic stresses. Laboratory-based molecular methods such as polymerase chain reaction (PCR) and quantitative (q)PCR are the most commonly and reliably used for plant pathogen detection, but rely on expensive equipment and skilled operators. Here, we describe a protocol combining a simplified DNA extraction, recombinase polymerase amplification (RPA), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a (RPA-Cas12a) for the detection of the invasive pathogen, Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4). The technique provides a simple single-tube detection alternative that is analytically robust with improved specificity compared to available molecular detection assays and negates the need for expensive and sophisticated laboratory equipment.},
}
@article {pmid41325824,
year = {2026},
author = {Uddin, N and Ullah, MW and Zhu, D and Li, X and Yang, S and Xie, X},
title = {Engineering lignin pathway, plant cell wall modification, and genome editing for advanced renewable bioenergy and material applications.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108772},
doi = {10.1016/j.biotechadv.2025.108772},
pmid = {41325824},
issn = {1873-1899},
mesh = {*Lignin/biosynthesis/genetics/metabolism ; *Cell Wall/metabolism/genetics ; *Gene Editing ; *Plants/genetics/metabolism ; *Metabolic Engineering ; *Plant Cells/metabolism ; *Biofuels ; CRISPR-Cas Systems ; },
abstract = {Lignin biosynthesis and plant cell wall engineering are central to plant structural integrity and biomass utility. Recent advances in molecular and synthetic biology have opened opportunities to tailor lignin contents, composition, and polymer structure for renewable bioenergy and sustainable biomaterial applications. This review provides an integrative perspective on biosynthesis, regulation, and engineering of lignin. It summarizes the current progress in understanding the genetic, transcriptional, epigenetic, and metabolic networks that control lignin formation, with a focus on emerging tools such as CRISPR/Cas genome editing, synthetic promoters, and metabolic rewiring. Beyond cataloguing current knowledge, it critically analyzes the trade-offs involved in lignin modification for biomaterials, addressing unresolved challenges such as monolignol transport, metabolic flux control, and species-specific regulatory divergence. Engineered lignin and modified plant cell walls hold significant potential for biorefineries, advanced polymers, pharmaceuticals, and carbon sequestration, yet their translation from the laboratory to the field remains limited. Engineered lignin offers real-world applications across diverse industries, including bioenergy, bioplastics, carbon fiber composites, pharmaceuticals, and sustainable construction materials, thereby reinforcing its pivotal role in advancing a circular bioeconomy. The review further proposes future research directions that integrate multi-omics, single-cell technologies, machine learning, and field-based validation to enable precision lignin engineering. Strategic advances in this field will support next-generation bioenergy systems, advanced biomaterials, and the transition to a circular bioeconomy.},
}
@article {pmid41325825,
year = {2026},
author = {Zhang, Y and Deng, S},
title = {Geminivirus vectors: From gene silencing to synthetic biology.},
journal = {Biotechnology advances},
volume = {87},
number = {},
pages = {108771},
doi = {10.1016/j.biotechadv.2025.108771},
pmid = {41325825},
issn = {1873-1899},
mesh = {*Geminiviridae/genetics ; *Synthetic Biology/methods ; *Genetic Vectors/genetics ; *Gene Silencing ; Gene Editing ; Genetic Engineering ; },
abstract = {Geminiviruses, the largest plant DNA virus family, cause devastating diseases in crops worldwide. These viruses possess distinctive features, such as the stem-loop structure and replication protein (Rep), which enable the creation of functional geminiviral replicons (GVRs) in plants. Over three decades, geminiviruses have been developed into vectors for virus-induced gene silencing (VIGS), high-level protein expression, and genome editing. This review introduces the genomic structure, Rep protein domains and functions, as well as the historical applications of geminiviruses, then highlights their prominent roles in VIGS and synthetic biology. As VIGS vectors, bipartite geminiviruses utilize AV1 gene replacement, while monopartite species rely on satellite DNAs to insert target sequences, enabling gene silencing in diverse plants. In synthetic biology, GVRs facilitate high-level protein expression through autonomous replication and enhance CRISPR/Cas genome editing efficiency in crops. Additionally, gene regulatory elements, including tissue-specific promoters and gene expression enhancement sequences from geminiviral genomes or satellite DNA expand their utility in genetic engineering. Finally, this review provides an outlook on the future development of geminivirus vectors. GVRs can work as plasmid-like DNAs for supporting diverse and creative designs in plant synthetic biology. The stem-loop structure and Rep are not unique to geminiviruses, a fact that suggests potential cross-kingdom applications of GVRs beyond plants. Vast viral resources enable further acceleration of GVR applications through resource mining and optimization. Moreover, attenuated or engineered geminiviral strains hold promise as "plant vaccines" via cross-protection. Collectively, geminivirus vectors bridge fundamental viral research with practical innovations in crop improvement, biomanufacturing, and synthetic biology.},
}
@article {pmid41326076,
year = {2026},
author = {Niu, RC and Zeng, QH and Wang, WJ and Hu, J and Liu, TX and Zhang, SZ},
title = {Multi-omics analyses identify the modulators COX6A1 and NAL as regulators of silk cocoon formation in Plutella xylostella.},
journal = {Pesticide biochemistry and physiology},
volume = {216},
number = {Pt 1},
pages = {106764},
doi = {10.1016/j.pestbp.2025.106764},
pmid = {41326076},
issn = {1095-9939},
mesh = {Animals ; *Moths/genetics/metabolism/growth &amp; development ; *Silk/biosynthesis/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Larva/metabolism/genetics/growth &amp; development ; Proteomics ; CRISPR-Cas Systems ; *Electron Transport Complex IV/genetics/metabolism ; Transcriptome ; Multiomics ; },
abstract = {The diamondback moth (DBM) is a major global pest of cruciferous crops. Silk production, essential for DBM larval locomotion and pupal attachment, is governed by fibroin heavy chain (FibH), fibroin light chain (FibL), and fibrohexamerin (P25). However, the regulatory mechanisms and downstream key genes involved in silk production in DBM remain poorly understood. To address this, we integrated transcriptomic and proteomic data from CRISPR/Cas9 generated PxFibH, PxFibL, and PxP25 mutants to investigate the impact of silk gene deletions in the silk gland and identify modulators of silk formation. In the transcriptomic analysis, we identified 1994, 913, and 1266 differentially expressed genes (DEGs) in the three mutant strains, respectively. GO and KEGG enrichment analysis revealed significant involvement in pathways such as oxidation-reduction process, transmembrane transport, enzyme activity, and extracellular matrix (ECM) receptor interaction. At the proteomic level, 604, 210, and 266 differentially expressed proteins (DEPs) were identified in the three mutants, respectively. GO and KEGG enrichment analysis of these DEPs consistently highlighted energy metabolism, hydrolase activity, and catalytic activity pathways. Integrated multi-omics analyses identified three conserved regulator genes: cytochrome c oxidase subunit 6A1 (COX6A1), N-acetylneuraminate lyase (NAL), and protein phosphatase 1 regulatory subunit 14B (PPP1R14B). CRISPR/Cas9 knockout of PxCOX6A1 resulted in incomplete cocoon formation, along with increased larval mortality, prolonged development, and reduced oviposition. PxNAL knockout was lethal, while heterozygotes exhibited decreased cocoon formation, pupal weight, and fecundity. This study reveals FibH/FibL/P25-dependent metabolic networks regulating silk production and identifies COX6A1 and NAL as novel targets for environmentally sustainable pest control strategies.},
}
@article {pmid41328347,
year = {2026},
author = {Li, Q and Bao, Q and Zhao, S and Wu, F and Li, Y and Wang, K and Li, W and Gao, H},
title = {Advancements in CRISPR-based therapies for ocular pathologies: from disease mechanisms to intervention strategies.},
journal = {Theranostics},
volume = {16},
number = {1},
pages = {156-192},
pmid = {41328347},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Eye Diseases/therapy/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; },
abstract = {Eye diseases caused by genetic mutations affect over 2.2 billion people worldwide. The development of CRISPR technology has opened exciting possibilities for how we diagnose and treat these conditions. However, designing effective CRISPR systems, managing potential risks, and considering the ethical questions around gene therapy in clinical practice are major challenges. To move forward successfully, it's important to evaluate how practical CRISPR-based treatments are for eye diseases from a clinical perspective, while also understanding how CRISPR systems work. In this review, we start by covering the basic principles behind CRISPR technology and explore its different types. Next, we look at various ways CRISPR is being used in eye research and treatments, from early studies to new clinical approaches. Lastly, we address the regulatory environment and ethical issues involved, discussing existing rules, safety concerns, and guidelines for genetic modifications in medical settings. Our goal is to share new insights into innovative treatments for eye diseases and to support the safe use of CRISPR in clinical eye care. This review aims to be a helpful resource for researchers, doctors, and regulators working on CRISPR-based therapies.},
}
@article {pmid41328409,
year = {2026},
author = {Zhang, H and Song, Y and Liu, W and Zheng, X and An, X and Li, C and Chen, W and Wang, H and Zhang, Y},
title = {Defect-complementation homologous recombination: A novel strategy for precise genome engineering of virulent phages.},
journal = {Synthetic and systems biotechnology},
volume = {12},
number = {},
pages = {59-70},
pmid = {41328409},
issn = {2405-805X},
abstract = {Engineered bacteriophages (phages) have been developed to overcome the limitations of natural phage therapies and serve as precision-targeted agents against drug-resistant bacterial infections. However, their application has been constrained by the low efficiency of existing genome-editing tools, largely because of the absence of effective selection markers. This study proposed a novel strategy, termed defect-complementation homologous recombination (DCHR), for precise phage genome editing. In this approach, CRISPR-Cas9 cleaves a donor plasmid in host cells to release a linear donor template carrying homology arms, an essential phage gene used as a selection marker, and two lox sites. The donor template undergoes homologous recombination with the genome of essential gene-deficient phage, thereby enabling targeted genome modifications. Using DCHR, we successfully generated large genomic deletions (1.48-kb gp0.4-0.7 and 1.02-kb gp4.3-4.7), achieved gene insertion (3.08-kb lacZ), and introduced a single-base substitution (TGA to TAA) in the stop codon of gp9 within the same T7 phage genome, all with 100 % accuracy. The significant advantages of DCHR are as follows: (i) High-efficiency screening: Only progeny phages derived from successful homologous recombination retain viability and replicative capacity, thereby greatly simplifying recombinant isolation. (ii) Editing flexibility: Unlike CRISPR-Cas systems, DCHR cannot be constrained by protospacer adjacent motif dependence and allows modifications across diverse genomic loci. (iii) High recombination efficiency: DCHR can achieve a recombinant phage titer of 3.1 × 10[5] PFU mL[-1] (plaque-forming units per mL) without relying on exogenous homologous recombination systems. In summary, DCHR demonstrates potential as a precise and efficient general genome-editing tool that facilitates design of engineered phages and advances functional genomic studies.},
}
@article {pmid41328592,
year = {2025},
author = {Geng, Y and Jiang, C and Zhang, H and Yang, H and Peng, Y and Chen, Y and Hu, C and Liu, H and Li, S and Chen, H and Xie, S and Guo, A},
title = {Genome-scale CRISPR screen identifies host factors associated with bovine parainfluenza virus 3 infection.},
journal = {Virulence},
volume = {16},
number = {1},
pages = {2589554},
pmid = {41328592},
issn = {2150-5608},
mesh = {Animals ; Cattle ; *Parainfluenza Virus 3, Bovine/physiology/genetics ; CRISPR-Cas Systems ; Virus Replication ; *Host-Pathogen Interactions/genetics ; Cell Line ; *Respirovirus Infections/virology/veterinary/genetics ; Wnt-5a Protein/genetics/metabolism ; *Cattle Diseases/virology ; Virus Internalization ; Gene Knockout Techniques ; },
abstract = {Bovine parainfluenza virus type 3 (BPIV-3) is a major pathogen associated with the bovine respiratory disease complex. However, the limited understanding of host factors crucial for BPIV-3 replication has hindered the development of effective preventive and therapeutic strategies. To tackle this critical issue, we constructed a bovine genome-wide CRISPR/Cas9 knockout library in Madin-Darby bovine kidney cells, which was then used to systematically identify and characterize the host genes essential for BPIV-3a replication. Subsequently, 10 genes were validated using both RT-qPCR and viral titration assays. Furthermore, through gene knockout or knockdown and rescue experiments, we identified three key genes required for BPIV-3a replication: Wnt family member 5A (WNT5A), solute carrier family 16 member 13 (SLC16A13), and selenoprotein N (SELENON). However, their effects on viral adhesion and internalization varied. WNT5A was involved in both processes, SLC16A13 participated solely in internalization, while SELENON had no significant impact on either. Beyond BPIV-3a, these three genes were also found to be essential for the infection of BPIV-3c and Bovine enterovirus. In conclusion, this study offers novel insights into the molecular mechanisms governing the replication and pathogenesis of BPIV-3a, BPIV-3c, and bovine enterovirus within host cells, thereby providing a foundation for identifying potential targets in the development of novel antiviral strategies.},
}
@article {pmid41328622,
year = {2026},
author = {Ma, C and Zhou, Y and Jiang, N and Ren, X and Xu, C and Su, N and Fan, Y and Liu, W},
title = {Development of an RPA-CRISPR-Cas12a Fluorescence Assay for Rapid and Sensitive Detection of Tilapia Parvovirus (TiPV).},
journal = {Journal of fish diseases},
volume = {49},
number = {5},
pages = {e70095},
doi = {10.1111/jfd.70095},
pmid = {41328622},
issn = {1365-2761},
support = {32202994//the National Natural Science Foundation of China/ ; 2023TD46//the Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; 2024XT0602//the Central Public-interest Scientific Institution Basal Research Fund, CAFS/ ; },
mesh = {Animals ; *Fish Diseases/diagnosis/virology ; Sensitivity and Specificity ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Tilapia ; *Parvovirus/isolation &amp; purification/genetics ; },
abstract = {Tilapia parvovirus (TiPV) is an emerging pathogen associated with high mortality rates in farmed tilapia, highlighting the urgent need for rapid and accurate diagnostic tools. In this study, we established an RPA-CRISPR/Cas12a detection system targeting the TiPV NS1 gene. The assay conditions were systematically optimised, including 15-min RPA amplification at 39°C, with reagent concentrations of 200 nM Cas12a, 250 nM crRNA and 200 nM ssDNA reporter. Specificity tests showed no cross-reactivity with other tilapia pathogens (TiLV, S. agalactiae) and other aquatic pathogens (LMBRaV, YcCV, GCRV II, WSSV, CyHV-2, SVCV). Sensitivity evaluation revealed a limit of detection (LoD) of 1.97 × 10[1]copies/μL, which was 100-fold more sensitive than PCR (1.97 × 10[3]copies/μL). Clinical validation with 20 tilapia samples demonstrated a 50% positive detection rate for RPA-CRISPR/Cas12a, 15% higher than PCR (35%). This integrated method combines the advantages of RPA and CRISPR-based signal transduction, offering a field-applicable solution for TiPV monitoring in resource-limited aquaculture environments.},
}
@article {pmid41328758,
year = {2026},
author = {Plewnia, A and Hoenig, BD and Lötters, S and Heine, C and Erens, J and Böning, P and Bending, GD and Krehenwinkel, H and Williams, MA},
title = {The Emergence of a CRISPR-Cas Revolution in Ecology: Applications, Challenges, and an Ecologist's Overview of the Toolbox.},
journal = {Molecular ecology resources},
volume = {26},
number = {1},
pages = {e70086},
pmid = {41328758},
issn = {1755-0998},
support = {//University of Warwick/ ; NE/S010270/1//Natural Environment Research Council/ ; },
mesh = {*CRISPR-Cas Systems ; *Ecology/methods ; *Gene Editing/methods ; },
abstract = {CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated nucleases) systems allow researchers to detect, capture, and even alter parts of an organism's genome. However, while the use of CRISPR-Cas has revolutionised many fields in the life sciences, its full potential remains underutilised in ecology and biodiversity research. Here we outline the emerging applications of CRISPR-Cas in ecological contexts, focusing on three main areas: nucleic acid detection, CRISPR-enhanced sequencing, and genome editing. CRISPR-based nucleic acid detection of environmental DNA samples is already reshaping species monitoring, providing highly sensitive and non-invasive tools for both scientists and the public alike, with reduced costs and minimal experience required. Further, CRISPR-enhanced sequencing, including Cas-mediated target enrichment, enables efficient recovery of ecologically relevant loci and supports diverse applications such as amplification-free metagenomics. Finally, while genome editing on wild species remains largely theoretical in ecology, these tools are already being used in controlled settings to study adaptation and resilience in the face of ongoing global stressors. Together, the applications of CRISPR-Cas are paving the way for more affordable, accessible, and impactful applications for species conservation, and promise to improve our ability to tackle the ongoing global biodiversity crisis.},
}
@article {pmid41329281,
year = {2025},
author = {Kolesov, DE and Orlova, NA and Vorobiev, II},
title = {Generation of Long-Lived CHO Cells Suitable for Production of Afucosylated Antibodies and Fc-Fusion Proteins.},
journal = {Doklady biological sciences : proceedings of the Academy of Sciences of the USSR, Biological sciences sections},
volume = {525},
number = {1},
pages = {359-367},
pmid = {41329281},
issn = {1608-3105},
mesh = {Animals ; CHO Cells ; Cricetulus ; *Immunoglobulin Fc Fragments/genetics/metabolism ; *Fucosyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; *Recombinant Fusion Proteins/genetics/biosynthesis ; *Antibodies/metabolism ; Gene Knockout Techniques ; },
abstract = {Using genome editing, we created a homozygous α-(1,6)-fucosyltransferase (FUT8[-]/[-]) knockout in apoptosis-resistant CHO 4BGD cells, yielding the new 4BGD-F cell line. Combining CRISPR/Cas9 with paired gRNAs and non-specific puromycin selection yielded a cell population with an exceptionally high FUT8 knockout frequency, obviating the need for metabolic enrichment with lentil lectin (Lens culinaris agglutinin, LCA). Despite impaired clonogenicity of the knockout cells, we successfully isolated multiple clonal cell lines harboring extensive biallelic FUT8 deletions. Isolated clones with biallelic deletions retained key parental line characteristics: viability >90% in 17-day fed-batch cultures at high densities (>15 × 10[6] cells/mL), and rapid selectability using both dihydrofolate reductase and glutamine synthetase systems. Mass spectrometric analysis of the test protein GLP1-Fc secreted by 4BGD-F cells confirmed the absence of N-glycan fucosylation. The CHO 4BGD-F cell line provides a valuable platform for producing afucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity.},
}
@article {pmid41329618,
year = {2025},
author = {Udemezue, VC and Shaikh, KM and Vorontsova, M and Valgepea, K},
title = {Optimization of Plasmid Curing from Genetically Engineered Clostridium autoethanogenum.},
journal = {ACS synthetic biology},
volume = {14},
number = {12},
pages = {4967-4972},
pmid = {41329618},
issn = {2161-5063},
mesh = {*Plasmids/genetics ; *Clostridium/genetics/metabolism ; CRISPR-Cas Systems/genetics ; *Metabolic Engineering/methods ; Gene Editing/methods ; *Genetic Engineering/methods ; Electroporation ; },
abstract = {Accumulation of greenhouse gases from combustion of fossil fuels drives climate change and threatens biosustainability on Earth. Microbial gas fermentation realizes the capture of CO2 toward biomanufacturing of value-added products. Acetogens are attractive biocatalysts here, as they use CO2 as their sole carbon source with H2. Metabolic engineering of novel cell factories is, however, hampered by the slow and complex genetic engineering workflows. Here, we developed different approaches to optimize plasmid curing from genetically engineered strains of the model acetogen Clostridium autoethanogenum. Interestingly, a CRISPR/Cas9-based curing plasmid (C-plasmid) targeting the origin of replication both in the target editing plasmid and in the C-plasmid did not improve curing over a non-targeting control plasmid. Strikingly, plasmid curing by making cells electrocompetent (ECCs) and by non-transformative electroporation of ECCs or buffer-washed glycerol stocks showed 14-100% curing efficiencies across the approaches for five different genetically engineered C. autoethanogenum strains. The most time-efficient approach with non-transformative electroporation of buffer-washed glycerol stocks also cured an editing plasmid from Escherichia coli, with ∼97% efficiency. This work both improves genetic engineering workflows for C. autoethanogenum by significantly accelerating plasmid curing and offers methods to potentially ease plasmid curing in other microbes.},
}
@article {pmid41330004,
year = {2025},
author = {Coşar, B and Kılıç, P and İşeri, &#xd6;D},
title = {The intersection of CAR-T immunotherapy with emerging technologies.},
journal = {Cytokine &amp; growth factor reviews},
volume = {86},
number = {},
pages = {238-259},
doi = {10.1016/j.cytogfr.2025.11.001},
pmid = {41330004},
issn = {1879-0305},
mesh = {Humans ; *Immunotherapy, Adoptive/methods ; *Receptors, Chimeric Antigen/immunology/genetics ; *Neoplasms/therapy/immunology ; Animals ; Cytokines/immunology ; *T-Lymphocytes/immunology ; CRISPR-Cas Systems ; Gene Editing ; Tumor Microenvironment/immunology ; },
abstract = {Chimeric antigen receptor (CAR) T-cell (CAR-T) therapy is a transformative modality in cancer immunotherapy that employs genetically engineered T-cells to eliminate malignant cells selectively. Its efficacy and limitations are governed by cytokine- and growth factor-mediated signaling networks that shape T-cell activation, proliferation, differentiation, and persistence. This review traces the molecular evolution of CAR-T architecture across generations, highlighting how synthetic modulation of cytokine and co-stimulatory pathways enhances potency while reducing exhaustion and toxicity. We discuss strategies that incorporate cytokine engineering, metabolic reprogramming, and logic-gated activation to counteract the immunosuppressive tumor microenvironment. Recent technological advances-such as clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9)-based cytokine pathway editing, induced pluripotent stem cell (iPSC)-derived "off-the-shelf" CAR-T platforms, and extracellular vesicle (EV)-mediated cytokine delivery-are reshaping adoptive immunotherapy. Framing CAR-T development through the lens of cytokine and growth factor biology, we outline how integrating these pathways enables safer, more durable, and scalable next-generation therapies for hematologic and solid tumors.},
}
@article {pmid41330077,
year = {2026},
author = {Fathpour, H and Fouladi, M and Jafarpour, F and Moradi-Hajidavaloo, R and Izadi, T and Shiralian-Esfahani, H and Kues, W and Nasr-Esfahani, MH and Hajian, M and Eghbalsaied, S},
title = {Crosstalk between myostatin and callipyge in CRISPR/Cas9-edited goat fibroblast cells.},
journal = {Research in veterinary science},
volume = {198},
number = {},
pages = {105992},
doi = {10.1016/j.rvsc.2025.105992},
pmid = {41330077},
issn = {1532-2661},
mesh = {Animals ; *Myostatin/genetics/metabolism ; *Goats/genetics ; *Fibroblasts/metabolism ; *CRISPR-Cas Systems ; Gene Editing/veterinary ; },
abstract = {Myostatin (MSTN) and Callipyge (CLPG) genes are key regulators of muscle growth. While MSTN inhibits muscle development, the CLPG mutation induces muscle hypertrophy through a specific imprinted genetic mechanism. The interaction between these genes remains of interest for improving livestock muscle traits. In this study, CRISPR/Cas9 was employed to edit MSTN and CLPG genes in goat fibroblast cells via electrotransfection. Cells were selected using puromycin antibiotic, and gene-editing efficiency was evaluated through Sanger sequencing. Gene expression changes were analyzed using RT-qPCR analysis. MSTN gene knockout resulted in significant downregulation of MSTN and CLPG, while GTL2 expression was upregulated by more than 50-fold. Additionally, myosin heavy chain genes (MYH1, MYH3, MYH4) were strongly upregulated, with MYH3 13-fold and MYH4 30-fold increase in the expression. In CLPG-edited cells, the expression of MSTN, TRIM28, and CLPG was reduced, while GTL2 was upregulated by 6-fold. MYH3 and MYH4 expression increased 4-fold in CLPG-edited cells, though the increase was less pronounced compared to MSTN-edited cells. DLK1 expression was undetectable in both non-edited control and gene-edited fibroblast cells. Our findings support the interaction between MSTN and CLPG, contributing to the regulation of muscle growth. Notably, the study also highlights the challenges associated with editing imprinted genes like CLPG and suggests that TRIM28 may play a role downstream of CLPG regulation. These results provide valuable insights into muscle development regulation, offering potential applications in livestock genetic improvement.},
}
@article {pmid41330274,
year = {2025},
author = {Macklin, BL and Runyon, WV and Feliciano, CM and Dierks, PH and Kelly, KR and Watry, HL and Judge, LM and Conklin, BR},
title = {Generation of WTD, a control human iPSC line for genetic research.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103872},
doi = {10.1016/j.scr.2025.103872},
pmid = {41330274},
issn = {1876-7753},
mesh = {Humans ; *Induced Pluripotent Stem Cells/metabolism/cytology ; Cell Line ; CRISPR-Cas Systems ; Cell Differentiation ; Mutation ; },
abstract = {The establishment of well characterized control iPSC lines is essential for robust, reproducible research across laboratories. We used CRISPR/Cas9 to derive an isogeneic control line from a patient-derived iPSC line carrying a mutation in the NEFL gene (E396K). After correction of the E396K mutation, UCSFi003-A (WTD) exhibits multi-lineage differentiation potential, a normal karyotype, no large genomic abnormalities, and has consents for public distribution of cells and genomic data.},
}
@article {pmid41330302,
year = {2026},
author = {Xu, W and Lin, Y and Huang, Z and Li, Y and Lu, Y and Liu, M and Cui, S and Zhang, T and Shi, N and Sheng, Y and Hu, J},
title = {Split proximity circuit initiated CRISPR-Cas12a system profiling exosomal surface proteins for early cancer detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118280},
doi = {10.1016/j.bios.2025.118280},
pmid = {41330302},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Breast Neoplasms/diagnosis/blood/genetics ; *Early Detection of Cancer/methods ; Female ; Epithelial Cell Adhesion Molecule/genetics/isolation &amp; purification ; *Biomarkers, Tumor/genetics/blood ; Limit of Detection ; Mucin-1/genetics ; Aptamers, Nucleotide/chemistry ; },
abstract = {Early diagnosis of breast cancer is critical for improving prognosis, but traditional methods have limitations. Herein, we propose an SPC-CRISPR system for the sensitive and specific detection of multiple breast cancer exosomal proteins without prior exosome isolation. This system couples CRISPR system with an enzyme-free amplification method to achieve dual-signal amplification. SPC-CRISPR is based on a split proximity circuit (SPC) that triggers catalytic hairpin assembly (CHA), converting protein signals on the surface of exosomes into nucleic acid signals, and the CRISPR-Cas12a system enabling further signal amplification and output. The system targets phosphatidylserine (PS), MUC1, and EpCAM on exosomes: Tim4-modified magnetic beads capture PS-expressing exosomes, and dual-aptamers recognize MUC1 and EpCAM, enabling SPC assembly and subsequent amplification. In buffer and cell-derived exosomes, the SPC-CRISPR system showed a detection limit of 10 particles/μL (R[2] = 0.990). Clinical tests utilizing merely 1 μL of serum samples successfully distinguished breast cancer patients from healthy donors (AUC = 0.9778, accuracy = 91.23 %), detected stage 0 breast cancer patients against healthy controls (accuracy = 92.59 %), and differentiated metastatic from non-metastatic cases (p < 0.001). The combination of high sensitivity, minimal sample requirements, and an exosome isolation-free workflow positions the SPC-CRISPR system as a promising tool for the clinical early detection and classification of breast cancer, with broader applicability to other cancers by swapping the corresponding aptamers.},
}
@article {pmid41330380,
year = {2026},
author = {Feng, C and Peets, EM and Zhou, Y and Crepaldi, L and Usluer, S and Dunham, A and Braunger, JM and Su, J and Strauss, ME and Muraro, D and Xian Cheam, KA and Bonder, MJ and Nogales, EG and Cooper, S and Bassett, A and Leonard, S and Gu, Y and Fussing, B and Burke, D and Parts, L and Stegle, O and Velten, B},
title = {A genome-scale single-cell CRISPRi map of trans gene regulation across human pluripotent stem cell lines.},
journal = {Cell genomics},
volume = {6},
number = {2},
pages = {101076},
pmid = {41330380},
issn = {2666-979X},
mesh = {Humans ; *Single-Cell Analysis/methods ; *Pluripotent Stem Cells/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Expression Regulation/genetics ; *Genome, Human/genetics ; Quantitative Trait Loci/genetics ; Cell Line ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Population-scale resources of genetic, molecular, and cellular information form the basis for understanding human genomes, charting the heritable basis of disease and tracing the effects of mutations. Pooled perturbation assays, probing the effect of many perturbations coupled with single-cell RNA sequencing (scRNA-seq) readout, are especially potent references for interpreting disease-linked mutations or gene-expression changes. However, the utility of existing maps has been limited by the comprehensiveness of perturbations conducted and the relevance of their cell-line context. Here, we present a genome-scale CRISPR interference perturbation map with scRNA-seq readout across many genetic backgrounds in human pluripotent cells. We map trans expression changes induced by knockdowns and characterize their variation across donors, with expression quantitative trait loci linked to higher genetic modulation of perturbation effects. This study pioneers population-scale CRISPR perturbations with high-dimensional readouts, which will fuel the future of effective modulation of cellular disease phenotypes.},
}
@article {pmid41330665,
year = {2026},
author = {Bao, Y and Ding, W and Zhang, L and Wang, W and Liu, J and Qu, Y and Zhu, L and Zhang, K and Zhong, G and Han, R and Shen, Q and Wang, B and Gu, X and Cao, Y and Sun, W},
title = {A novel multiplex RPA/CRISPR-Cas12a integrated biosensor for on-site detection of high-risk HPV genotypes.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344830},
doi = {10.1016/j.aca.2025.344830},
pmid = {41330665},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; Humans ; Genotype ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation &amp; purification ; *Recombinases/metabolism ; Papillomavirus Infections/virology/diagnosis ; DNA, Viral/genetics ; },
abstract = {Accurate genotyping of high-risk human papillomavirus (HR-HPV) at the point of care is critical for global cervical cancer elimination, but its application remains limited by the need for complex equipment and specialized procedures, particularly in resource-limited regions. Here, we develop H-MRC12a-an integrated platform based on degenerate primers and type-specific crRNAs that combines multiplex recombinase polymerase amplification (RPA) with CRISPR-Cas12a trans-cleavage activity-for ultrasensitive detection of eight key HR-HPV genotypes (16, 18, 31, 33, 52, 53, 58, 66). By introducing a degenerate primer strategy coupled with type-specific crRNAs, the system overcomes interference from primer dimer formation and achieves single-copy sensitivity within 50 min under isothermal conditions (37 °C). Clinical validation demonstrated 100 % concordance with qPCR and identified three additional low viral-load positives (Ct > 35) that were missed by conventional methods. Crucially, the platform enables visual readout under UV light and eliminates the need for specialized instruments. This 'broad-spectrum capture and precision typing' paradigm establishes a versatile framework for multiplexed pathogen detection, advancing accessible molecular diagnostics for global health equity.},
}
@article {pmid41330674,
year = {2026},
author = {Hou, L and Ruan, F and Zhao, K and Li, B},
title = {Boosting split-crRNA CRISPR/Cas12a activity by 3'-end extension of DNA activator for direct microRNA sensing.},
journal = {Analytica chimica acta},
volume = {1382},
number = {},
pages = {344841},
doi = {10.1016/j.aca.2025.344841},
pmid = {41330674},
issn = {1873-4324},
mesh = {*MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Humans ; *DNA/chemistry/metabolism/genetics ; Limit of Detection ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {BACKGROUND: The unique trans-cleavage activity of CRISPR/Cas12a has been extensively utilized in the domain of biosensing. Nevertheless, the detection of miRNAs using the traditional CRISPR/Cas12a system requires nucleic acid amplification or reverse transcription to convert miRNA into DNA, which increases reaction time and the risk of contamination.<br><br>RESULTS: This study presents a split-crRNA CRISPR/Cas12a-based biosensing for direct detection of miRNA. The target miRNA-375 was utilized as the spacer region of the crRNA, facilitating its binding to the truncated scaffold RNA, thereby resulting in the formation of a complete crRNA. More importantly, we discovered that the cleavage activity of split-crRNA CRISPR/Cas12a was significantly enhanced by extending sequences at the 3'-end of the DNA activator. Compared with the conventional split-crRNA CRISPR/Cas12a system, the split-crRNA CRISPR/Cas12a with 24-nucleotide random sequence extension at the 3'-end of the DNA activator exhibited a 6.4-fold increase in activity. The enhancement mechanism of 3'-end extension of DNA activator was discussed. This proposed split-crRNA CRISPR/Cas12a system was applied to detect miRNA-375 with a linear range of 5 pM-1&#xa0;nM, and the detection limit was estimated to be 0.6 pM (3&#x3c3;). Furthermore, this system was used to detect miRNA-375 in 10&#xa0;% diluted human serum, achieving satisfactory recovery rate (98&#xa0;%-106&#xa0;%).<br><br>SIGNIFICANCE: This finding indicates that it is feasible to enhance the activity of the split-crRNA CRISPR/Cas12a by extending the 3'-end of the DNA activator, thereby achieving highly sensitive direct detection of miRNA. It is a simple yet effective strategy for enhancing the sensitivity of direct miRNA detection.},
}
@article {pmid41330849,
year = {2026},
author = {Boob, AG and Zhang, C and Pan, Y and Zaidi, A and Whitaker, RJ and Zhao, H},
title = {Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic archaeon Sulfolobus islandicus.},
journal = {Trends in biotechnology},
volume = {44},
number = {4},
pages = {1167-1186},
doi = {10.1016/j.tibtech.2025.11.003},
pmid = {41330849},
issn = {1879-3096},
mesh = {*Sulfolobus/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Metabolic Engineering/methods ; *Chromosomes, Archaeal/genetics ; Synthetic Biology ; },
abstract = {Sulfolobus islandicus, an emerging archaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs targeting eight integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. We leveraged the endogenous CRISPR-Cas system to integrate the reporter gene lacS and validated heterologous expression through a β-galactosidase assay, revealing significant positional effects. As a proof of concept, we utilized these sites to genetically manipulate lipid ether composition by overexpressing glycerol dibiphytanyl glycerol tetraether (GDGT) ring synthase B (GrsB). This study expands the genetic toolbox for S. islandicus and advances its potential as a robust platform for archaeal synthetic biology and industrial biotechnology.},
}
@article {pmid41330932,
year = {2025},
author = {Van Vu, T and Thi Nguyen, N and Kim, J and Sung, YW and Chung, WS and Kim, JY},
title = {The evolving landscape of precise DNA insertion in plants.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10428},
pmid = {41330932},
issn = {2041-1723},
support = {RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2021-NR060105//National Research Foundation of Korea (NRF)/ ; RS-2025-02263262//National Research Foundation of Korea (NRF)/ ; RS-2022-NR070609//National Research Foundation of Korea (NRF)/ ; RS-2020-NR049590//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Gene Editing/methods ; Genome, Plant ; Plants, Genetically Modified/genetics ; *DNA, Plant/genetics ; *Plants/genetics ; *Mutagenesis, Insertional/methods ; Gene Targeting/methods ; },
abstract = {Precise DNA insertion into plant genomes is central to advancing crop improvement and synthetic biology. CRISPR-Cas systems have enabled programmable DNA integration using tools such as gene targeting (GT), prime editing (PE), and recombinase- or transposase-based platforms. These tools are transitioned from theoretical concepts to practical applications, supporting applications like in-locus protein tagging, regulatory element engineering, and multi-gene stacking. Key challenges persist, such as inefficient large-fragment insertion, delivery barriers, and regulatory hurdles. This review traces the evolution from random to CRISPR-Cas-based systems, analyzes current limitations, and discusses emerging solutions paving the way for predictable DNA insertion in modern plant biotechnology.},
}
@article {pmid41331675,
year = {2025},
author = {Wang, M and Zhang, Y and Bi, C and Li, M},
title = {CRISPR-Cas9-induced double-strand breaks disrupt maintenance of epigenetic information.},
journal = {Genome biology},
volume = {26},
number = {1},
pages = {411},
pmid = {41331675},
issn = {1474-760X},
support = {BAS/1/1080-01-01//KAUST Office of Sponsored Research/ ; 5932//KAUST Center of Excellence for Smart Health/ ; },
mesh = {Humans ; *DNA Breaks, Double-Stranded ; *CRISPR-Cas Systems ; DNA Methylation ; *Epigenesis, Genetic ; Gene Editing ; MutL Protein Homolog 1/genetics ; Human Embryonic Stem Cells/metabolism ; Genomic Imprinting ; DNA Repair ; },
abstract = {BACKGROUND: CRISPR-Cas9 genome editing enables precise genetic modifications by introducing targeted DNA double-strand breaks (DSBs). While Cas9-induced DSBs are known to cause unintended on-target mutations, their impact on the epigenetic landscape remains unexplored.<br><br>RESULTS: Here, we investigate how Cas9-induced DSBs affect DNA methylation patterns in human embryonic stem cells (hESCs). We induce DSBs at differentially methylated regions of imprinted genomic loci and perform high-coverage, long-read native DNA sequencing to simultaneously obtain genetic variant and base-resolution methylation data in a haplotype-resolved manner. Our findings reveal that DSBs cause significant changes in DNA methylation at target sites through mechanisms including homologous recombination, large structural variations, or defective methylation maintenance during DNA repair. Notably, these epigenetic changes can occur either together with or independently of genetic alterations. Beyond imprinted loci, Cas9-induced DSBs significantly disrupt DNA methylation patterns of the MLH1 epimutation alleles in colorectal cancer cells, and hypermethylated heterochromatin loci in hESCs. Clonal analysis indicates that the aberrant methylation changes are stable during in vitro passaging. Intriguingly, significant changes in DNA methylation levels are also detected around endogenous deletions in unedited genomic regions, suggesting that methylation alterations are not unique to Cas9 nuclease activity but represent a general outcome of DSB repair in human cells.<br><br>CONCLUSIONS: This study underscores the importance of assessing and mitigating unintended epigenetic consequences in genome editing applications, as such changes can profoundly affect gene regulation and cellular function.},
}
@article {pmid41331925,
year = {2025},
author = {Matsumoto, D and Kubota, K and Sato, Y and Kato-Inui, T and Nigorikawa, K and Miyaoka, Y and Nomura, W},
title = {Screening strategy to identify Cas9 variants with higher HDR activity based on diphtheria toxin.},
journal = {Journal of biomedical science},
volume = {32},
number = {1},
pages = {102},
pmid = {41331925},
issn = {1423-0127},
support = {JP23K13844//KAKENHI/ ; JP24K09445//KAKENHI/ ; JP20H03442//KAKENHI/ ; JP20K21253//KAKENHI/ ; JP22H02201//KAKENHI/ ; JP23K23468//KAKENHI/ ; },
mesh = {Humans ; *Diphtheria Toxin/genetics ; *CRISPR-Associated Protein 9/genetics ; *Gene Editing/methods ; *Recombinational DNA Repair/genetics ; *CRISPR-Cas Systems ; HEK293 Cells ; Mutation ; },
abstract = {BACKGROUND: In gene therapy via genome editing, it is essential to precisely repair disease-associated gene sequences without introducing random mutations. However, achieving highly accurate genome editing remains challenging owing to the low efficiency of homology-directed repair (HDR)-mediated gene repair, which relies on template DNA. Therefore, if Cas9 mutants capable of enhancing HDR can be identified, they could enable more precise gene therapies.<br><br>METHOD: In this research project, we developed a screening system that uses the acquisition of diphtheria toxin resistance as an indicator of HDR efficiency in human cells and EGFP disruption as an indicator of off-target effect.<br><br>RESULTS: By screening a library of SpCas9 variants with random mutations introduced into its nuclease domain, we identified a novel SpCas9 mutant with higher HDR efficiency than wild-type Cas9.<br><br>CONCLUSION: We explored the possibility of obtaining Cas9 mutants with high HDR efficiency via this screening system.},
}
@article {pmid41332134,
year = {2026},
author = {Inuzuka, T and Mouzannar, K and Zhang, M and Umarova, R and Park, SB and Uchida, T and Ma, CD and Liang, TJ},
title = {A CRISPR-based genome-wide loss-of-function screen defines a role of host metabolism in regulating hepatitis B virus infection.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1616-1632},
pmid = {41332134},
issn = {1525-0024},
support = {Z01 DK054500/ImNIH/Intramural NIH HHS/United States ; Z01 DK054504/ImNIH/Intramural NIH HHS/United States ; Z99 DK999999/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Hepatitis B virus/genetics/physiology ; *Hepatitis B/metabolism/virology/genetics ; *CRISPR-Cas Systems ; Hep G2 Cells ; Virus Replication/genetics ; Hepatocytes/metabolism/virology ; *Host-Pathogen Interactions/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Hepatitis B virus (HBV) co-opts and interacts with an extensive array of host factors for productive infection. Herein, we develop an HBV reporter virus expressing red fluorescent protein (HBV-RFP) that is suitable for a CRISPR-based genome-wide screen for HBV host dependency factors. HepG2[NTCP/Cas9] cells were transduced with a pooled lentiviral library of single-guide RNA (sgRNA) targeting 19,114 human genes, edited and infected with HBV-RFP. RFP-low cells were sorted using fluorescence-activated cell sorting. The sorted cells were expanded and underwent two additional rounds of infection and sorting to enrich for sgRNA-targeted proviral host factors. By next-generation sequencing and bioinformatic analyses, we identified 63 genes as candidate host proviral factors, including known HBV proviral factors: RXRA, POLL, LDLR, and NTCP. Among the novel candidate genes, knockout of 12 genes significantly decreased HBV replication markers. Validation using siRNA knockdown in primary human hepatocytes confirmed several factors including the monoacylglycerol acyltransferase 2 (MOGAT2) gene as a bona fide HBV proviral factor. Further analysis with MGAT2 inhibitors demonstrated that inhibition of MOGAT2 activity impairs HBV transcription and replication. Our study demonstrates the value of the HBV reporter system in identifying previously unrecognized host metabolic factors important for HBV infection, offering a potential avenue for therapeutic development.},
}
@article {pmid41332531,
year = {2025},
author = {Zhang, P and Xue, B and Xie, Y and Li, K and Yang, H and Sun, P and Zhang, L},
title = {OSM-11 modulates salinity-stress tolerance in Caenorhabditis elegans.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41332531},
issn = {2692-8205},
support = {P40 OD010440/OD/NIH HHS/United States ; },
abstract = {Most terrestrial animals exhibit narrow salinity tolerance compared to their marine counterparts. Previous studies identified osm-11 (which encodes a Notch co-ligand) mutations as a driver of hyper-saline tolerance in Caenorhabditis elegans, but mechanistic insights remained unclear. This study employs RNA sequencing and CRISPR/Cas-9 genome editing to demonstrate that osm-11 mutations enhance salinity stress resistance through upregulation of fatty acid metabolism (acdh-12, acs-17) and cytochrome P450 pathways (ugt-15), while suppressing calcium signaling. Furthermore, we demonstrated that acdh-12 mutation impairs salinity-stress tolerance by activating ferroptosis and mitophagy, accompanied by down-regulated oxidative phosphorylation and up-regulated autophagic pathways. Morphological observations show that mitochondrial fragmentation contributes to wild-type nematode mortality under high salinity, while enlarged lipid droplets in wild-types correlate with reduced β-oxidation gene expression (dhs-28, daf-22), whose knockout disrupts tolerance in mutants. These findings unravel the multi-pathway regulatory network of osm-11-mediated salinity tolerance, providing mechanistic insights for developing protective strategies against environmental salinity stressors impacting animal survival.},
}
@article {pmid41336948,
year = {2025},
author = {Yu, ES and Jang, H and Kwon, J and Jeong, H and Park, J and Kang, T and Jeong, KH},
title = {On-chip Nanoplasmonic RT-RPA and CRISPR/Cas12a Assay for Point-of-care Molecular Diagnostics.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-4},
doi = {10.1109/EMBC58623.2025.11253682},
pmid = {41336948},
issn = {2694-0604},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; SARS-CoV-2/genetics ; *COVID-19/diagnosis ; *Point-of-Care Systems ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques ; *Pathology, Molecular/methods ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Rapid and accurate nucleic acid detection at point-of-care (POC) is essential for advancing effective disease diagnosis and management. Here, we report a handheld nanoplasmonic all-in-one setup for on-chip recombinase polymerase amplification (RPA) and real-time fluorescence detection by CRISPR/Cas12a reaction. The all-in-one setup consists of AuNIs-based nanoplasmonic cavity (AuNIs-NC), a disposable plastic-on-polymer (PoP) cartridge, and fluorescence microlens array (FMLA) camera. The AuNIs-NC allows uniform and efficient photothermal heating under white LED illumination due to strong broadband light absorption and internal reflection by randomly distributed AuNIs and thin Al film. This setup allows the RPA and CRISPR/Cas 12a reactions in a single chamber of PoP cartridge, with fluorescence signals monitored by a FMLA camera. The experimental result demonstrates rapid SARS-CoV-2 E gene plasmid DNA detection within 20 min, achieving a detection sensitivity of 10 copies/ul. Testing with 16 clinical samples shows a linear trend with RT-qPCR, indicating the platform's reliable sensitivity and specificity. This compact platform offers affordable and reliable molecular diagnosis, facilitating rapid and scalable POC testing for a range of infectious diseases.Clinical Relevance- This on-chip real-time RT-RPA and CRISPR/Cas12a assay provides rapid and precise molecular diagnostics at POC using fully integrated plasmonic system.},
}
@article {pmid41337296,
year = {2025},
author = {Cimolato, C and Letrari, S and Chiacchiera, AF and Del Favero, S and Schenato, L and Pasotti, L and Bellato, M},
title = {Modeling of Phage-Mediated CRISPRi System to Inhibit Antibiotic Resistances in Bacteria.},
journal = {Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference},
volume = {2025},
number = {},
pages = {1-7},
doi = {10.1109/EMBC58623.2025.11253443},
pmid = {41337296},
issn = {2694-0604},
mesh = {*Bacteriophages/genetics ; *CRISPR-Cas Systems/genetics ; *Bacteria/genetics/drug effects/virology ; *Drug Resistance, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Humans ; },
abstract = {Antimicrobial resistance (AMR) poses a critical threat to global health, rendering traditional antibiotics increasingly ineffective and amplifying the urgency for innovative solutions. Among promising alternatives, synthetic biology emerges as a powerful tool to combat AMR. This work proposes an innovative strategy based on engineering bacteriophages to deliver CRISPR interference (CRISPRi) systems into antibiotic-resistant pathogens to precisely silence target resistance genes. A comprehensive mathematical model is developed and simulated to capture the dynamics of phage-mediated CRISPRi delivery. By explicitly incorporating mutations that affect CRISPRi functionality, the study evaluates system performance and its potential for long-term therapeutic efficacy. This model serves as a critical framework for optimizing future CRISPRi-based interventions and advancing synthetic biology-driven approaches to tackle AMR.Clinical relevance- This paper provides a quantitative modeling framework to evaluate key parameters affecting engineered phage therapy efficiency, supporting rational design and phage posology optimization.},
}
@article {pmid41338106,
year = {2026},
author = {Paenkaew, S and Euppayo, T and Tungtrakanpoung, R and Teapunvong, W and Nganvongpanit, K and Buddhachat, K},
title = {Rapid and specific detection of Babesia vogeli using RPA/CRISPR-Cas12a: A feasible field-friendly diagnostic for canine babesiosis.},
journal = {Veterinary parasitology},
volume = {342},
number = {},
pages = {110660},
doi = {10.1016/j.vetpar.2025.110660},
pmid = {41338106},
issn = {1873-2550},
mesh = {Animals ; Dogs ; *Babesiosis/diagnosis/parasitology ; *Dog Diseases/diagnosis/parasitology ; *Babesia/isolation &amp; purification/genetics ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Recombinases/metabolism ; },
abstract = {Babesia vogeli is a protozoan parasite causing canine babesiosis, a tick-borne disease prevalent in tropical and subtropical regions. Its microscopic identification is challenging due to morphological similarity with other Babesia spp., and serological assays often yield inaccurate results. To address this issue, we developed a rapid, equipment-minimal diagnostic method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a (RPA/CRISPR-cas12a) for B. vogeli-specific detection. The RPA assay enables DNA amplification for both B. vogeli and Hepatozoon canis, while CRISPR/Cas12a using gRNA_Bab ensures specificity for B. vogeli, even in co-infections and other pathogens. This approach detects as few as 10[5] copies within two hours for both readout platforms such as fluorescence and lateral flow dipstick (LFD). Forty canine blood samples were detected by RPA/CRISPR-cas12a to examine its performance. Results showed high concordance with qPCR-high resolution melting (HRM) (Cohen's kappa: 0.93 for fluorescence, 0.81 for LFD), outperforming conventional PCR. The clinical sensitivity and specificity of RPA/CRISPR-cas12a were 100 % and 96.8 %, respectively and the concordance with qPCR-HRM was 97.5 %. RPA/CRISPR-cas12a for Babesia spp. detection provided a simple, rapid, and accurate method, demonstrating promise for point-of-care diagnosis of canine babesiosis in resource-limited settings. This method showed high potential as a practical diagnostic tool in veterinary clinics, with accelerated surveillance to control outbreaks of Babesia-associated canine babesiosis.},
}
@article {pmid41338219,
year = {2026},
author = {Yang, Z and Zhang, L and Jiang, X and Yang, X and Ma, K and Yoo, D and Lu, Y and Zhang, S and Chen, J and Nie, Y and Bian, X and Han, J and Fu, L and Zhang, J and Ventura, M and Zhang, G and Sun, Q and Eichler, EE and Mao, Y},
title = {Incomplete lineage sorting of segmental duplications defines the human chromosome 2 fusion site early during African great ape speciation.},
journal = {Cell genomics},
volume = {6},
number = {1},
pages = {101079},
pmid = {41338219},
issn = {2666-979X},
support = {R01 HG002385/HG/NHGRI NIH HHS/United States ; },
mesh = {Animals ; Humans ; *Hominidae/genetics ; *Segmental Duplications, Genomic/genetics ; *Chromosomes, Human, Pair 2/genetics ; *Genetic Speciation ; Chromosome Inversion/genetics ; Evolution, Molecular ; CRISPR-Cas Systems ; },
abstract = {All great apes differ karyotypically from humans due to the fusion of chromosomes 2a and 2b, resulting in human chromosome 2. Here, we show that the fusion was associated with multiple pericentric inversions, segmental duplications (SDs), and the turnover of subterminal repetitive DNA. We characterized the fusion site at the single-base-pair resolution and identified three distinct SDs that originated more than 5 million years ago. These three distinct SDs were differentially distributed among African great apes as a result of incomplete lineage sorting (ILS) and lineage-specific duplication. One of these SDs shares homology to a hypomethylated SD spacer sequence present in the subterminal heterochromatin of Pan but is completely absent subtelomerically in both humans and orangutans. CRISPR-Cas9-mediated depletion of the fusion site in human neural progenitor cells alters the expression of genes, indicating a potential regulatory consequence to this human-specific karyotypic change. Overall, this study offers insights into how complex regions subject to ILS may contribute to speciation.},
}
@article {pmid41338874,
year = {2026},
author = {Villegas, NK and Tran, MH and Keller, A and Plesa, C},
title = {BAR-CAT: Targeted Recovery of Synthetic Genes via Barcode-Directed CRISPR-dCas9 Enrichment.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {9-20},
doi = {10.1177/25731599251401526},
pmid = {41338874},
issn = {2573-1602},
mesh = {*CRISPR-Cas Systems/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Library ; *Genes, Synthetic/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Gene Editing/methods ; Plasmids/genetics ; },
abstract = {Modern gene synthesis platforms enable investigations of protein function and genome biology at an unprecedented scale. Yet, the proportion of error-free constructs in diverse gene libraries decreases with length due to the propagation of oligo synthesis errors. To rescue these error-free constructs, we developed Barcode-Assisted Retrieval CRISPR-Activated Targeting (BAR-CAT), an in vitro method that uses multiplexed dCas9-single-guide RNA (sgRNA) complexes to extract barcodes corresponding to error-free constructs. After a 15-min incubation and wash regimen, three low-bundance targets in a 300,000-member test library were enriched 600-fold, greatly reducing downstream requirements. When applied to a 384-gene DropSynth gene library, BAR-CAT enriched 12 targets up to 122-fold and revealed practical limits imposed by sgRNA competition and library complexity, which now guide ongoing protocol scaling. By eliminating laborious clone-by-clone validation and working directly on plasmid libraries, BAR-CAT provides a platform for recovering perfect synthetic genes, subsetting large libraries, and ultimately lowering the cost of functional genomics at scale.},
}
@article {pmid41339636,
year = {2025},
author = {Agnarelli, A and Buckley-Benbow, L and Ozgencil, M and Lad, M and Ampah, KK and Kalinka, A and Belan, O and Maslen, S and Skehel, MJ and Walter, D and Day, M and Bellelli, R},
title = {The genetic and biochemical basis of human leading strand synthesis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {412},
pmid = {41339636},
issn = {2041-1723},
mesh = {Humans ; *DNA Replication/genetics ; Genomic Instability ; *DNA Polymerase II/metabolism/genetics/chemistry ; Proliferating Cell Nuclear Antigen/metabolism ; DNA/biosynthesis/genetics ; Iron/metabolism ; Iron-Sulfur Proteins/metabolism ; CRISPR-Cas Systems ; },
abstract = {The maintenance of genome stability requires efficient leading strand synthesis by DNA Polymerase Epsilon (Polε). By performing CRISPR genetic screens in cells lacking the POLE4 subunit of Polε we define a genetic map of the factors required to support Polε function in the absence of its accessory subunits. A set of genes involved in iron metabolism emerge as required to sustain Iron Sulphur Cluster (ISC)-dependent Polε activity. We then dissect a synthetic lethal interaction between POLE3-POLE4 and the CHTF18-RFC2/5 complex. By combining cell biology, structural modelling and biochemistry, we define the existence of two tiers of regulation of Polε processivity: leading strand-specific loading of PCNA by CHTF18-RFC2/5 and "gripping" of newly synthesised dsDNA by POLE3-POLE4. The combined loss of these functions is incompatible with leading strand synthesis and viability. In summary, we describe the biochemical basis of human leading strand synthesis and the consequence of its dysfunction in genome stability.},
}
@article {pmid41339642,
year = {2025},
author = {Yang, WJ and Liu, BY and Xue, L},
title = {Knockout of protein arginine methyltransferase 1 inhibited cell growth and promoted cell migration in human bronchial epithelial cells.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {43069},
pmid = {41339642},
issn = {2045-2322},
support = {2018BFC360//Fund for Key Laboratory Construction of Hubei Province/ ; 31101047//National Natural Science Foundation of China/ ; CZQ22013//"the Fundamental Research Funds for the Central Universities", South-Central MinZu University/ ; PTZ24018//i Medical Biology International Science and Technology Cooperation Base/ ; },
mesh = {Humans ; *Cell Movement/genetics ; *Cell Proliferation/genetics ; *Protein-Arginine N-Methyltransferases/genetics/metabolism ; *Epithelial Cells/metabolism/cytology ; *Bronchi/cytology/metabolism ; Cell Line ; Apoptosis/genetics ; Gene Knockout Techniques ; *Repressor Proteins/genetics/metabolism ; Cell Cycle/genetics ; CRISPR-Cas Systems ; },
abstract = {Previous studies have demonstrated that PRMT1 was involved in the progression of multiple lung diseases. However, its specific function within the bronchial epithelium was still limited and needed further exploration. In the present study, human bronchial epithelial cell line 16HBE was chosen to elucidate the biological role of PRMT1 in lung epithelium. Cell proliferation, cell-cycle distribution, cell apoptosis, and cell motility capacity were systematically evaluated following CRISPR/Cas9-mediated knockout of PRMT1. We showed that knockout of PRMT1 in 16HBE inhibited cell proliferation, redistributed cell cycle, promoted cell apoptosis, and accelerated cell migration via a series of regulated cyclins, cyclin-dependent kinase regulators, and EMT markers. Taken together, these findings identify PRMT1 as a potential modulator of epithelial cell proliferation, survival, and motility in the human bronchial epithelium, offering new insights into its possible role in epithelial remodeling during pulmonary disorders.},
}
@article {pmid41340056,
year = {2025},
author = {Braun, S and Knackfuß, K and Ziesmann, T and Mlinzk, L and Goerg, A and Frankenheim, J and Walter, A and Schneider-Brachert, W and Distler, U and Fritsch, J},
title = {Loss of ADAM15 prevents necroptosis induction by partial RIPK1 degradation due to enhanced TNF-R1 surface expression and basal caspase-8 activation.},
journal = {Cell communication and signaling : CCS},
volume = {23},
number = {1},
pages = {520},
pmid = {41340056},
issn = {1478-811X},
mesh = {Humans ; *Caspase 8/metabolism ; *ADAM Proteins/metabolism/genetics/deficiency ; *Receptor-Interacting Protein Serine-Threonine Kinases/metabolism ; *Proteolysis ; *Membrane Proteins/metabolism/genetics/deficiency ; *Necroptosis ; *Receptors, Tumor Necrosis Factor, Type I/metabolism ; Enzyme Activation ; Jurkat Cells ; U937 Cells ; Signal Transduction ; },
abstract = {BACKGROUND: Cell death and survival processes must be tightly regulated to ensure proper tissue homeostasis and prevent excessive inflammation and tissue damage. Death receptors, including TNF-R1, can induce either immunogenic (necroptosis) or non-immunogenic (apoptosis) cell death and relay proliferative / cell survival signaling by activating NFκB and MAPK cascades. In a recent report, we identified the metalloproteinase ADAM15 as a possible TNF-responding enzyme, leading to the hypothesis that it regulates either cell survival or death cascades.<br><br>METHODS: CRISPR/Cas-9 was used to knock out the adam15 gene. Loss of gene expression was validated by Western blot and flow cytometry in U937 and Jurkat cells. NF&#x3ba;B, MAPK signaling, and cell death cascades were monitored by Western blot, flow cytometry, and enzyme assays. A bottom-up proteome analysis was performed to elucidate cellular processes affected by ADAM15 loss. The subcellular localization of ADAM15 was monitored by microscopy and immuno-magnetic fractionation.<br><br>RESULTS: We identified ADAM15 as a regulator of necroptosis, leaving apoptosis and cell survival signaling unaffected. Loss of ADAM15 resulted in abrogated necroptosis, as evidenced by the application of death ligands TNF, TRAIL, FasL, and TL1a, as well as the BH3 mimetic Obatoclax. We observed enhanced basal Caspase-8 activity, which was not cytotoxic, and partial RIPK1 proteolysis. The loss of ADAM15 was verified in a proteome screen, which revealed alterations in various molecular pathways, including autophagy, organelle trafficking, and sorting. We observed ADAM15 in intracellular compartments, which in part have a lysosomal protein signature. We observed enhanced surface expression of TNF-R1, proposing it as a possible ADAM15 substrate.<br><br>CONCLUSIONS: ADAM15 is a previously unknown regulator of necroptosis, likely due to its role in modulating intracellular organelle sorting processes. Its proteolytic activity and possible scaffolding capacity for recruiting adaptor molecules make it a veritable drug target. The activation or deactivation of ADAM15 may be exploited to modulate various disease conditions.},
}
@article {pmid41341502,
year = {2025},
author = {Gao, Y and Chen, J},
title = {Fast but accurate: a systematic review and meta-analysis on diagnostic performance of MRSA detection in clinical samples by using CRISPR-based rapid molecular methods.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1703247},
pmid = {41341502},
issn = {1664-302X},
abstract = {BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant global health threat due to its multidrug resistance and association with severe infections. Conventional culture methods are time-consuming, usually requiring 48-72 h to obtain results, while conventional molecular methods such as PCR or qPCR, though faster, still require trained personnel and specialized instruments, which may delay timely clinical treatment and infection control. CRISPR-based methods have emerged as promising alternative tools for MRSA detection, but their real-world performance still requires comprehensive assessment. This meta-analysis aimed to systematically evaluate the diagnostic accuracy and timeliness of CRISPR/Cas systems for MRSA detection in clinical samples.<br><br>METHODS: A systematic search of PubMed, Embase, Web of Science, and Cochrane Library was conducted using search terms related to MRSA, CRISPR/Cas, diagnostic accuracy, and rapid detection. Studies reporting sensitivity and specificity with extractable 2&#x202f;&#xd7;&#x202f;2 contingency tables were included. Quality was assessed via QUADAS-2. Meta-disc 1.4.0 and Stata 16.0 were used for statistical analysis, including pooled sensitivity, specificity, likelihood ratios, diagnostic odds ratios (DOR) and summary receiver operating characteristic (SROC). Median detection time and subgroup analyses were also conducted.<br><br>RESULTS: Twelve studies were included. The results showed that the CRISPR-based methods showed a pooled sensitivity of 99% (95% CI: 97-100%) and specificity of 100% (95% CI: 99-100%), with a PLR of 32.68 (95% CI: 15.45-69.15), NLR of 0.03 (95% CI: 0.02-0.07), and DOR of 664.25 (95% CI: 234.59-1880.84). The median detection time across included studies was 60&#x202f;min (IQR: 41.25-98.75&#x202f;min).<br><br>CONCLUSION: CRISPR-based molecular assays demonstrated exceptional accuracy and rapid detection capability for MRSA in clinical settings, significantly outperforming conventional methods. However, potential publication bias and methodological limitations warrant cautious interpretation of these results.<br><br>PROSPERO ID: CRD420251115439.},
}
@article {pmid41341583,
year = {2025},
author = {Jiang, Z and Jia, B and Hu, N and Zhang, M and Xiao, H and Chen, G and Yu, J and Li, X and Shen, B and Feng, J and Wang, J},
title = {In Vivo engineering of transgenic mice for systemic human neutralizing antibody production against staphylococcal enterotoxin B.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1679421},
pmid = {41341583},
issn = {1664-3224},
mesh = {Animals ; Mice, Transgenic ; Humans ; Mice ; *Enterotoxins/immunology ; *Antibodies, Neutralizing/immunology/genetics/biosynthesis ; Female ; CRISPR-Cas Systems ; Genetic Engineering ; Antibodies, Monoclonal/immunology/genetics ; Glycosylation ; },
abstract = {Transgenic animal bioreactors provide a complementary strategy to traditional mammalian cell culture systems for the production of therapeutic human monoclonal antibodies (mAbs). Here we present a CRISPR/Cas9-mediated breakthrough in creating two novel genetically engineered (GE) mouse models with species-specific chromosomal integration of human anti-staphylococcal enterotoxin B (SEB) mAb genes at either the ROSA26 or Hipp11 (H11) safe-harbor loci - evolutionarily conserved genomic safe harbors (GSH). These genetically optimized animals demonstrated broad tissue capability for glycosylation-competent human antibodies, achieving exceptional secretion levels reaching 208 mg/L in serum, 43 mg/L in mammary secretions, 24 mg/L in saliva on average. The transgenic lines maintained this antibody production stability for >140 weeks without compromising animal viability, while preserving germline transmission fidelity through six successive generations. Furthermore, the highly glycosylated human antibodies derived from these genetic engineered mice exhibited high binding affinity to SEB (KD=0.108 nM for ROSA26; 0.154 nM for H11), providing comprehensive protection against SEB intoxication in vivo. This study opens avenues for utilizing transgenic animal bioreactors for large-scale production of fully human antibodies or disease-resistant livestock in the foreseeable future.},
}
@article {pmid41342880,
year = {2026},
author = {Kama, Y and Hirano, KI and Masuhara, K and Endo, Y and Suzuki, Y and Fujimoto, M and Matsuda, T and Yahata, T and Kato, M and Hozumi, K and Tanaka, T and Hosokawa, H},
title = {Notch interaction with RUNX factors regulates initiation of the T-lineage program.},
journal = {The Journal of experimental medicine},
volume = {223},
number = {2},
pages = {},
pmid = {41342880},
issn = {1540-9538},
support = {JP19H03692//Japan Society for the Promotion of Science/ ; JP24K22062//Japan Society for the Promotion of Science/ ; JP24K02485//Japan Society for the Promotion of Science/ ; JP23K15307//Japan Society for the Promotion of Science/ ; //Tokai University/ ; //Chugai Foundation for Innovative Drug Discovery Science/ ; //Uehara Memorial Foundation/ ; //Naito Foundation/ ; //Takeda Science Foundation/ ; //SENSHIN Medical Research Foundation/ ; //Princess Takamatsu Cancer Research Fund/ ; //Foundation for Promotion of Cancer Research/ ; //Chemo-Sero-Therapeutic Research Institute/ ; //Vehicle Racing Commemorative Foundation/ ; //Kobayashi Foundation/ ; //Ichiro Kanehara Foundation for the Promotion of Medical Science and Medical Care/ ; //TERUMO Life Science Foundation/ ; //Mitsubishi Foundation/ ; 35-y10//Kawano Masanori Memorial Public Interest Incorporated Foundation for Promotion of Pediatrics/ ; //Tokai University School of Medicine/ ; //Kyushu University/ ; },
mesh = {*T-Lymphocytes/metabolism/physiology ; *Cell Lineage/genetics ; Gene Knock-In Techniques ; Mice, Transgenic ; *CCCTC-Binding Factor ; *Core Binding Factor Alpha 2 Subunit/metabolism ; CRISPR-Cas Systems ; *Receptor, Notch1/metabolism ; Lymphoid Progenitor Cells/metabolism/physiology ; Mediator Complex/metabolism ; E1A-Associated p300 Protein/metabolism ; Transcriptional Activation/physiology ; Protein Binding ; Protein Domains ; Male ; Female ; Animals ; Mice ; Trans-Activators ; },
abstract = {Runt-related transcription (RUNX) factors play a key role in T cell development. At the T-lineage commitment checkpoint, RUNX1 undergoes dynamic partner switching, resulting in its redeployment. Here, we investigated the functional differences in RUNX factors between the lymphoid progenitor (LP)- and Notch-stimulated earliest T progenitor stages (Phase 1). We identified CCCTC-binding factor (CTCF) as an LP-specific RUNX1-interacting partner, with LP-specific RUNX1-binding genomic sites significantly enriched for CTCF consensus motifs and co-occupied by CTCF. On Notch stimulation, Notch1 intracellular domain directly interacts with RUNX1 and recruits the RUNX1/Mediator/p300 transcriptional activation complex to Notch-regulated T-signature gene loci. CRISPR/Cas9-mediated stage-specific deletion of RUNX factors and their binding partners revealed that the RUNX1/CTCF complex in LP negatively regulates T-signature gene expression, whereas the RUNX1/Mediator/p300 complex in Phase 1 promotes it. Our findings highlight the crucial role of Notch-mediated functional conversion of RUNX factors, including protein complex reorganization and genomic redeployment in initiating T-lineage program.},
}
@article {pmid41344099,
year = {2025},
author = {Ryu, YC and Bao, G and Hwang, BH},
title = {Peptide-assisted lipofection enables efficient non-viral delivery of large CRISPR/Cas9 constructs for genome editing applications.},
journal = {Biomedicine &amp; pharmacotherapy = Biomedecine &amp; pharmacotherapie},
volume = {193},
number = {},
pages = {118838},
doi = {10.1016/j.biopha.2025.118838},
pmid = {41344099},
issn = {1950-6007},
mesh = {*Gene Editing/methods ; Humans ; *CRISPR-Cas Systems/genetics ; HEK293 Cells ; Animals ; *Transfection/methods ; Plasmids/genetics ; *Gene Transfer Techniques ; *Peptides/chemistry ; Mice ; *Lipids/chemistry ; Genetic Therapy/methods ; Cell Survival ; Genetic Vectors ; },
abstract = {Efficient and safe delivery of large genetic constructs such as CRISPR/Cas9 plasmids remains a critical bottleneck in gene therapy. In this work, the peptide-assisted lipofection (PAL) system was developed as a breakthrough non-viral vector for gene delivery, specifically designed for large plasmids including CRISPR/Cas9 constructs. This system achieved exceptional transfection efficiency up to 98.7 % in HEK293T cells, surpassing conventional delivery methods such as electroporation and standard lipofections. PAL successfully delivered large plasmids up to 29 kb while maintaining high cell viability and achieved 44.1 % indel formation efficiency in gene editing experiments. Fluorescence microscopy verified PAL's efficient endosomal escape and nuclear targeting abilities. The superior performance of the PAL is attributed to its cellular uptake and endosomal escape enhanced by transfection-assisting peptide. In vivo studies in mouse models showed sustained gene expression in liver tissue, demonstrating superior performance compared to naked plasmid delivery. These results establish PAL as a versatile and promising platform for gene therapy and genome editing, offering a safer alternative to viral vectors for large genetic payload delivery.},
}
@article {pmid41344241,
year = {2026},
author = {Peng, Y and Xu, J and Chen, B and Zeng, D and Yu, X and Chen, W},
title = {Ultrasensitive detection of lead ion in tea samples using a versatile and robust multi-DNAzyme DNA machine mediated CRISPR/Cas12a signal amplification system.},
journal = {Food chemistry},
volume = {499},
number = {},
pages = {147352},
doi = {10.1016/j.foodchem.2025.147352},
pmid = {41344241},
issn = {1873-7072},
mesh = {*DNA, Catalytic/genetics/chemistry ; *Tea/chemistry ; *Lead/analysis ; *Food Contamination/analysis ; CRISPR-Cas Systems ; *Biosensing Techniques/methods/instrumentation ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *Camellia sinensis/chemistry/genetics ; *Endodeoxyribonucleases/genetics/chemistry/metabolism ; Ions/analysis ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Lead ion (Pb[2+]) contamination in tea poses serious threats to food safety. We present a novel detection platform integrating a multifunctional DNAzyme machine with the CRISPR/Cas12a system. Upon target Pb[2+] binding, the multi-DNAzyme activates the DNA machine, initiating multiple isothermal cycles that generate a poly-A sequence. This sequence activates Cas12a, triggering trans-cleavage of the hairpin fluorescent probes and significant signal amplification. The self-sustained amplification of DNA machine eliminates the need for any auxiliary probes, greatly simplifying the assay design. This platform achieves excellent sensing perfromances with detection limit of 67.53 pM, wide linear range (0.01-100 nM), satisfied specificity and high recovery rates above 96 % in real tea samples. This integration of DNAzyme recognition with CRISPR/Cas12a signal enhancement provides a rapid, and cost-effective method for Pb[2+] detection. This strategy offers practical potential for food safety monitoring and environmental assessment, and future research may expand its applicability to other heavy metal contaminants.},
}
@article {pmid41344285,
year = {2026},
author = {Dasanayaka, BP and Pathirana, SL and Jayawardana, A and Handunnetti, SM and Fernando, N and Galhena, BP and Weerasena, SJ and Nitsche, A and Iddamaldeniya, SS and Dietzsch, AK},
title = {A roadmap in detecting frequently reported bovine babesiosis: From blood smear to CRISPR.},
journal = {Veterinary parasitology},
volume = {342},
number = {},
pages = {110662},
doi = {10.1016/j.vetpar.2025.110662},
pmid = {41344285},
issn = {1873-2550},
mesh = {Animals ; Cattle ; *Babesiosis/diagnosis/parasitology/blood ; *Cattle Diseases/diagnosis/parasitology/blood ; *CRISPR-Cas Systems ; *Babesia/isolation &amp; purification/genetics ; Babesia bovis/isolation &amp; purification/genetics ; Real-Time Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity ; },
abstract = {Current diagnosis of Babesia bovis and B. bigemina relies on direct microscopy, nucleic acid detection, and serology. Light-microscopic analysis of Giemsa-stained smears still serves as the primary diagnostic modality at the point of care. However, carrier cattle, particularly those harbouring B. bovis, often carry parasites at levels far below the detection threshold, and such levels are sensitive only to DNA-based detection approaches. Early probe-hybridization techniques have been largely replaced by conventional Polymerase Chain Reaction (PCR), nested formats, and real-time quantitative PCR (qPCR)-which enable species-specific discrimination within closed-tube systems, thereby minimizing contamination risk. Species‑level identification is essential for clinical management, surveillance, and experimental studies. Duplex TaqMan qPCRs simultaneously distinguish B. bovis from B. bigemina, surpassing the analytical sensitivity offered by nested PCR (nPCR) methodologies. Reverse-line-blot (RLB) hybridisation broadens the diagnostic scope by concurrently detecting co-infections of B. bovis and B. bigemina and mixed haemoparasitic species in one workflow. However, the analytical sensitivity of RLB remains inferior to that of qPCR in detecting low-density and carrier-state infections of B. bigemina. RLB remains useful for retrospective genotyping when amplification is impractical or fails. Field-ready isothermal approaches have expanded the scope of molecular diagnostics beyond laboratory settings, facilitating field-level application and rapid on-site detection. Coupling Loop-mediated isothermal amplification (LAMP) with a lateral-flow dipstick (LFD) (LAMP-LFD) enables pen-side direct visual detection. Antibody tests are essential tools for herd-level surveillance. A recent chimeric Enzyme-Linked Immunosorbent Assay (ELISA) that combines three immunodominant B. bovis antigens broaden strain coverage and boosts diagnostic reliability. Recombinase polymerase amplification coupled to CRISPR-Cas12a cleavage has achieved single-target detection of B. bigemina from tick salivary-gland DNA, paving the way for innovative pen-side platforms, once cost and technical hurdles are overcome. Importantly, vector-based detection using appropriate tissues bearing an optimum level of ticks is species-dependent. B. bigemina sporozoites concentrate in nymph/adult salivary glands, whereas B. bovis sporozoites are produced mainly in larval salivary glands, so monitoring programs should stratify sampling accordingly.},
}
@article {pmid41344296,
year = {2025},
author = {Landi, E and Zondag, R and Dehnen, JA and Albert, S and Dickman, MM and LaPointe, VLS and van Bokhoven, H},
title = {Biallelic excision of the CTG18.1 expansion in two Fuchs endothelial corneal dystrophy-derived iPSC lines and one control (SCTCi046-A-1, SCTCi047-A-1 and SCTCi041-A-1) using an episomal vector-based CRISPR/Cas9 approach.},
journal = {Stem cell research},
volume = {89},
number = {},
pages = {103881},
doi = {10.1016/j.scr.2025.103881},
pmid = {41344296},
issn = {1876-7753},
mesh = {Humans ; *Fuchs' Endothelial Dystrophy/genetics/pathology/metabolism ; *Induced Pluripotent Stem Cells/metabolism/pathology ; *CRISPR-Cas Systems/genetics ; *Trinucleotide Repeat Expansion/genetics ; Cell Line ; Transcription Factor 4/genetics ; Alleles ; Plasmids/genetics ; },
abstract = {An expanded CTG repeat in intron 2 of the transcription factor 4 (TCF4) gene is the main cause of Fuchs endothelial corneal dystrophy (FECD), a complex corneal disease. The prevailing paradigm is that the expanded repeat exerts toxic effects, resulting in corneal endothelium degeneration. Here we explored the use of CRISPR/Cas9-mediated, non-homologous end-joining (NHEJ) for disease-modeling purposes, by performing a biallelic excision of the CTG18.1 expansion in two FECD- and one control-derived induced pluripotent stem cell lines (iPSCs). The three Δ/Δ CTG18.1 lines generated by this study provide a platform to investigate the CTG18.1 contribution to FECD pathogenesis.},
}
@article {pmid41344324,
year = {2026},
author = {Djamshidi, M and Hill, A and Heshmatzad, K and Langley, J and Krowicki, H and Ali, M and Yang, Y and Tanida, R and Abdul-Careem, MF and Billon, P and Riabowol, K},
title = {FAME-CRISPR improves CRISPR-Cas9 genome editing via HDAC inhibition and engineered virus-like particle delivery.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101248},
pmid = {41344324},
issn = {2667-2375},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Histone Deacetylase Inhibitors/pharmacology ; *Virion/genetics ; DNA Breaks, Double-Stranded ; HEK293 Cells ; },
abstract = {CRISPR-mediated gene editing using engineered virus-like particles (eVLPs) can achieve high efficiency, but performance varies with reduced effectiveness often seen in primary cells or when generating polyclonal models at scale. We developed a faster, accurate and 4-fold more efficient CRISPR-Cas9 (FAME-CRISPR) method using pan-histone deacetylase inhibitors with eVLP transduction compared to previous reports using other histone deacetylase inhibitors. Combined optimization of pan-HDACi treatment with eVLP enhanced double-strand break (DSB)-mediated CRISPR and base editing gave significantly edited populations within 2- to 3-cell mean population doublings, reducing the need for post-editing selection in immortalized cancer cells and in primary diploid fibroblasts that have limited replicative lifespans.},
}
@article {pmid41344457,
year = {2026},
author = {Liu, L and Huang, X and Wan, S and Gao, Z and Liu, H},
title = {Ectodysplasin A regulates the development of scale and intermuscular bone in teleosts.},
journal = {International journal of biological macromolecules},
volume = {337},
number = {Pt 2},
pages = {149465},
doi = {10.1016/j.ijbiomac.2025.149465},
pmid = {41344457},
issn = {1879-0003},
mesh = {Animals ; Zebrafish/genetics/growth &amp; development ; *Ectodysplasins/genetics/metabolism ; *Fishes/genetics/growth &amp; development ; *Bone and Bones/metabolism ; Phylogeny ; *Animal Scales/growth &amp; development/metabolism ; Signal Transduction ; Gene Expression Regulation, Developmental ; *Fish Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Most bony fish are extensively covered by scales, which play crucial roles in locomotion, balance, and sensory perception. However, the molecular mechanisms underlying fish scales development remain poorly understood. In this study, comparative genomic analyses were performed between scaled and scaleless fish species, leading to the identification of key genes (eda, wnt3a, gsk3bb, etc.) involved in scale formation. Phylogenetic and sequence analyses of the eda gene revealed that the Eda protein in Monopterus albus lacks a transmembrane domain, disrupting Eda/Edar binding and potentially driving scale degeneration. Using CRISPR/Cas9 technology, we generated zebrafish eda[-/-] mutants, which exhibited a complete absence of scales, fin rays, pharyngeal teeth, and gill rakers. Notably, the intermuscular bones in these mutants showed significantly reduced length, and simplified morphology, indicating impaired growth. Furthermore, quantitative PCR (qPCR) analysis demonstrated that eda deficiency disrupts the Eda/Edar/NF-κB signaling pathway. Our findings provide significant insights into the molecular regulatory mechanisms underlying the development of skin appendages (e.g., scales) and intermuscular bones.},
}
@article {pmid41344486,
year = {2026},
author = {Huang, Y and Li, L and Do, CW and Luo, Q and Zheng, Z and Xiong, W},
title = {Lipid nanoparticle-mediated CRISPR/Cas9 delivery enables efficient trabecular meshwork gene editing in mice.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {389},
number = {},
pages = {114499},
doi = {10.1016/j.jconrel.2025.114499},
pmid = {41344486},
issn = {1873-4995},
mesh = {Animals ; *Trabecular Meshwork/metabolism ; *Gene Editing/methods ; *Nanoparticles/administration &amp; dosage/chemistry ; *CRISPR-Cas Systems ; *Lipids/chemistry/administration &amp; dosage ; Mice, Inbred C57BL ; Mice ; RNA, Messenger/administration &amp; dosage/genetics ; *Gene Transfer Techniques ; Humans ; Calcium-Binding Proteins/genetics ; Green Fluorescent Proteins/genetics ; Extracellular Matrix Proteins/genetics ; Male ; CRISPR-Associated Protein 9/genetics ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) enable efficient mRNA delivery, yet their potential for ocular gene editing remains largely unexplored. Here, we systematically evaluated three LNP formulations containing distinct ionizable lipids, DLin-MC3-DMA, ALC0315, and SM102, for gene delivery to ocular tissues. Among them, SM102-based LNP encapsulating GFP mRNA (SM102-GFP) exhibited the highest transfection efficiency across three cultured ocular cells in vitro. Following intravitreal injection in mice, SM102-GFP achieved selective and robust expression in the trabecular meshwork (TM) without detectable retinal transfection. GFP expression in TM peaked at one week post-injection, declined by three weeks, and could be effectively re-induced by a second dosing of the same vector. Compared with adeno-associated viral (AAV) and adenoviral (Ad) vectors, SM102-GFP showed superior TM specificity and reduced retinal inflammation. Co-delivery of SpCas9 mRNA and sgRNA via SM102-based LNPs enabled efficient CRISPR-mediated knockout of Matrix Gla Protein (Mgp), a key inhibitor of TM calcification. Mgp knockout induced sustained intraocular pressure elevation and anterior chamber deepening with open angles, recapitulating features of primary open-angle glaucoma. Chronic ocular hypertension further led to Müller gliosis and ganglion cell complex thinning, indicative of progressive retinal stress. These findings establish SM102-based LNPs as a safe and efficient platform for TM-targeted gene editing and glaucoma modeling.},
}
@article {pmid41344769,
year = {2026},
author = {Liu, X and Zheng, Y and Chen, Z and Wang, S and Liao, H and Jia, J and Wang, G and Wang, J and Yuan, C and Guo, X and Yin, Y and Hu, Q},
title = {Rapid and visual detection of Listeria monocytogenes by combining one-pot LAMP-CRISPR/Cas12b with lateral flow assay.},
journal = {Food microbiology},
volume = {135},
number = {},
pages = {104977},
doi = {10.1016/j.fm.2025.104977},
pmid = {41344769},
issn = {1095-9998},
mesh = {*Listeria monocytogenes/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Animals ; Food Contamination/analysis ; Food Microbiology/methods ; Swine ; *CRISPR-Cas Systems ; *Molecular Diagnostic Techniques/methods ; Sensitivity and Specificity ; Limit of Detection ; },
abstract = {Listeria monocytogenes, the leading cause of fatalities worldwide among foodborne pathogens, poses serious risks to food safety and public health. Therefore, a rapid and accurate detection method is crucial for early interception and effective management. In this study, a one-pot LAMP-CRISPR/Cas12b detection system based on the lmo0753 gene was developed for rapid detection of L. monocytogenes by combining loop-mediated isothermal amplification (LAMP) with a CRISPR/Cas12b assay. Further integration of a lateral flow assay (LFA) to develop a LAMP-CRISPR/Cas12b-LFA assay enabled direct detection of the results on the strips with the naked eye. Nine L. monocytogenes strains belonging to eight serotypes tested positive with both the one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays. Two assays did not show cross-reactivity with L. innocua and eight other foodborne bacteria. The limits of detection were 10 CFU/mL for pure culture and 20 CFU/g for spiked pork samples. Moreover, the enrichment time was substantially shortened to 3 h for pork samples spiked with only L. monocytogenes F2365, and 4-5 h for pork samples spiked with mixed bacteria. In addition, with one-pot LAMP-CRISPR/Cas12b detection, 5 of 66 fresh pork samples, 1 of 20 ready-to-eat food samples, and 2 of 24 raw milk samples tested positive for L. monocytogenes, in agreement with the results obtained through a culture based standard method. Thus, this study established one-pot LAMP-CRISPR/Cas12b and LAMP-CRISPR/Cas12b-LFA assays for rapid, visual detection of L. monocytogenes in food samples.},
}
@article {pmid41345100,
year = {2025},
author = {Bai, M and Zhang, J and Lin, W and Zhou, Y and Jiang, M and Wu, H and Peng, C and Lin, J and He, F and Kuang, H and Guan, Y},
title = {A flanking-nicks prime editor (FLICK-PE) system to boost prime editing in dicots.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {337},
pmid = {41345100},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *Glycine max/genetics/drug effects ; *Nicotiana/genetics ; Plants, Genetically Modified/genetics ; Glyphosate ; Glycine/analogs &amp; derivatives/pharmacology ; CRISPR-Cas Systems/genetics ; Herbicide Resistance/genetics ; 3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {Prime editing (PE) enables precise genome modifications to mammalian cells and monocot staple crops, but remains relatively challenging in dicot plants. Here, we develop a Flanking-Nicks Prime Editor (FLICK-PE) system that boosts editing efficiency in soybean and tobacco. We show that optimization for PE by adding a nicking sgRNA could dramatically enhance intended-editing efficiency in soybean. Inspired by this observation, we design a FLICK-PE strategy to confer a pair of nicks flanking the target site. In soybean, FLICK-PE achieves on average a 15.7-fold increase in intended-editing efficiency compared to PE2, and a 2.2-fold increase compared to PE3. Using FLICK-PE, we efficiently engineer glyphosate resistance in soybean by introducing TAP-IVS mutations in EPSPS1a, achieving three amino-acid substitutions and an intended editing efficiency of 21.1%. This approach yields stable edited soybean varieties with vigorous glyphosate tolerance and minimal growth penalties in a field trial. FLICK-PE also demonstrates efficacy in tobacco, underscoring its broad applicability and versatility for rapid, precision breeding in agriculturally vital crops.},
}
@article {pmid41345278,
year = {2026},
author = {de la Rosa, C and Kendirli, A and Baygün, S and Bauernschmitt, F and Thomann, AS and Kisioglu, I and Beckmann, D and Carpentier Solorio, Y and Pfaffenstaller, V and Tai, YH and Mehraein, N and Sanchez, P and Spieth, L and Gerdes, LA and Beltran, E and Dornmair, K and Simons, M and Peters, A and Schmidt-Supprian, M and Kerschensteiner, M},
title = {In vivo CRISPR screen reveals regulation of macrophage states in neuroinflammation.},
journal = {Nature neuroscience},
volume = {29},
number = {2},
pages = {493-509},
pmid = {41345278},
issn = {1546-1726},
support = {259373024//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 408885537//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 239283807//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {Animals ; *Macrophages/metabolism ; Mice ; Cytokines/metabolism/genetics ; Mice, Inbred C57BL ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *Multiple Sclerosis/immunology/genetics ; Humans ; *Neuroinflammatory Diseases/genetics ; CRISPR-Cas Systems ; *Encephalomyelitis, Autoimmune, Experimental/immunology ; },
abstract = {Here we established an in vivo CRISPR screening pipeline using genetically editable progenitor cells to dissect macrophage regulation in mouse models of multiple sclerosis (MS). Screening over 100 cytokine receptors and signaling molecules identified interferon-γ, tumor necrosis factor, granulocyte-macrophage colony-stimulating factor and transforming growth factor-β as essential regulators of macrophage polarization in vivo. Single-cell transcriptomics confirmed that transferred progenitor cells generate all blood-derived CNS myeloid cell populations, enabling Perturb-seq analysis of cytokine actions in neuroinflammation. Combined with biosensor expression, our approach allows monitoring cytokine effects on myeloid cell migration, debris phagocytosis and oxidative activity in vivo. Comparative transcriptomic analyses revealed conserved neuroinflammatory cytokine signatures across myeloid populations, CNS compartments and species, elucidating cytokine cues shaping myeloid function in the cerebrospinal fluid and parenchyma of individuals with MS. This versatile pipeline thus provides a scalable framework for high-resolution analysis of macrophage states and uncovers the cytokine signals that underlie their regulation in MS and MS models.},
}
@article {pmid41345283,
year = {2025},
author = {Saydam, S and Dinçer, P},
title = {Precision rewriting of muscle genetics: therapeutic horizons of base and prime editing in skeletal muscle disorders.},
journal = {Gene therapy},
volume = {},
number = {},
pages = {},
pmid = {41345283},
issn = {1476-5462},
abstract = {Base Editing (BE) and Prime Editing (PE), novel precision tools of the CRISPR/Cas toolbox, have emerged as transformative technologies that enable highly specific genetic modifications. Their compatibility with post-mitotic cell types makes them invaluable for treating genetic skeletal muscle disorders. Despite their severity and progressive nature, monogenic muscle diseases remain without definitive treatments. They are caused by diverse mutations in critical muscle proteins, for which gene editing offers a promising therapeutic avenue. However, traditional CRISPR/Cas9 applications face challenges such as genotoxicity and inefficiency in post-mitotic tissues. BE and PE technologies overcome these limitations by enabling safe and efficient modifications without causing double-strand breaks or requiring homology-directed repair. Their therapeutic potential comes from two key features: their ability to work in non-dividing cells such as myotubes and cardiomyocytes, and their capacity to target a broad range of mutations found in genetic muscle diseases. In this review, we explore mechanisms of BE and PE and summarize their current applications in monogenic skeletal muscle disorders. We discuss the challenges of in vivo application in skeletal muscle and highlight innovations to bypass them. Collectively, both systems offer flexible precision solutions with immense potential for mutation-specific and personalized gene therapy approaches for monogenic skeletal muscle disorders.},
}
@article {pmid41346237,
year = {2025},
author = {Pandit, B and Hanson, E and Dagci, H and Yang, Q and Yigit, MV and Royzen, M},
title = {Effects of N[6]-Methyladenosine (m[6]A) and 5-Methylcytosine (m[5]C) Modifications in the Guide Region of CRISPR RNA on Cas12a Nuclease Activity.},
journal = {Bioconjugate chemistry},
volume = {36},
number = {12},
pages = {2551-2556},
pmid = {41346237},
issn = {1520-4812},
support = {R21 HG012257/HG/NHGRI NIH HHS/United States ; R35 GM156250/GM/NIGMS NIH HHS/United States ; },
mesh = {*5-Methylcytosine/chemistry/metabolism ; *Adenosine/analogs &amp; derivatives/chemistry/metabolism ; *RNA, Guide, CRISPR-Cas Systems/chemistry/metabolism/genetics ; *CRISPR-Associated Proteins/metabolism/chemistry ; *CRISPR-Cas Systems ; *Endodeoxyribonucleases/metabolism ; *Bacterial Proteins/metabolism ; },
abstract = {CRISPR-Cas12a is a versatile biosensing platform that detects sequence-specific DNA or RNA targets via a CRISPR RNA (crRNA) guide. While Cas12a's specificity is dictated by its crRNA, chemical modifications within the crRNA can influence nuclease performance. Here, we examined the effects of two well-known RNA modifications, N[6]-methyladenosine (m[6]A) and 5-methylcytosine (m[5]C), introduced into the different positions of the guide region of a crRNA. Melting temperature (Tm) analysis showed that m[6]A had a minimal impact on RNA-DNA duplex stability. In contrast, the incorporation of m[5]C residues stabilized the duplex. Using a fluorescence recovery assay, we found that both modifications preserved Cas12a's nuclease activity, indicating that small thermodynamic shifts in duplex formation are insufficient to disrupt its catalytic function. Despite the greater Tm increase with m[5]C, m[6]A incorporation led to a faster fluorescence recovery rate than that with m[5]C.},
}
@article {pmid41346247,
year = {2026},
author = {Singh, V and Mishra, M and Singla-Pareek, SL and Roy, JK and Pareek, A},
title = {Lysine Matters: Genetic and Biotechnological Innovations to Combat Protein Malnutrition.},
journal = {Plant, cell &amp; environment},
volume = {49},
number = {3},
pages = {1509-1529},
doi = {10.1111/pce.70316},
pmid = {41346247},
issn = {1365-3040},
support = {//This study was supported by Department of Biotechnology, Ministry of Science and Technology, India./ ; },
mesh = {*Lysine/metabolism/deficiency ; Crops, Agricultural/genetics/metabolism ; *Biotechnology/methods ; Gene Editing ; Metabolic Engineering ; Plants, Genetically Modified ; Plant Breeding ; *Protein Deficiency/prevention &amp; control ; CRISPR-Cas Systems ; Biofortification ; },
abstract = {Lysine deficiency in staple crops like maize, rice, and wheat remains a major cause for global protein malnutrition, underscoring the urgent need for effective biofortification strategies. This review critically examines recent advances in enhancing lysine content, spanning conventional breeding and metabolic engineering to cutting-edge precision genome editing. While conventional breeding, exemplified by Quality Protein Maize, has improved lysine levels, it is often constrained by yield and quality trade-offs. Metabolic engineering strategies, including overexpression of lysine biosynthetic genes, suppression of catabolic genes, and modification of storage proteins, have achieved substantial lysine enrichment but face regulatory and consumer acceptance challenges due to their transgenic nature. The advent of CRISPR/Cas technology now enables precise, transgene-free editing of key enzymes such as DHDPS, AK, and LKR/SDH offering a powerful alternative, though concerns regarding off-target effects and pleiotropy remain. While integrating multi-omics with AI-driven predictive modelling can optimise metabolic flux for higher lysine yield, coupling next-generation genome editing with speed breeding offers a transformative route to develop high-lysine, high-yielding crops for sustainable nutritional security.},
}
@article {pmid41346702,
year = {2026},
author = {Patra, C and Hussein, Z and Ace, VD and Misnik, EV and Rybalko, DS and Salimova, AA and Ereshko, DS and Dubovichenko, MV and Nour, MAY and Drozd, VS and Kolpashchikov, DM},
title = {The efficacy of oligonucleotide-based gene therapeutics in gene silencing.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {599-616},
pmid = {41346702},
issn = {1838-7640},
mesh = {Humans ; *Genetic Therapy/methods ; *Gene Silencing ; *Oligonucleotides, Antisense/therapeutic use/genetics ; RNA, Small Interfering/genetics/therapeutic use ; Animals ; *Oligonucleotides/genetics/therapeutic use ; },
abstract = {Oligonucleotide-based gene therapeutics (OGTs) have emerged as a promising strategy for treating a variety of diseases, offering a tool for gene modulation at the mRNA level. Despite significant progress in OGTs development, their efficacy in both experimental and clinical settings has often fallen short of expectations. Current estimates suggest that less than 1% of transfected OGTs are released into the cytosol, significantly limiting the interaction with target RNA. Moreover, data suggests that only about 2% of the tested siRNAs achieve the expected 70% target gene knockdown in vitro. Clinically approved OGTs appear to be effective only against genetic disorders that lack effective alternative treatment, and even in these cases their therapeutic contribution remains marginal. Notably, the majority of approved OGTs, as well as those currently in clinical trials, are antisense oligonucleotides (ASOs) despite cell culture data showing that small interfering RNAs (siRNAs) exhibit greater potency. The delayed commercialization of siRNAs, despite high research interest, may be attributed to passenger stand-dependent off target effect and the immaturity of their design and modification strategies. This review critically evaluates the factors influencing therapeutic efficacy of OGTs and highlights the persistent gap between theoretical promise and clinical reality.},
}
@article {pmid41347244,
year = {2025},
author = {Daraghmeh, DN and AbuIriban, RW and Nawawreh, N and Abuamro, AM and Alassar, MM and Daraghma, SN and Alhajahmed, NM and Thandar, Y},
title = {Advancements in alternative approaches to address antimicrobial resistance in bacterial pneumonia: a comprehensive review.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1704931},
pmid = {41347244},
issn = {1664-302X},
abstract = {PURPOSE: This review explores both current and emerging alternative treatment approaches to combat AMR specifically in the context of bacterial pneumonia, highlighting therapies that extend beyond conventional antibiotics.<br><br>METHODS: PubMed, Embase, and Google Scholar were searched for full-text, English-language articles, with emphasis on publications from 2020 to 2025. Earlier seminal studies were also included when necessary to provide historical, mechanistic, or conceptual context. The review focuses was on alternative strategies that have shown effectiveness in preclinical or clinical settings to combat AMR in relation to bacterial pneumonia.<br><br>RESULTS: Emerging strategies to tackle AMR in bacterial pneumonia involve several innovative approaches including stem cells, bacteriophage therapy, metal based nanoparticles (e.g., silver, copper, and gold). The adjunctive use of probiotics and herbal medicine has demonstrated potential in enhancing clinical outcomes and modulating host immunity. Moreover, gene editing technologies like CRISPR-CAS and various vaccination programs are being investigated for their roles in prevention and resistance management. While these methods show promise, many are still in the early stages of development and encounter challenges related to standardization, safety, and regulatory approval.<br><br>CONCLUSION: Alternative therapies present exciting possibilities for addressing AMR in bacterial pneumonia. However, to effectively translate these innovations into clinical practice, we need thorough research, international collaboration, and supportive policy frameworks. By combining these strategies with antimicrobial stewardship initiatives, we can help maintain antibiotic effectiveness and enhance patient outcomes.},
}
@article {pmid41348151,
year = {2025},
author = {Cheng, Y and Gao, W and Shi, S and Han, F and Dong, H},
title = {Identification of the orange pigment in Nonomuraea gerenzanensis and development of a pigment-free mutant.},
journal = {AMB Express},
volume = {16},
number = {1},
pages = {4},
pmid = {41348151},
issn = {2191-0855},
support = {2024TSGC0896//Shandong Province Science and Technology-based Small and Medium-sized Enterprises Innovation Capacity Enhancement Project/ ; },
abstract = {The secondary metabolite A40926, a precursor to the glycopeptide antibiotic dalbavancin, is synthesized by the rare actinomycete Nonomuraea gerenzanensis (N. gerenzanensis) within the pharmaceutical industry. The biosynthesis of A40926 is accompanied by the production of an orange pigment, which poses significant challenges and incurs high costs in the purification process of A40926. To identify this orange pigment, a comprehensive analysis was conducted, including the examination of the biosynthetic gene cluster, potential biosynthetic pathways, purification processes, and structural identification. Additionally, the ispF gene, which encodes the enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase and is implicated in the biosynthesis of orange pigment, was deleted using the CRISPR/Cas9 system. To enhance A40926 production in the ΔIspF mutant, the overexpression of the cyclic AMP receptor protein (Crp) was implemented to assess its regulatory impact on A40926 biosynthesis. Consequently, the orange pigment produced by N. gerenzanensis was identified as lycopene, synthesized via the methylerythritol phosphate (MEP) pathway. Although the ΔIspF mutant was unable to biosynthesize the orange pigment, its production of A40926 was adversely affected and was lower than that of the original strain. Consequently, the overexpression of the global regulator Crp significantly enhanced A40926 production, achieving a yield of 841.1 mg/L. The investigation of pigment-free mutants presented in this study offers valuable insights for effectively reducing production costs within the microbial pharmaceutical industry.},
}
@article {pmid41348871,
year = {2025},
author = {Puppala, AK and Nielsen, AC and Regan, M and Mancinelli, GE and De Pooter, RF and Arnovitz, S and Harding, C and McGregor, M and Balanis, NG and Clarke, R and Merrill, BJ},
title = {Programmable multistep CRISPR gene activation via control of RNA polymerase III termination.},
journal = {Science advances},
volume = {11},
number = {49},
pages = {eadt1532},
pmid = {41348871},
issn = {2375-2548},
support = {R01 GM139894/GM/NIGMS NIH HHS/United States ; },
mesh = {Humans ; *RNA Polymerase III/metabolism/genetics ; *CRISPR-Cas Systems ; *Transcriptional Activation ; Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; },
abstract = {Although genomes encode instructions for mammalian cell differentiation with rich syntactic relationships, existing methods for genetically programming cells have only modest capabilities for stepwise gene regulation. Here, we develop a sequential genetic system that transcriptionally activates endogenous genes in a preprogrammed, stepwise manner. This system uses the removal of an RNA polymerase III termination sequence to trigger both the transcriptional activation and DNA endonuclease activities of a Cas9-VPR protein, driving progression through a cascade of gene activation events. The system's functionality in human cells, including iPSCs, enables the development of a path for cellular programming by controlling the sequential order of gene activation to influence cellular states.},
}
@article {pmid41349356,
year = {2025},
author = {Shahid, N and Hammond, JR},
title = {Characterization of genetically modified human embryonic kidney 293 cells lacking equilibrative nucleoside transporter subtype 2, or both subtypes 1 and 2, and the impact of their loss on sensitivity to chemotherapeutic purine/pyrimidine analogs.},
journal = {Drug metabolism and disposition: the biological fate of chemicals},
volume = {53},
number = {12},
pages = {100203},
pmid = {41349356},
issn = {1521-009X},
mesh = {Humans ; HEK293 Cells ; *Equilibrative Nucleoside Transporter 1/genetics/metabolism ; *Equilibrative-Nucleoside Transporter 2/genetics/metabolism ; CRISPR-Cas Systems ; *Purines/pharmacology/metabolism ; *Pyrimidines/pharmacology/metabolism ; *Antineoplastic Agents/pharmacology ; Biological Transport ; Gene Knockout Techniques ; },
abstract = {Equilibrative nucleoside transporters (ENTs) 1 and 2 are considered critical to the cellular uptake of purine and pyrimidine analogs used to treat cancer and viral infections. However, a detailed understanding of the discrete and overlapping roles of these ENT subtypes in drug activity remains limited. A significant barrier to progress has been the absence of model systems that enable functional characterization of individual nucleoside transporters in the context of their native environment. To address this, we developed and characterized a panel of CRISPR/cas9-engineered human embryonic kidney 293 cell lines with selective deletion of ENT subtypes: ENT1 knockout, ENT2 knockout, and dual knockout. These models were used to dissect subtype-specific roles of ENT1 and ENT2 in nucleoside/nucleobase analog uptake and cytotoxicity. Our data show that ENT1 and ENT2 in their endogenous environment have a similar affinity for a range of both endogenous and chemotherapeutic nucleoside and nucleobase analogs. Deletion of ENT1 generally enhanced the sensitivity of cells to these drugs, particularly the nucleobase analogs, likely due to reduced nucleoside salvage by the cells via ENT1. Deletion of ENT2, on the other hand, dramatically reduced the ability of a number of the tested drugs to impact cell viability, by mechanisms beyond those related to reduced cellular uptake of the drugs. This study highlights distinctive roles of ENT1 and ENT2 in the actions of nucleoside/nucleobase analog drugs. SIGNIFICANCE STATEMENT: A panel of genetically modified human embryonic kidney 293 cells has been created as a model to screen novel nucleoside transporter inhibitors and substrates. Using these cell lines, it was revealed that ENT2 may play a more functionally significant role in nucleoside analog chemotherapeutic drug activity than previously appreciated.},
}
@article {pmid41349512,
year = {2025},
author = {Kosaka, Y and Lopez, B and Kishimoto, N and Jacob, S and Montenont, E and Huallanca, R and Coughenour, G and Di Paola, J and Ross, J and Lee, K and Rondina, MT and Bray, PF and Rowley, JW},
title = {Functional classification of platelet gene variants using CRISPR HDR in CD34[+] cell-derived megakaryocytes.},
journal = {American journal of human genetics},
volume = {112},
number = {12},
pages = {2888-2901},
pmid = {41349512},
issn = {1537-6605},
support = {R01 HL166805/HL/NHLBI NIH HHS/United States ; R01 HL142804/HL/NHLBI NIH HHS/United States ; R01 HL139825/HL/NHLBI NIH HHS/United States ; I01 CX001696/CX/CSRD VA/United States ; K24 HL155856/HL/NHLBI NIH HHS/United States ; R01 HL144957/HL/NHLBI NIH HHS/United States ; U54 DK106829/DK/NIDDK NIH HHS/United States ; },
mesh = {Humans ; *Megakaryocytes/metabolism ; *Blood Platelets/metabolism ; *CRISPR-Cas Systems/genetics ; *Antigens, CD34/metabolism/genetics ; Integrin beta3/genetics ; Gene Editing/methods ; Integrin alpha2/genetics ; *Genetic Variation ; Hematopoietic Stem Cells/metabolism ; Thrombasthenia/genetics ; },
abstract = {The interpretation of genetic variants in inherited diseases, such as inherited platelet disorders (IPDs), remains a major clinical challenge, as most are classified as variants of uncertain significance (VUSs). A key barrier to functional evaluation is the lack of accessible, lineage-appropriate assays that reliably reflect native gene regulation and cell-specific biology. To address this gap, we developed CRIMSON HD (CRISPR-edited megakaryocytes [MKs] for surveying platelet variant functions through homology-directed repair [HDR]), a CRISPR-Cas9 HDR-based genome-editing platform applicable to CD34[+] cell-derived blood lineages and optimized for evaluating platelet-associated variants. Using this system, we modeled known and candidate disease-associated variants in integrin alpha 2b (ITGA2B) and integrin beta 3 (ITGB3), which encode the platelet αIIb/β3 integrin and are causative in Glanzmann thrombasthenia (GT). We introduced precise variants into primary human MKs derived from CD34[+] hematopoietic stem and progenitor cells, achieving >90% editing efficiency. Edited MKs faithfully recapitulated both expression and functional phenotypes of known type I, II, and III GT variants. CRIMSON HD enabled functional evaluation and reclassification of several GT VUSs, including αIIb Gly201Ala, a population variant now shown to cause near-complete loss of αIIb/β3 expression; αIIb Ala777Asp, which results in intermediate αIIb/β3 expression and impaired agonist-induced integrin binding; and β3 Arg119Gln, previously linked to the loss of anti-HPA1a antibody binding in fetal and neonatal alloimmune thrombocytopenia (FNAIT), now shown to impair integrin surface expression. These findings demonstrate the importance of lineage-specific, physiologically relevant assays for the functional classification of platelet-related variants, providing mechanistic information and clinically meaningful insights for individuals with IPDs.},
}
@article {pmid41349515,
year = {2025},
author = {Kim, I and Suh, JY},
title = {Capture first, then deliver!.},
journal = {Structure (London, England : 1993)},
volume = {33},
number = {12},
pages = {2008-2009},
doi = {10.1016/j.str.2025.11.001},
pmid = {41349515},
issn = {1878-4186},
mesh = {*CRISPR-Cas Systems ; *CRISPR-Associated Proteins/chemistry/metabolism ; DNA/metabolism/chemistry ; *Integrases/chemistry/metabolism ; Protein Binding ; },
abstract = {In this issue of Structure, Henriques et al.[1] present structural snapshots that capture distinct conformational states of the type I-F Cas1-Cas2/3 integrase complex, illustrating that foreign DNA binding triggers a large-scale domain rearrangement that enables prespacer delivery to the CRISPR array.},
}
@article {pmid41350682,
year = {2025},
author = {Lyu, G and Li, P and Lang, W},
title = {A review of recent studies on CRISPR/Cas9-mediated genome editing in a variety of muscle-related genetic disorders.},
journal = {Journal of translational medicine},
volume = {23},
number = {1},
pages = {1381},
pmid = {41350682},
issn = {1479-5876},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing ; Animals ; *Muscular Diseases/genetics/therapy ; *Genetic Diseases, Inborn/genetics ; Genetic Therapy ; Muscular Dystrophies/genetics ; },
abstract = {The human body is capable of mutating a single gene to produce a wide range of debilitating disorders. Genomic editing for disease prevention via phenotypic reversal was a significant challenge prior to the development of clustered regulatory interspaced short tandem repeats (CRISPR) and CRISPR-associated protein (Cas) systems. Gene therapy-editing a patient's DNA to correct a particular mutation-and treating human diseases that have not responded to conventional medicine are two areas where CRISPR/Cas9 technology shows the most promise as a therapeutic tool. This powerful instrument has shown great promise in muscle-related illnesses, offering new insights into muscle biology and developing more effective treatment techniques. Discoveries about the hereditary causes of the majority of inherited myopathies and muscular dystrophies (MDs) have emerged over the last two decades. Additionally, skeletal muscles weaken and degenerate over time due to a group of hereditary disorders known as MDs. The field of skeletal muscle diseases and associated genetic alterations is seeing remarkable progress in developing therapeutic vectors to fix these mutations. Myopathies, MDs, and neuromuscular disorders are just a few examples of the many genetic abnormalities related to muscles that have sparked renewed interest in the potential of genome editing as a therapeutic tool due to its efficiency, adaptability, and relative ease of use in targeted genome editing. Consequently, CRISPR/Cas9 has garnered much interest and is used more often in therapeutic techniques due to its potential capacity to cure various human ailments. To pave the way for more effective and personalized therapies, this review article provides a thorough overview of the revolutionary role of CRISPR/Cas9 in improving our understanding and treatment of genetic disorders related to muscles by combining present knowledge with future perspectives.},
}
@article {pmid41351274,
year = {2025},
author = {Hou, H and Li, Y and Su, N and Ding, Y and Shang, C and Li, X and Xiong, Z and Sun, Y and Zhan, W and Wang, Y and Zhang, X and Pan, Y and Wu, L and Li, J},
title = {Slmsh1-induced heritable enhancement of traits for tomato breeding improvement.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {5},
pages = {e70607},
doi = {10.1111/tpj.70607},
pmid = {41351274},
issn = {1365-313X},
support = {CSTB2023TIAD-KPX0026//Special Key Project of Technological Innovation and Application Development of Chongqing/ ; 31872123//National Natural Science Foundation of China/ ; 32172597//National Natural Science Foundation of China/ ; CARS-23-B08//China Agriculture Research System/ ; SWU-KF25027//Fundamental Research Funds for the Central Universities/ ; },
mesh = {*Solanum lycopersicum/genetics/physiology/growth &amp; development ; *Plant Breeding/methods ; *Plant Proteins/genetics/metabolism ; Fruit/genetics/growth &amp; development ; Droughts ; Quantitative Trait, Heritable ; Phenotype ; CRISPR-Cas Systems ; Gene Expression Regulation, Plant ; },
abstract = {Vegetable grafting is a horticultural technique employed to develop specialized plant varieties by effectively enhancing resistance to both biotic and abiotic stresses, as well as improving fruit quality and yield. However, these advantageous traits are generally non-heritable. The MSH1 gene induced heritable enhancement-through-grafting (HEG) effect on growth vigor, demonstrating promising application potential. In this study, we employed the msh1 mutant tomato as a rootstock to induce heritable superior traits and combined this approach with hybridization techniques to enhance tomato cultivars. Three Slmsh1 mutants were generated using CRISPR/Cas9 which exhibited a dwarf phenotype with whitened spots. By grafting several distinct inbred lines onto Slmsh1, we observed significant HEG, drought stress tolerance, and fruit quality. Under drought conditions, Slmsh1-grafted tomato seedlings exhibited increased biomass and enhanced drought tolerance through the regulation of antioxidant enzyme activities. Differential expression and methylation analyses of the graft progeny revealed that these heritable enhanced traits (HETs) are likely attributable to epigenetic modifications in the expression of ROS-scavenging- and hormone-related genes. Furthermore, to explore practical applications, we crossed inbred lines with HETs and evaluated the growth, yield, and fruit quality of the resulting hybrid combinations. The results indicated that these hybrid combinations improved fruit yield and quality, enhancing the total soluble solids, soluble sugar, and soluble protein content. These findings suggest that Slmsh1-grafted progenies enhanced plant biomass and drought resistance, while their hybrid combinations positively influenced root growth, yield, and fruit quality, providing new insights into the synergistic integration of genome editing and conventional breeding.},
}
@article {pmid41352420,
year = {2026},
author = {Tang, M and Liang, R and Wu, Z and Chen, C and He, B and Zhou, N and Wang, S and Xiao, X and Li, G and Jiang, Y and Gong, G and Zhou, Y},
title = {Deciphering OCT4A-dose-dependent transcriptional profiles associated with tumorigenic potential in somatic cancer cells.},
journal = {SLAS technology},
volume = {36},
number = {},
pages = {100381},
doi = {10.1016/j.slast.2025.100381},
pmid = {41352420},
issn = {2472-6311},
mesh = {*Octamer Transcription Factor-3/genetics/metabolism ; Humans ; *Neoplasms/genetics/pathology ; Cell Line, Tumor ; *Carcinogenesis/genetics ; Gene Expression Regulation, Neoplastic ; Gene Expression Profiling ; *Transcriptome ; CRISPR-Cas Systems ; Gene Regulatory Networks ; Prognosis ; },
abstract = {AIMS: The transcription factor OCT4A, a well-established master pluripotency factor, exerts regulatory effects on cell fate determination that are closely associated with its protein levels. This study aims to uncover the downstream gene profile features relevant to tumorigenic potential mediated by OCT4A under varying protein abundance in somatic cancer cells (SCCs).<br><br>MATERIALS AND METHODS: CRISPR-Cas9-mediated knockout and doxycycline-inducible OCT4A expression systems were established in cervical (HeLa) and hepatocellular (HepG2, Huh7) cancer cells. Single-cell sequencing, spatial transcriptomic and survival analysis data were used to elucidate the expression pattern of OCT4 in somatic cancer tissues and its prognostic relevance. The plate colony formation assay was performed to assess the tumorigenic capacity of SCCs, and Bulk RNA sequencing coupled with weighted gene co-expression network analysis (WGCNA) identified dose-relevant downstream pathways. Functional enrichment, survival modeling, and RT-qPCR validation were used to construct OCT4A-dose-dependent transcriptional regulatory networks.<br><br>KEY FINDINGS: OCT4 transcript, is heterogeneously present and confined to a small subset of tumor cells within somatic cancer tissues, with a significantly higher proportion of OCT4-positive cells in tumor tissues compared to paired paraneoplastic tissues and is significantly correlated with poor prognosis in SCCs. Endogenous low-level OCT4A positively regulates tumorigenic capacity predominantly through targeting non-coding genes, whereas high-level OCT4A suppresses tumorigenic capacity primarily via protein-coding genes in SCCs. A prognostic model based on high-level OCT4A-regulated protein-coding genes was associated with favorable clinical outcomes, aligning with in vitro phenotypic results.<br><br>SIGNIFICANCE: Our findings further confirm in SCCs that the functional pleiotropy of OCT4A is closely linked to its protein abundance, and further systematically elucidate the molecular signatures of OCT4A-regulated downstream gene networks associated with tumorigenic phenotypes at differential protein levels, providing novel insights for its translational exploitation in both oncological intervention and regenerative medicine strategies.},
}
@article {pmid41352695,
year = {2026},
author = {Pradhan, K and Anoop, S},
title = {CRISPR 2.0: Expanding the genome engineering Toolbox for epigenetics, RNA editing, and molecular diagnostics.},
journal = {Gene},
volume = {979},
number = {},
pages = {149938},
doi = {10.1016/j.gene.2025.149938},
pmid = {41352695},
issn = {1879-0038},
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Epigenesis, Genetic ; *RNA Editing/genetics ; *Pathology, Molecular/methods ; Epigenomics/methods ; Animals ; Genetic Engineering/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {Non-canonical CRISPR systems adaptation has led to genome editing through nucleases, and the development of transcriptional and epigenetic regulation, transcriptome editing, and molecular diagnostics has resulted in a diversified set of tools-CRISPR 2.0. In this review, the author summarizes the mechanisms and recent engineering advances of (i) dCas9-based epigenetic effectors, (ii) RNA-targeting Cas13 systems and engineered RNA editors, (iii) DNA base editors and prime editors, and (iv) CRISPR-powered diagnostic platforms and their translational readiness. There is a critical comparison of the various approaches (e.g., RNAi/ASO versus Cas13-based methods; base editing versus prime editing) along with practical translational considerations such as delivery technologies, safety (off-target/edit windows, mosaicism), and regulatory pathways which are evaluated. Three concise case studies refer to map laboratory evidence to clinical or near-clinical outcomes and the ethical and governance discussion is widened to include global access, intellectual property and equity in deployment. Finally, the authors classify technologies according to their level of readiness - diagnostics and some ex-vivo therapeutic approaches are already in or very close to clinical use, chosen in-vivo editing methods are undergoing early trials, and AI-assisted nuclease design is still mostly theoretical but is getting better fast. This comprehensive viewpoint is intended to help researchers and physicians understand which CRISPR tools are most likely to be translated soon and where more validation is required.},
}
@article {pmid41352906,
year = {2026},
author = {Fakhr, ZA and Xie, W and Zeng, S and Cai, S},
title = {Site accessibility-driven CRISPR/Cas13a activation for amplification-free RNA biosensing.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344858},
doi = {10.1016/j.aca.2025.344858},
pmid = {41352906},
issn = {1873-4324},
mesh = {*Biosensing Techniques/methods ; *CRISPR-Cas Systems ; *RNA/analysis ; *RNA, Guide, CRISPR-Cas Systems/metabolism/genetics/chemistry ; Kinetics ; *CRISPR-Associated Proteins/metabolism ; },
abstract = {BACKGROUND: CRISPR-Cas13a biosensing enables rapid, amplification-free RNA diagnostics, yet assay sensitivity varies widely because guide RNAs (gRNAs) differ in their ability to activate the enzyme. Two factors, including the gRNA-target binding affinity and the structural accessibility of the target site, have been proposed to govern activation efficiency, but their relative importance remains unclear. In this study, we systematically disentangle these contributions by measuring binding affinities for gRNAs that span a spectrum of site accessibilities and by comparing their Michaelis-Menten kinetic parameters.<br><br>RESULTS: Three ciRS-7-specific gRNAs were designed with high, intermediate, and low spacer accessibility. Isothermal titration calorimetry (ITC) quantified site accessibility through entropy changes (&#x394;S&#xa0;=&#xa0;-862, -813, and -615&#xa0;cal/mol/K), confirming greater structural exposure for less structured spacers, and also determined binding affinity for each gRNA-target pair. Michaelis-Menten analysis showed kcat values of 1.39, 1.31, and 1.16 s[-1] for the high, intermediate, and low-accessibility guides, respectively, establishing a clear relationship between structural accessibility and catalytic turnover. Importantly, the most structured gRNA exhibited lower activation efficiency compared with the gRNA that had higher site accessibility and lower binding affinity, demonstrating that site accessibility drives Cas13a activation. Detection-limit experiments also confirmed these results, showing that gRNAs with greater spacer accessibility yielded stronger signals and superior sensitivity.<br><br>SIGNIFICANCE: Our data establish site accessibility as a critical determinant of Cas13a activation for amplification-free RNA sensing. Prioritizing unstructured spacer regions enables improved enzyme activation efficiency, providing a clear design rule for next-generation CRISPR diagnostics. This accessibility-driven strategy will facilitate the development of faster, simpler, and more sensitive point-of-care assays for diverse RNA biomarkers.},
}
@article {pmid41352908,
year = {2026},
author = {Li, L and Tang, Z and Xu, H and Zhou, F and Ji, X and He, Z},
title = {Investigation on CRISPR-Cas12a-split crRNA system for successively detecting DNA and RNA in one tube.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344860},
doi = {10.1016/j.aca.2025.344860},
pmid = {41352908},
issn = {1873-4324},
mesh = {*CRISPR-Cas Systems ; *DNA, Viral/analysis/genetics ; *RNA, Viral/analysis/genetics ; Hepatitis B virus/genetics ; *CRISPR-Associated Proteins/metabolism/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; HIV/genetics ; },
abstract = {Recently, CRISPR/Cas system has been proposed as a novel tool with simplicity and high accuracy. The CRISPR RNA (crRNA) can be divided into spacer crRNA and handle crRNA without losing its original function. In this work, we have investigated CRISPR Cas12a with split crRNA to detect HBV DNA and HIV RNA in a single tube. In the first step, Cas12a can recognize HBV DNA and initiate its trans-cleavage on FAM-BHQ1 reporter, after 1 h incubation, the fluorescence intensity was correlated with the concentration of HBV DNA. In the second step, the TAMRA BHQ2 ds DNA reporter was introduced in the same tube to bind with remained Cas12a proteins, HIV RNA and handle crRNA. The trans-cleavage from the first step would not interfere with HIV RNA and dsDNA reporter. With the incubation for another hour, HIV RNA can be quantified by the cis-cleavage of TAMRA BHQ2 reporter. we can successively identify the two nucleic acids with the limit of detection of 0.70 pM for HBV DNA, and 0.47 nM for HIV RNA, respectively. This special designed split crRNA can simplify detecting procedure and only need Cas12a protein in a single tube. Next, we expand this strategy in semi-quantifying two kinds of DNA in one tube. Overall, this study overcomes the limitation of conventional CRISPR-based methods and provides a new, inexpensive, and low-threshold approach based on Cas12a with split crRNA.},
}
@article {pmid41352919,
year = {2026},
author = {Guan, X and Wang, S and Wang, P and Zhang, J and Sun, S},
title = {Enhanced chemiluminescence aptasensing with triple cascade amplification for sensitive detection of tumor-derived exosomes.},
journal = {Analytica chimica acta},
volume = {1383},
number = {},
pages = {344873},
doi = {10.1016/j.aca.2025.344873},
pmid = {41352919},
issn = {1873-4324},
mesh = {*Exosomes/chemistry/metabolism ; Humans ; *Aptamers, Nucleotide/chemistry/metabolism ; *Luminescent Measurements/methods ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Limit of Detection ; Mucin-1 ; Alkaline Phosphatase/chemistry/metabolism ; Nucleic Acid Amplification Techniques ; CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Biomarkers, Tumor ; Tetraspanin 30 ; },
abstract = {BACKGROUND: Tumor-associated exosomes hold significant clinical promise as liquid biopsy biomarkers. However, the accurate detection of these rare exosome subpopulations in clinical samples demands analytical platforms with exceptionally high sensitivity and specificity. While conventional nucleic acid amplification-based methods provide considerable detection sensitivity, they are often hampered by time-consuming procedures, operational complexity, and susceptibility to contamination. Therefore, it is imperative to develop practical exosome measurement platforms that combine high sensitivity, robustness, and rapid analysis capabilities to provide reliable evidence-based support for precision oncology.<br><br>RESULTS: In this work, a triple cascade-amplified aptasensor (TCAA) via functionalized gold nanoparticle (fAuNP), CRISPR/Cas12a, and alkaline phosphatase (ALP) was developed for enhanced chemiluminescence (CL) assay of tumor-derived exosomes without nucleic acid amplification. The target exosomes were initially recognized by CD63 and MUC1 aptamers. fAuNP-conjugated Trigger sequences then activated CRISPR/Cas12a to cleave single-stranded DNA and release ALP. Consequently, the ALP catalyzed substrate to produce CL signals correlating with the concentration of the analyte. By simultaneously integrating the signal amplification capabilities of multiple techniques, this TCAA achieved a limit of detection of 44 particles/&#x3bc;L for MUC1-positive exosomes within 60&#xa0;min with excellent robustness. Compared with the single- and dual-amplification methods, the sensitivity was increased by 40-fold and 6-fold, respectively. Clinical trials showed that the area under the curve of this approach was 0.96, which was higher than that of the commercialized chemiluminescence immunoassay and effectively distinguished breast cancer-derived specimens.<br><br>SIGNIFICANCE: These findings indicate that the TCAA strategy provides a highly sensitive, rapid, and robust tool for the detection of low-abundance tumor exosome subpopulations without nucleic acid amplification. It effectively addresses the limitations of conventional methods and demonstrates high clinical utility. This work offers a reliable and practical platform for non-invasive liquid biopsy, holding great potential for trace-level detection of diverse biomarkers.},
}
@article {pmid41353207,
year = {2025},
author = {Sun, H and Teng, Q and Liu, W and Guo, R and Li, M and Xiong, W and Huang, Q and Yu, Q and Luo, N and Li, Y and Song, J and Gong, S and Shi, X and Yi, C and Liu, K},
title = {CRISPR-free RNA base editing mediated PTC-readthrough restores hearing in mice with Otof nonsense mutation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {413},
pmid = {41353207},
issn = {2041-1723},
support = {no. 81770997//National Natural Science Foundation of China (National Science Foundation of China)/ ; no. 82460223//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; *Codon, Nonsense/genetics ; Mice ; *Deafness/genetics/therapy ; *Genetic Therapy/methods ; Disease Models, Animal ; *Membrane Proteins/genetics/metabolism ; *RNA Editing/genetics ; *Hearing/genetics ; Humans ; Cochlea/metabolism ; Gene Editing ; CRISPR-Cas Systems ; Male ; Female ; Mice, Inbred C57BL ; },
abstract = {The gene therapy achieved by AAV-mediated otoferlin-overexpression is an effective therapeutic strategy for congenital deafness. However, achieving its physiological and endogenous patterns of expression remains challenging. Here, we generate the homologous mutation Otof c.1315 C > T (p.R439*), equivalent to OTOF c.1273 C > T (p.R425*) found in humans with profound deafness, to create a nonsense mutation-induced deaf mouse model. We then deliver the 'RESTART v3' system, which is a CRISPR-free RNA base editor for nonsense mutation suppression, into the cochlea of the mice. We achieve physiological otoferlin expression, and the edited premature termination codon is reverse-mutated to the original amino acid. We observe significant hearing restoration and enhancement of the behavioral auditory startle reflex. Thus, our study presents a successful RNA editing strategy to significantly restore hereditary deafness in mice carrying the specific Otof nonsense mutation, which holds great promise for future clinical translation.},
}
@article {pmid41353342,
year = {2025},
author = {He, L and Yao, Y and You, Y and Wei, X and Ma, Y and Yuan, W and Lang, Z and Zhu, JK},
title = {Versatile molecular tools enabling customizable DNA methylation editing in Arabidopsis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {251},
pmid = {41353342},
issn = {2041-1723},
support = {32188102//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100458//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Arabidopsis/genetics ; *DNA Methylation/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Genome, Plant ; },
abstract = {Tools to edit DNA methylation in a targeted manner are vital for establishing causal relationships between DNA methylation and its function, as well as for plant breeding and gene therapy. Here, by constructing dCas9 fusions to a panel of effectors and cofactors, we develop a range of highly effective tools for editing DNA methylation in Arabidopsis, including five tools for DNA methylation and six tools for DNA demethylation. Our tools show a diversity of performance features in terms of specificity and efficiency, offering either the capacity to edit DNA methylation in a target-specific manner or the ability to edit DNA methylation genome-wide due to potent off-target effect. Importantly, DNA methylation edited by these tools is inherited in the absence of transgene. These versatile tools pave the way for diverse applications of DNA methylation editing in not only research but also epigenetic breeding of crops.},
}
@article {pmid41353404,
year = {2025},
author = {Burgold, T and Karakoc, E and Gonçalves, E and Barrio-Hernandez, I and Dwane, L and Silva, R and Souster, E and Sharma, M and Beck, A and Koh, GCC and Zalmas, LP and Garnett, MJ and Bassett, AR},
title = {A next-generation dual guide CRISPR system for genetic interaction library screening.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {561},
pmid = {41353404},
issn = {2041-1723},
support = {/WT_/Wellcome Trust/United Kingdom ; 220540/Z/20/A//Wellcome Trust (Wellcome)/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Library ; Cell Line, Tumor ; Streptococcus pyogenes/genetics ; Colorectal Neoplasms/genetics ; CRISPR-Associated Protein 9/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Transfer/genetics ; },
abstract = {Pairwise perturbation of gene function using the CRISPR/Cas9 system has potential in screening for genetic interactions and synthetic lethal gene pairs to identify combination therapies for cancer. However, existing dual guide expression systems are cumbersome to clone, often result in a large proportion of undesired guide pairs and have an imbalance of guide expression from the two positions. Here, we demonstrate a next-generation system for dual guide delivery based around a tRNA spacer that allows a single-step cloning strategy, as little as 2% of undesired guide pairs, and highly balanced expression of the two guides. This system allows efficient library-scale screening for hundreds of thousands of genetic interactions using the well-understood Streptococcus pyogenes Cas9 (SpCas9) system. We use this to screen a 100,136 guide pair library in colorectal cancer cells and successfully identify synthetic lethal genetic interactions between paralogs or other known interacting genes, establishing our method for performing efficient large-scale genetic interaction screens. This system is versatile and could be used with most guide RNA vector systems, and for other uses of paired guide delivery, such as improving single gene knockout efficiency or improving guide detection in single cell or optical CRISPR screens.},
}
@article {pmid41353974,
year = {2026},
author = {Madny, MA and Yadav, KS},
title = {Biomimetic oral drug delivery: Translating nature's design into therapeutic innovation.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {259},
number = {},
pages = {115348},
doi = {10.1016/j.colsurfb.2025.115348},
pmid = {41353974},
issn = {1873-4367},
mesh = {Humans ; Administration, Oral ; *Drug Delivery Systems/methods ; *Biomimetic Materials/chemistry ; *Biomimetics/methods ; Animals ; Nanoparticles/chemistry ; },
abstract = {Oral drug delivery, the most patient friendly administration route offers convenience and compliance but faces formidable biological barriers. Enzymatic degradation, mucosal entrapment, efflux transport and extensive first-pass metabolism drastically reduce the effectiveness of sensitive therapeutics including peptides, proteins, nucleic acids and vaccines. Conventional formulations often fail to overcome these challenges highlighting the need for innovative approaches. Biomimetic drug delivery has emerged as a transformative strategy. By emulating structures and functions from cells, membranes, exosomes, viruses and gut microbiota these systems achieve immune evasion, mucus penetration, site-specific targeting and stimulus-responsive release. Such approaches improve formulation stability and in vivo absorption but also promise precise and patient centric therapies. This review provides a comprehensive overview of biomimetic oral systems highlighting their mechanisms, design principles and translational potential. Recent advances include cell membrane-coated nanoparticles for tumor targeting and immune modulation, exosome-inspired carriers for protein and RNA transport, virus-like particles (VLPs) for oral vaccines, and mucoadhesive or mucus-penetrating polymers modeled on pathogen strategies. Complementary pH, enzyme and redox-responsive platforms exploit gastrointestinal (GI) microenvironments to ensure controlled release. Emerging tools such as bioinspired computational modeling, 3D/4D printing, organoid-on-chip models and CRISPR/Cas-based platforms accelerate optimization and clinical translation. Although most technologies remain in preclinical development, early findings demonstrate superior pharmacokinetics, therapeutic efficacy, and safety over conventional systems. This article critically examines biomimetic oral drug delivery addressing advances and underlying mechanisms including regulatory considerations and future directions. They stand poised to form the foundation of next-generation precision therapeutics.},
}
@article {pmid41354630,
year = {2026},
author = {Wang, W and Chen, K and Wang, Z},
title = {Genome-Wide CRISPR Screen Reveals PIK3CA Inhibition Enhances Lipid Nanoparticle-Mediated siRNA Delivery.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {11},
pages = {e17617},
pmid = {41354630},
issn = {2198-3844},
support = {//International Society for Advancement of Cytometry/ ; //Chan Zuckerberg Biohub/ ; //Chan Zuckerberg Initiative/ ; },
mesh = {Humans ; *RNA, Small Interfering/genetics/administration &amp; dosage ; *Nanoparticles/chemistry ; *Class I Phosphatidylinositol 3-Kinases/genetics/antagonists &amp; inhibitors/metabolism ; Animals ; Mice ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Lipids/chemistry ; CRISPR-Cas Systems/genetics ; Liposomes ; },
abstract = {Lipid nanoparticles (LNPs) are useful carriers for therapeutic siRNA delivery, yet their clinical efficacy remains constrained by insufficient cellular uptake. Here, using a genome-wide CRISPR knockout screen, multiple genetic modulators of LNP uptake is uncovered, with PIK3CA emerging as a top druggable target. Pharmacologic inhibition of PIK3CA with BAY1082439 - a clinically evaluated small molecule - significantly enhances LNP uptake, siRNA delivery, and gene silencing across diverse epithelial cancer cell lines in vitro. Co-administration of BAY1082439 with siRNA-loaded LNPs also better suppressed tumor growth and reduced liver inflammation in vivo, respectively. These findings establish PIK3CA inhibition as a broadly applicable strategy to boost LNP-mediated RNA interference and highlight the promise of combining functional genomics with nanomaterials to advance RNA-based therapeutics.},
}
@article {pmid41354953,
year = {2025},
author = {Nguyen, VT and Van, BTT and Uyen, TN and Tong, NX and Pham, TL and Vy, NHT and Thuy, DT and Thuy, NP and Kobayashi, M},
title = {Functional divergence of zebrafish keap1 paralogs revealed by CRISPR/Cas9-mediated gene editing: a specialized role for keap1b in inflammation.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {53},
pmid = {41354953},
issn = {1573-9368},
support = {108.06-2020.19//National Foundation for Science and Technology Development/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Zebrafish Proteins/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; Oxidative Stress/genetics ; *Inflammation/genetics ; Kelch-Like ECH-Associated Protein 1/genetics ; NF-E2-Related Factor 2/genetics ; Signal Transduction/genetics ; Gene Editing ; Carrier Proteins ; },
abstract = {The Keap1/Nrf2 signaling pathway is a master regulator of cellular defense against oxidative and electrophilic stress. In teleosts like zebrafish (Danio rerio), whole-genome duplication resulted in two keap1 paralogs, keap1a and keap1b, whose functional specificities remain incompletely understood. This study investigates the divergent roles of these paralogs by comparing the responses of established keap1a and novel keap1b knockout larvae to distinct chemical stressors. By comparing the responses of keap1b[dl40], keap1a[dl07], and nfe2l2a[dl703] (Nrf2a) larvae to these stressors, we uncovered a striking functional dichotomy. While loss of either paralog conferred resistance to H2O2-induced oxidative stress, keap1b[dl40] larvae, unlike their keap1a[dl07] counterparts, exhibited extreme sensitivity to the lethal effects of CuSO4 exposure, with survival rates plummeting to ~ 25%. This heightened sensitivity to copper sulfate was associated with a blunted transcriptional response of inflammatory markers tnf-a and c3a, suggesting that Keap1b is critical for modulating the Nrf2a-mediated response to inflammatory stress in orchestrating a viable inflammatory response. This work clarifies the non-redundant, vital function of Keap1b in the response to heavy metal-induced stress and provides a valuable genetic resource (keap1b[dl40] null allele) for future studies.},
}
@article {pmid41354981,
year = {2025},
author = {Das, T and Barman, T and Prasad, A},
title = {Precision editing to improve fruit traits: CRISPR/Cas into the picture.},
journal = {Protoplasma},
volume = {},
number = {},
pages = {},
pmid = {41354981},
issn = {1615-6102},
abstract = {Crop growth, quality, and yield can be adversely affected by various biotic and abiotic stresses. Crop characteristics can be improved with conventional breeding and other variation-based breeding strategies. However, these strategies are time as well as resource consuming and to overcome this, novel approaches are necessary. CRISPR/Cas technique allows to improve desired traits more efficiently and accurately by targeting specific genes. Genome editing has become more versatile with CRISPR/Cas systems and is a valuable tool to protect food security by developing commercial crops optimized for yield and nutritional quality. Researchers are able to target and edit stress response pathway genes to develop crops with increased tolerance to stress. A lack of regeneration protocols and sufficient genome sequencing data has restricted fruit editing to only a few fruits (tomatoes, citrus, apple, kiwi, banana, grapes, strawberries, watermelon, etc.). This review is focused on CRISPR/Cas applications on the nutritional aspects of fruit engineering along with the challenges and opportunities. Another aspect which will be covered is the use of CRISPR/Cas technology to improve fruit resilience to biotic and abiotic stress, but not at the cost of yield. We discuss the pros and cons of using this technology, such as unintended effects on fruit traits or public concerns about GMOs. We conclude that the application of CRISPR/Cas9 technology has the potential to be of great benefit to the agricultural industry not only to improve nutritional aspects but also to help reduce crop losses.},
}
@article {pmid41355773,
year = {2026},
author = {Tian, S and Yao, L and Gong, F and Li, Y and Zhao, Y and Yang, Y},
title = {Rapid and sensitive detection of circulating tumor DNA via a CRISPR/Cas12a-based catalytic hairpin assembly.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {1},
pages = {115-123},
doi = {10.1039/d5ay01624j},
pmid = {41355773},
issn = {1759-9679},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/blood/genetics ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; Biomarkers, Tumor/blood/genetics ; Biosensing Techniques/methods ; Neoplasms/blood/diagnosis/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Cancer is one of the major diseases that endanger the human health. Circulating tumor DNA (ctDNA) is an ideal biomarker for the real-time monitoring of cancer. In the present work, a rapid and sensitive assay coupled with CRISPR/Cas12a and CHA (Cas12a-CHA) was constructed for the detection of ctDNA. We designed and prepared a trigger, which was the substrate of Cas12a. On the addition of ctDNA, crRNA-guided ctDNA activated the trans-endonuclease activity of Cas12a. After being activated, Cas12a exhibited a high trans-cleavage activity on the trigger, which resulted in a decrease in fluorescence. Owing to this design, the Cas12a-CHA assay enabled the sensitive detection of ctDNA with a linear range of 10 fM to 50 pM. Furthermore, a limit-of-detection of 5.8 fM was achieved within 40 min. Besides, the proposed assay had an excellent base mismatch recognition ability and worked well in human serum samples. Conclusively, this detection platform holds significant potential for application in early cancer diagnosis.},
}
@article {pmid41356190,
year = {2026},
author = {Zhong, XY and Yang, YX and Xiong, YF and Ye, GC and Gong, X and Zhong, ML and He, HD and Wang, SG and Xia, QD},
title = {Programmable molecular microscopy: CRISPR/Cas fluorescent probes revolutionizing spatiotemporal genomic imaging.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1877-1904},
pmid = {41356190},
issn = {1838-7640},
mesh = {*CRISPR-Cas Systems/genetics ; *Fluorescent Dyes ; Humans ; Animals ; *Molecular Imaging/methods ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Bioimaging technologies visually resolve spatiotemporal dynamics of biomolecules, cells, and tissues, enabling essential insights into gene regulation, disease mechanisms, and drug metabolism. CRISPR/Cas-based fluorescent probes transform CRISPR from "genetic scissors" into "molecular microscopes," providing an indispensable tool for in situ decoding of molecular events in living systems. Their high nucleic acid specificity establishes CRISPR/Cas as a pivotal technology for dynamically monitoring genomic and transcriptomic events at live-cell and in vivo levels. This work systematically outlines design strategies and functional mechanisms of mainstream CRISPR/Cas fluorescent probes for bioimaging, encompassing five categories: fluorescent proteins, synthetic dyes, smart gated probes, nanomaterials, and multimodal integrated probes. Recent advances and persistent challenges in achieving high-sensitivity targeted imaging, effective signal amplification, and precise delivery control are comprehensively examined, including analysis of their advantages, limitations, and adaptability in complex biological environments. Building on breakthroughs in in vivo delivery systems, diverse carriers demonstrate significant potential for enhancing CRISPR/Cas transport efficiency, improving tissue penetration, and enabling spatiotemporal controlled release. Continued innovation drives CRISPR/Cas imaging platforms toward higher sensitivity, enhanced biocompatibility, and multifunctional integration, thereby fostering the convergence and broad application of gene editing and molecular diagnostics.},
}
@article {pmid41356196,
year = {2026},
author = {Zhang, Y and Deng, Q and Xu, Y and Wu, W and Wu, T and Huang, J and Hu, Y and Lin, W and Xu, X and Wu, J},
title = {ROS-responsive cellular vesicles with ferroptosis-targeting siACMSD delivery for acute kidney injury therapy.},
journal = {Theranostics},
volume = {16},
number = {4},
pages = {1941-1958},
pmid = {41356196},
issn = {1838-7640},
mesh = {*Ferroptosis/drug effects ; Animals ; *Reactive Oxygen Species/metabolism ; *Acute Kidney Injury/therapy/metabolism/drug therapy/pathology ; Mice ; Humans ; Cisplatin/pharmacology ; *Carboxy-Lyases/genetics/metabolism ; Disease Models, Animal ; Cell Line ; Male ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Mitochondria/metabolism/drug effects ; },
abstract = {Background: Acute kidney injury (AKI) is a severe and prevalent nephrotic syndrome which lack of definitive therapies. Alpha-amino-β-carboxymuconic acid-ε-semialdehyde decarboxylase (ACMSD) is a metabolic enzyme mainly expressed in the kidney which exacerbated AKI injury by promoting TCA cycle and inhibiting nicotinamide adenine dinucleotide (NAD[+]) production, whereas lack of effective intervention strategies for ACMSD-targeted therapy. Methods: Herein, we knocked out ACMSD in vitro through CRISPR-Cas9 method, and developed a reactive oxygen species (ROS)-responsive neutrophil-derived cellular vesicles (CVs) drugs (RNAi@ROS-CVs), which efficiently mediated ACMSD knockdown in vivo, exploring the mechanism of ACMSD-induced ferroptosis process in AKI. Results: ACMSD knockout effectively alleviated cisplatin (CP)-induced mitochondrial damage, suppressed TCA cycle progression, promoted NAD[+] synthesis, and inhibited ferroptosis in HK2 cells. In mice AKI model, RNAi@ROS-CVs effectively targeted the injured kidneys, downregulated ACMSD expression in renal tubular epithelial cells, reduced ROS production and lipid peroxidation, and alleviated CP or ischemia/reperfusion (I/R)-induced ferroptosis. Conclusion: These findings highlight the therapeutic potential of ACMSD-targeted knockout in AKI intervention and introduce a versatile and efficient controlled-release drug delivery platform for AKI-targeted therapy, with potential applicability to other acute renal diseases.},
}
@article {pmid41356473,
year = {2025},
author = {Wei, C and Chen, Z},
title = {Comprehensive analysis of phage genomes from diverse environments reveals their diversity, potential applications, and interactions with hosts and other phages.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1686402},
pmid = {41356473},
issn = {1664-302X},
abstract = {Phages are ubiquitous and diverse, playing a key role in maintaining microbial ecosystem balance. However, their diversity, potential applications, and their interactions with hosts and other phages remain largely unexplored. To address this, we collected 59,652,008 putative viral genomes from our laboratory, 45 public viral datasets, and an integrated public viral genome database (IGN), covering seven habitats. We obtained 741,692 phage genomes with completeness ≥50% (PGD50), and most (93.83%, 695,938/741,692) of these phage genomes were classified into the Caudoviricetes class. We found that 158,522 species-level viral clusters that contained 28.96% (214,814/741,692) phage genomes without any known phage genomes in the IGN, indicating substantial novelty. Global phylogenetic trees for five iterations based on complete phage genomes significantly expanded the known diversity of the virosphere. Genome analysis revealed phage potential divergence with habitat types and highlighted the utilization of alternative genetic codes. Furthermore, 3D structural similarity searches demonstrated significant potential for annotating previously uncharacterized viral proteins. Analysis of CRISPR spacer inferred potential hosts of phages and competitive networks among phages, highlighting virulent phages as promising candidates for phage therapy against pathogenic bacteria. Intriguingly, diverse CRISPR-Cas systems were detected within phage genomes themselves, suggesting their enormous potential as novel gene editing tools. Collectively, this study provides a comprehensive phage genome resource, foundational for future research into phage-host and phage-phage interactions, phage therapy development, and the mining of next-generation genetic tools.},
}
@article {pmid41356798,
year = {2026},
author = {Birappa, G and Perumalsamy, H and Hong, SH and Gowda, DAA and Chandrasekaran, AP and Karapurkar, JK and Rajkumar, S and Balusamy, SR and Jayachandran, A and Baek, KH and Lee, J and Matam, V and Kim, WJ and Kim, KS and Ramakrishna, S and Suresh, B},
title = {Single-cell RNA sequence analysis reveals USP32 as a therapeutic target to mitigate PD-L1-driven colorectal tumorigenesis in vitro and in vivo.},
journal = {Theranostics},
volume = {16},
number = {2},
pages = {986-1005},
pmid = {41356798},
issn = {1838-7640},
mesh = {Humans ; Animals ; *B7-H1 Antigen/metabolism/genetics ; *Colorectal Neoplasms/genetics/pathology/metabolism ; Mice ; *Ubiquitin Thiolesterase/genetics/metabolism ; Single-Cell Analysis/methods ; Ubiquitination ; *Carcinogenesis/genetics ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Sequence Analysis, RNA ; },
abstract = {Background: The expression levels of the programmed death-ligand 1 (PD-L1) protein serves as a prognostic indicator for patients with colorectal cancer (CRC). Advancement of CRC is facilitated by deubiquitinating enzymes (DUBs), which regulate oncoprotein levels via the ubiquitin-proteasomal pathway. The post-translational regulatory mechanisms governing PD-L1 protein abundance on CRC, in relation to different tumor grades and their clinical relevance, remains unknown. Methods: We analyzed single-cell RNA sequencing (scRNA-seq) data to identify DUB genes associated with PD-L1 expression in CRC. We used a loss-of-function-based CRISPR/Cas9 library to identify putative DUB genes that regulate the PD-L1 protein level. Immunoprecipitation was used to confirm the interaction between the USP32 and PD-L1 along with its ubiquitination status. A series of in vitro and in vivo carcinogenesis-related experiments were conducted to determine the clinical relevance between USP32 and PD-L1 expression in CRC progression. Results: In this study, we analyzed scRNA-seq data from extensive cohorts of human and mice at the single-cell level to identify DUB genes associated with PD-L1 expression in CRC. Our analysis identified multiple putative DUBs, including USP32 and USP12, as prognostic markers associated with PD-L1 expression, which was found to be elevated in T cells, macrophages, and classical monocytes cell types in patients with CRC. A secondary screening using CRISPR/Cas9-mediated loss-of-function analysis for DUBs found that USP32 modulates PD-L1 protein levels in CRC. Furthermore, we demonstrated that USP32 interacts with, stabilizes, and extends the half-life of PD-L1 by preventing its K-48-linked polyubiquitination as an underlying mechanism that contributes for tumorigenesis. Conclusion: A combination of scRNA-seq analysis and wet-lab experimental validation confirmed that USP32 mediates PD-L1 protein stabilization in colon cancer, identifying it as a potential therapeutic target for CRC. CRISPR/Cas9-mediated targeted knockout of the USP32 gene reduced PD-L1 protein levels and significantly mitigated colorectal cell proliferation and tumorigenesis, both in vitro and in vivo, in a xenograft mouse model, underscoring a novel and alternative approach to the treatment of CRC.},
}
@article {pmid41358836,
year = {2025},
author = {Li, Q and Xu, J and Jiang, J and Gong, L and Mao, X and Wang, F and Yao, P},
title = {Nucleic acid detection method for Chlamydia psittaci based on RPA-CRISPR/Cas12a.},
journal = {Letters in applied microbiology},
volume = {78},
number = {12},
pages = {},
doi = {10.1093/lambio/ovaf138},
pmid = {41358836},
issn = {1472-765X},
support = {BE2023694//Key Research and Development Project of Jiangsu Province/ ; H2023060//Jiangsu Provincial Commission of Health Project/ ; K2024003//Key Project of the Jiangsu Provincial Commission of Health/ ; Ym2023015//Jiangsu Province Preventive Medicine Research Project/ ; Ym2023073//Jiangsu Province Preventive Medicine Research Project/ ; x202339//Jiangsu Provincial Blood Parasite and Endemic Disease Prevention Research Project of China/ ; CE20225041//Changzhou science and technology Foundation/ ; CJ20253132//Changzhou science and technology Foundation/ ; CJ20253133//Changzhou science and technology Foundation/ ; CPHM202401//Nanjing Medical University/ ; CPHM202303//Nanjing Medical University/ ; },
mesh = {*Chlamydophila psittaci/genetics/isolation &amp; purification ; Humans ; *Psittacosis/diagnosis/microbiology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {In recent years, misdiagnosis or delayed diagnosis of Chlamydia psittaci (C. psittaci) infections has led to frequent outbreaks of severe public health events, such as severe pneumonia and respiratory distress, drawing increasing attention. Rapid and simple detection methods are vital for early intervention to reduce severity and mortality. In this study, we designed highly specific RPA primers and crRNA (CRISPR RNA) based on the highly conserved CPSIT_0429 gene in the C. psittaci genome, and preliminarily established a nucleic acid detection method for C. psittaci using the RPA-CRISPR/Cas12a system. In the two-step assay, the combination of the CPSIT_0429-F1/R1 primer pair and CPSIT_0429-crRNA2 achieved a detection limit of 2 × 10° copies/μL. Incorporating 20% glycerol enabled a one-tube assay with a limit of 2 × 102 copies/μL. Furthermore, the method showed no cross-reactivity with common respiratory pathogens such as influenza virus, SARS-CoV-2, and Streptococcus pneumoniae, demonstrating excellent specificity. Both the two-step and one-tube methods were compared with qPCR-verified C. psittaci positive samples. The results indicated that both assays showed high consistency with qPCR results. The RPA-CRISPR/Cas12a detection method is rapid, accurate, highly sensitive, and specific, providing a reliable platform for early diagnosis and clinical management of C. psittaci infections.},
}
@article {pmid41359128,
year = {2025},
author = {Li, Z and Cheng, Y and Li, C and Wu, Q and Xin, Y},
title = {Harnessing microalgae for bioproducts: innovations in synthetic biology.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {41},
number = {12},
pages = {500},
pmid = {41359128},
issn = {1573-0972},
support = {32560020 and 31600059//National Natural Science Foundation of China/ ; RZ2300002678//Start-Up Funds of Hainan University/ ; DC2300001799//Open Project of State Key Laboratory of Marine Resource Utilization in South China Sea/ ; 2018YFA0902500//National Key Research and Development Program of China/ ; },
mesh = {*Microalgae/metabolism/genetics ; *Synthetic Biology/methods ; Biofuels ; *Metabolic Engineering/methods ; Gene Editing ; Lipids/biosynthesis ; CRISPR-Cas Systems ; Metabolic Networks and Pathways ; Photobioreactors ; },
abstract = {Microalgae are increasingly recognized as versatile platforms for sustainable production of biofuels and high-value bioproducts such as lipids, carotenoids and polyunsaturated fatty acids. Rapid progress in synthetic biology is transforming microalgal engineering by enabling precise rewiring of metabolic pathways and overcoming long-standing technical bottlenecks, particularly those related to transformation efficiency, genetic stability and strain scalability. Recent innovations (including CRISPR/Cas genome editing, modular cloning systems, synthetic promoter libraries and dynamic, environment-responsive regulatory circuits) have greatly expanded the genetic toolset available for both model and recalcitrant species. These advances support targeted control of lipid and pigment biosynthesis, improved flux distribution and more robust performance under industrially relevant conditions. When integrated with progress in photobioreactor design, automated cultivation, and process intensification, synthetic biology unlocks new potential for scalable, economically viable microalgal biomanufacturing. This review summarizes these developments, highlights remaining challenges in strain robustness and bioprocess translation, and outlines future pathways toward high-performance microalgal biofactories that can contribute meaningfully to a low-carbon, bio-based economy.},
}
@article {pmid41359384,
year = {2025},
author = {Ichinose, M and Ohta, M and Shimajiri, Y and Akaiwa, Y and Nakamura, I and Shimamoto, M and Makinoda, R and Ozaki, S and Tamai, T and Maekawa, N and Tonomoto, M and Nakamura, T and Yagi, Y and Gutmann, B},
title = {RECODE: a programmable guide-free C-to-U RNA editing tool.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41359384},
issn = {1362-4962},
support = {//EditForce, Inc/ ; },
mesh = {*RNA Editing ; Humans ; Animals ; Mice ; *Cytidine Deaminase/genetics/metabolism/chemistry ; RNA, Guide, CRISPR-Cas Systems/genetics ; CRISPR-Cas Systems ; *Uridine/genetics/metabolism ; HEK293 Cells ; *Cytidine/metabolism/genetics ; RNA-Binding Proteins/genetics/metabolism ; Gene Editing/methods ; },
abstract = {Programmable RNA cytidine deaminase tools have been developed to convert cytidine-to-uridine (C-to-U) using CRISPR systems with guide RNAs. These tools, however, have limitations such as low editing efficiency, limited targetable sequence flexibility, and off-target RNA editing. Here, we present a novel guide-free C-to-U editing tool, named RECODE (RNA Editor for C-to-U with an Optimized DYW Enzyme), based on the RNA-binding pentatricopeptide repeat proteins, naturally fused to a C-terminal DYW cytidine deaminase domain. The RECODE specificity domain was engineered to enable retargeting, while its length and sequence were optimized to reduce off-target effects. Further optimization of the C-terminal catalytic region increased both the editing activity and the translation of the edited RNA. We showed that RECODE efficiently edits a wide range of targets in human cells, without affecting adjacent cytidines. It achieved over 50% editing efficiency for most sites, except those with an upstream guanine. Furthermore, we showed that RECODE is functional in mice, with high editing efficiency observed in specific tissues such as skeletal muscles using an AAV delivery system, suggesting its therapeutic potential for various diseases.},
}
@article {pmid41359835,
year = {2025},
author = {Roura-Martinez, D and Popa, N and Jaouen, F and Rombaut, C and Lepolard, C and Bachar, D and Borges, A and Cazorla, M and Villet, M and Moreno, S and Marie, H and Gascon, E},
title = {Combination of Cas9 and adeno-associated vectors enables efficient in vivo knockdown of precise miRNAs in the rodent and primate brain.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {50},
pages = {e2513076122},
pmid = {41359835},
issn = {1091-6490},
support = {ANR-22-CE17-0034//Association Nationale de la Recherche et de la Technologie (ANRT)/ ; 6239//Fondation France Alzheimer/ ; 2022//Fondation Recherche Alzheimer/ ; },
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; *CRISPR-Cas Systems/genetics ; *Brain/metabolism ; Mice ; Genetic Vectors/genetics ; *Gene Knockdown Techniques/methods ; *Dependovirus/genetics ; Rats ; RNA, Guide, CRISPR-Cas Systems/genetics ; Neural Stem Cells/metabolism ; Neurons/metabolism ; Primates ; Receptors, AMPA/metabolism ; Olfactory Bulb/metabolism ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {microRNAs (miRNAs) are key regulators of multiple biological functions. Although intensively studied, inactivating miRNAs in vivo is particularly challenging, especially in the brain. Here, we designed cell-specific tools aiming at downregulating defined miRNA species in vivo and investigating their function in discrete neuronal networks. Focusing on miR-124, a miRNA highly expressed in the mammalian brain and transcribed from three independent chromosomal loci, we designed and validated different guide RNAs. In vivo, our CRISPR-Cas9 designs strongly downregulate miR-124 levels without affecting the expression of other miRNAs. As a result, levels of endogenous miR-124 targets exhibit a significant increase supporting the release of its silencing activity. We provide evidence that specific deletion of miR-124 in neural stem cells of the subventricular zone altered migration of newly generated neurons into the olfactory bulb. We also showed that our vectors modified the Ca[2+] permeability of AMPA receptors, a robust functional output downstream of miR-124. We also extended our approach to other miRNAs, mammalian species, and Cas9 proteins, confirming the versatility of CRISPR-Cas9. These tool properties support their potential for elucidating miRNA functions in complex experimental in vivo settings such as brain networks.},
}
@article {pmid41360396,
year = {2025},
author = {Jiang, Q and Ramachandran, A and Avaro, AS and Huyke, DA and Santiago, JG},
title = {Reaction Kinetics of CRISPR trans-Cleavage Controlled Using Isotachophoresis.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {27646-27653},
doi = {10.1021/acs.analchem.5c04301},
pmid = {41360396},
issn = {1520-6882},
mesh = {*Isotachophoresis/methods ; Kinetics ; *CRISPR-Cas Systems ; },
abstract = {CRISPR-based diagnostics are powerful tools for nucleic acid detection due to their high specificity and programmability. However, assay sensitivity is often limited by the slow kinetics of the trans-cleavage reaction, which typically proceeds at a rate of ∼0.1 to 1 turnover per second. Here, we present a reaction-transport model and experimental study that analyze and accelerate this limiting step using electric-field-driven isotachophoresis (ITP). Building on the work of Ramachandran and Santiago, we develop a model that captures the coupling among ITP focusing, mixing, and preconcentration with CRISPR enzymatic reaction kinetics. Our analysis identifies two key regimes in ITP-coupled CRISPR reactions and derives analytical approximations for the limiting behaviors in each. Compared to a standard, well-mixed assay, we predict a 10- to 100-fold reduction in reaction duration using ITP. We validate the model with experiments across a range of target concentrations. Our work offers a quantitative framework for understanding and optimizing CRISPR trans-cleavage dynamics and provides guidance to design assays that use electric-field-mediated transport.},
}
@article {pmid41361167,
year = {2025},
author = {Fast, L and Omar, M and Kanis, P and Schaffer, T and Chowdhury, D and Rakava, E and Pääbo, S and Riesenberg, S},
title = {Search-and-remove genome editing allows selection of cells by DNA sequence.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10985},
pmid = {41361167},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; Humans ; Animals ; CRISPR-Cas Systems/genetics ; Neanderthals/genetics ; Mutation, Missense ; CRISPR-Associated Protein 9/metabolism ; Base Sequence ; Cell Line, Tumor ; },
abstract = {The selection of cells that have acquired a desired gene edit is often done by the introduction of additional genes that confer drug resistance or encode fluorophores. However, such marker genes can have unintended physiological effects and are not compatible with editing of single nucleotides. Here, we present SNIPE, a method that allows the marker-free selection of edited cells based on single nucleotide differences to unedited cells. SNIPE drastically enriches for cells, which have been precisely edited (median 7-fold). We validate the approach for 42 different edits using Cas9 or Cas12a in different cell types and species. We use it to enrich for combinations of substitutions that change missense mutations carried by all people today back to the ancestral state seen in Neandertals and Denisovans. We also show that it can be used to kill cultured tumor cells with aberrant genotypes and to repair heterozygous tumorigenic mutations.},
}
@article {pmid41361700,
year = {2025},
author = {Ain, QU and McCarthy, A and Nadeem, A and Javed, M and Niakan, K and Nashta, AF},
title = {CRISPR/Cas9-mediated generation of GATA3 knockout in Bovine Fibroblast and MDBK cell lines to assess sgRNAs targeting efficiency.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {269},
pmid = {41361700},
issn = {1438-7948},
mesh = {Animals ; Cattle ; *CRISPR-Cas Systems ; *GATA3 Transcription Factor/genetics/metabolism ; Fibroblasts/metabolism/cytology ; Cell Line ; *Gene Knockout Techniques ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing/methods ; },
abstract = {GATA3 is expressed in the outer cells of the morula stage during embryonic development and is considered a key driver of the regulation of early lineage development in bovines. This research presents an optimised somatic cell validation resource, successfully generating GATA3 knockout (KO) Bovine Fetal Fibroblasts and MDBK cells using CRISPR/Cas9-mediated genome editing for their future implications in vivo studies designed to definitively understand the role of GATA3 in cell lineage specification and bovine embryo development. This involved designing single-guide RNAs (sgRNAs) targeting different regions of the GATA3 gene, cloning them into the px459 plasmid, delivering the CRISPR clone into bovine fibroblast cells and the MDBK cell line, screening for successful targeting and knockouts, and MiSeq analysis to verify successful disruption of the GATA3 gene. A total of eleven guides were designed targeting the functional domains in Exons 4 and 5 and the transcription initiation site in Exon 2. Designed guides were first optimized and screened using an in vitro cleavage assay. The guides with the best cutting efficiencies were then tested in vivo by targeting bovine fetal fibroblast (BFFs) and MDBK cell line followed by MiSeq analysis to verify the successful knockouts. A total of two effective guides were identified targeting the zinc-finger (ZnF) functional domains of the GATA3 gene (sgRNA#5 and sgRNA#8 in Exon 4 and Exon 5, respectively) and one in Exon 2 (sgRNA#1) targeting the transcription initiation site of the GATA3 gene. MiSeq data from targeted bovine cells showed indel frequency of 47.40%, 55.5%, and 42.4% in bovine fetal fibroblasts, 11.03%, 28.9% and 7.3% for MDBK cells for top three sgRNAs. Overall, MiSeq data for 3 selected sgRNAs showed successful disruption of the GATA3 gene, inserting a base pair 2-3 bp upstream of the PAM site, ultimately resulting in a premature stop codon TAA in the downstream region. This study established and validated highly efficient sgRNAs targeting the GATA3 gene, forming a molecular basis for forthcoming functional investigations in bovine embryos to explore gene function and protein-level effects.},
}
@article {pmid41361988,
year = {2026},
author = {Min, YH and Lee, DG and Lee, HY and Yoo, JH and Lee, KH and Shin, YB and Byun, JY},
title = {CRISPR/Cas12a with Antisense Oligonucleotide-Regulated Translational Amplification for Ultrasensitive Nucleic Acid Detection.},
journal = {ACS sensors},
volume = {11},
number = {1},
pages = {394-404},
doi = {10.1021/acssensors.5c03081},
pmid = {41361988},
issn = {2379-3694},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Oligonucleotides, Antisense/genetics/chemistry ; *Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; *DNA, Viral/analysis/genetics ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics ; CRISPR-Associated Proteins ; },
abstract = {Highly sensitive nucleic acid testing-assisted early disease detection is crucial for effective disease prevention and management, particularly when targeting low-abundance genetic materials in molecular diagnostics. This study describes CRATE (CRISPR/Cas controlled antisense oligonucleotide (ASO)-mediated translational signal enhancement), a novel ultrasensitive approach for nucleic acid detection by integrating Cas12a trans-cleavage, ASO-controlled gene expression, and cell-free signal protein amplification. This assay leverages the target-induced trans-cleavage of ASO-controlled gene expression for the amplification of signal proteins, with luminescent signal allowing for attomolar-level target DNA detection, as well as antigenic protein application enabling visual detection by lateral flow assay. The CRATE assay improves sensitivity using ASO-modified locked nucleic acid, achieving a 10-aM-level DNA detection. The proof of concept demonstrates 0.1 copies/μL detection of HPV genomic DNA from HPV-positive cancer cells as well as colorimetric lateral flow tests with ∼10 copies/μL sensitivity. The CRATE assay can detect the HBV target in plasma from HBV-positive patients with 100% sensitivity and specificity. With high specificity and accuracy, the CRATE assay retains the potential for detecting any nucleic acid of interest. By integration of precise CRISPR-based cleavage, ASO regulation, and efficient protein signal amplification, this approach provides a promising solution for highly selective and sensitive nucleic acid detection and potential applications in clinical diagnostics and point-of-care testing.},
}
@article {pmid41361999,
year = {2025},
author = {Cheng, Y and Zhang, X and Zhao, P and Zhu, D and Meng, YH and Fu, X and Wang, X},
title = {Enhanced Mannan Production of Saccharomyces cerevisiae by CRISPR/Cas9 and Mannoproteins Characteristics on Wine Astringency Modulation.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {50},
pages = {32195-32208},
doi = {10.1021/acs.jafc.5c10790},
pmid = {41361999},
issn = {1520-5118},
mesh = {*Saccharomyces cerevisiae/metabolism/genetics ; *Wine/analysis/microbiology ; *Mannans/metabolism/chemistry ; *Membrane Glycoproteins/metabolism/genetics/chemistry ; CRISPR-Cas Systems ; Taste ; Humans ; *Saccharomyces cerevisiae Proteins/genetics/metabolism ; Mannose/metabolism/analysis ; },
abstract = {Mannoproteins are critical in modulating wine astringency, yet the specific impacts of their monosaccharide ratio and side-chain structure remain insufficiently explored. This study employed CRISPR/Cas9 to engineer yeast strains producing mannoproteins with either a high mannose-to-glucose ratio (high-yield-mannan strain BSFA12) or a nonbranched N-glycan structure (BY4741-ΔMNN2). The resulting mannoproteins (MPBSFA, MPBY2) were compared against controls (MPBY extracted from Saccharomyces cerevisiae BY4741, a commercial product MP60) using physicochemical analyses (fluorescence quenching, dynamic light scattering, and isothermal titration calorimetry) and sensory evaluation. Both engineered variants demonstrated superior astringency reduction in model and red wines. Our results establish that an increased mannose-to-glucose ratio and reduced N-glycosylation significantly enhance the astringency-mitigating effect, providing a foundation for the industrial-scale production and application of mannoprotein additives.},
}
@article {pmid41362221,
year = {2026},
author = {Yao, X and Wang, L and Su, L and Rao, W and Luo, Z and Li, Y},
title = {Localized CRISPR/Cas13a powered DNA walker for sensitive and high-throughput detection of norovirus.},
journal = {Journal of materials chemistry. B},
volume = {14},
number = {2},
pages = {572-579},
doi = {10.1039/d5tb02131f},
pmid = {41362221},
issn = {2050-7518},
mesh = {*Norovirus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; *DNA/chemistry/genetics ; Metal Nanoparticles/chemistry ; Gold/chemistry ; High-Throughput Screening Assays ; Limit of Detection ; RNA, Viral/analysis ; },
abstract = {The development of sensitive and high-throughput methods for detecting foodborne viruses is crucial for disease prevention and public health protection. In this study, we present a novel localized Cas13a-based DNA walker (LCas13a-DNA walker) for the ultrasensitive, stable, and rapid detection of norovirus (NoV). When the DNA walker was confined in AuNPs, the spatial confinement effect improved the local concentration of reaction substrates, accelerated the reaction speed, and enhanced the sensitivity of the DNA walker. Besides, an original design of uracil-rich hairpin (UH)-modified AuNPs as the walking track significantly improves the stability of the detection system. Meanwhile, employing CRISPR/Cas13a as the driving force streamlines viral RNA recognition and substantially reduces the reaction time down to 30 minutes by eliminating the reverse transcription step. Additionally, a biomimetic array, formed by photonic crystals (PCs), enabled high-throughput signal acquisition with a microplate reader, and concurrently amplified the fluorescence signal. The proposed assay realized ultra-sensitivity of NoV with a detection limit as low as 4.1 pM and a wide linear range from 10 pM to 5 nM. Due to the advantages of high sensitivity, high-throughput, stability, and rapid analysis, this proposed method provides a potential strategy for point-of-care detection of pathogenic viruses in food safety monitoring and disease diagnosis.},
}
@article {pmid41362674,
year = {2026},
author = {Toofan, P and Singh, M and Brooks, A and McLuckie, K},
title = {Non-clinical safety considerations on genome editing using the CRISPR/Cas system.},
journal = {Genes &amp; diseases},
volume = {13},
number = {2},
pages = {101785},
pmid = {41362674},
issn = {2352-3042},
abstract = {Recent advances in gene editing using the CRISPR/Cas system have revolutionized genome editing, opening new horizons for human cellular and gene therapy products. Genome editing technologies are rapidly being adopted in clinical trials. However, critical non-clinical safety considerations are required to address challenges in translating research to the clinic. Here, we review current ex vivo and in vivo genome editing approaches using the CRISPR/Cas system and discuss the practical use of these methods in pre-clinical studies and in the clinic. We also discuss known limitations of genome editing in humans and the mitigation of risk factors associated with it from a non-clinical safety perspective. This review aims to aid researchers in acquiring a perspective that is essential for the safe translation of genome editing to the clinic.},
}
@article {pmid41363172,
year = {2025},
author = {Wen, TT and Xu, L and Jin, R and Liu, Z and Liu, MQ and Dong, CH and Sun, L and Wang, HY and Sun, WJ and Cui, FJ},
title = {Functional Characterization of a β-1,3-Glucanosyltransferase CmGel4 in Cordyceps militaris Using a Precise CRISPR-Cas9 Genome-Editing System.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {50},
pages = {32078-32093},
doi = {10.1021/acs.jafc.5c08653},
pmid = {41363172},
issn = {1520-5118},
mesh = {CRISPR-Cas Systems ; *Cordyceps/genetics/enzymology/chemistry ; Gene Editing ; *Fungal Proteins/genetics/metabolism/chemistry ; beta-Glucans/metabolism ; Amino Acid Sequence ; Glucan Endo-1,3-beta-D-Glucosidase ; },
abstract = {Cordyceps militaris polysaccharides, especially β-glucans, have presented significant antitumor, hypoglycemic, and immunomodulatory activities. However, the enzymes involved in the branching formation of C. militaris β-glucans remain to be elucidated. In the present study, a 1.69-kb β-1,3-glucanosyltransferase CmGel4 gene putatively involved in β-glucan branching was cloned from C. militaris mycelia and bioinformatically analyzed. The encoded 54.12 kDa CmGel4p consisted of 515 amino acid residues and contained a typical GH72[+] structural characteristic of a signal peptide (1-19aa), a GH72 conserved domain (20-334aa), a GPI-anchor site (485aa), and a CBM43/X8 domain (382-458aa). Using the established CRISPR-Cas9 genome-editing system, the full length of 1.69-kb CmGel4 was precisely inserted at a genomic safe-harbor site CmSh1, and the GH72 conserved domain of CmGel4 was successfully deleted in C. militaris genome for the first time. By comparing the mycelial growth and fermentation performance of WT, control, and CmGel4-overexpressed/knockout mutants, β-1,3-glucanosyltransferase gene CmGel4 was shown to play key roles in cell growth and branching of exo-polysaccharides of C. militaris, accompanied by the transcriptional changes of genes such as CmGel4, CmUgp, and CmPgm. These findings provided the proof of β-1,3-glucanosyltransferases vital for formatting cell walls and maintaining cellular integrity, and a fine regulation strategy for precisely remodeling the β-1,3-glucan with high-branched structures in edible fungi.},
}
@article {pmid41364162,
year = {2025},
author = {Murtaza, M and Gupta, P and Choudhary, P and Manzoor, M and Sharma, S and Jaglan, S},
title = {Strategies to decipher silent biosynthetic gene clusters in actinomycetes.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {53},
pmid = {41364162},
issn = {1432-072X},
mesh = {*Actinobacteria/genetics/metabolism ; *Multigene Family/genetics ; *Biosynthetic Pathways/genetics ; Anti-Bacterial Agents/biosynthesis ; CRISPR-Cas Systems ; },
abstract = {Actinobacteria have a huge, mainly untapped potential for the production of secondary metabolites. These metabolites are an important source of bioactive compounds. However, a majority of biosynthetic gene clusters (BGCs) are either under-expressed or fully silent under standard laboratory conditions, limiting their potential. The present review article aims to explore the biosynthetic gene clusters (BGCs) of actinobacteria using strategies that aid in unlocking these silent BGCs. The strategies discussed are PCR-Targeted Gene Replacement (PCR-TR); Cre-LoxP recombination system; Transcription factor decoys, Ribosome engineering, and CRISPR/Cas technologies. Besides, elicitors also helped with the identification of these cryptic or silent BGCs and advanced our ability to explore these natural products. Combining experimental and computational platforms provides an opportunity to unlock the hidden chemical diversity in nature, thereby accelerating the identification of new bioactive substances. The new antibiotics explored by all the strategies could help in the fight against antimicrobial resistance (AMR).},
}
@article {pmid41365122,
year = {2026},
author = {Kim, WN and Kim, HU},
title = {Precise DGAT1 base editing and in-frame deletion reveal motif-specific regulation of seed oil biosynthesis in Arabidopsis.},
journal = {Plant physiology and biochemistry : PPB},
volume = {230},
number = {},
pages = {110861},
doi = {10.1016/j.plaphy.2025.110861},
pmid = {41365122},
issn = {1873-2690},
mesh = {*Arabidopsis/genetics/metabolism ; *Diacylglycerol O-Acyltransferase/genetics/metabolism ; *Seeds/metabolism/genetics ; *Arabidopsis Proteins/genetics/metabolism ; *Gene Editing ; *Plant Oils/metabolism ; Triglycerides/biosynthesis ; *Sequence Deletion ; Gene Expression Regulation, Plant ; CRISPR-Cas Systems ; },
abstract = {Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in triacylglycerol (TAG) biosynthesis and is a key determinant of seed oil content and composition. To dissect the functional contribution of the conserved DGAT1 domains, we employed adenine and cytosine base editors and CRISPR/Cas9-mediated in-frame deletion to generate targeted alleles in Arabidopsis thaliana. A total of 25 single guide RNAs were designed to introduce precise nucleotide substitutions across functional domains, and the edited lines were screened using seed fluorescence and Sanger sequencing. Five base-edited (BE) DGAT1 mutants affecting acyl-CoA/CoA allosteric binding site (S124F, S123R/S124L), thiolase acyl-enzyme intermediate signature motif (L229P), diacylglycerol (DAG)-binding motif (W416C/R419Q, V418I), and an in-frame deletion in the intrinsically disordered N-terminal region (Δ49-76H) were characterized. Amino acid substitutions in the conserved domains led to distinct shifts in seed fatty acid profiles. Loss-of-function-like mutants (S123R/S124L, L229P, W416C/R419Q) reduced 18:1 and 20:1 levels and increased 18:3 levels, whereas putative gain-of-function mutants (S124F, V418I) enhanced 20:1 incorporation and elevated total oil content. Overexpression of DGAT1[S124F] and DGAT1[V418I] in the dgat1 mutant background further increased seed oil accumulation beyond that achieved with wild-type DGAT1. Structural modeling of DGAT1 proteins revealed the location of substituted amino acids and their interactions with surrounding residues, as well as the absence of putative N-terminal regulatory segment. These results demonstrate that precise base editing can modulate DGAT1 activity and TAG composition by targeting functional motifs, providing insights into the structure-function relationships of this key enzyme and offering strategies for metabolic engineering of seed oils.},
}
@article {pmid41365143,
year = {2025},
author = {Queffeulou, M and Fakhfakh, R and Fani, F and Dos Santos, A and Reis Ferreira, G and Bigot, S and Godin, C and Leprohon, P and Papadopoulou, B and Ouellette, M},
title = {CRISPR-Cas13b mediated gene knockdowns in Leishmania infantum.},
journal = {International journal for parasitology. Drugs and drug resistance},
volume = {29},
number = {},
pages = {100629},
pmid = {41365143},
issn = {2211-3207},
mesh = {*Leishmania infantum/genetics/drug effects ; *CRISPR-Cas Systems/genetics ; *Gene Knockdown Techniques/methods ; Antiprotozoal Agents/pharmacology ; Phosphorylcholine/analogs &amp; derivatives/pharmacology ; Protozoan Proteins/genetics ; Antimony/pharmacology ; Luciferases, Firefly/genetics ; RNA, Messenger/genetics ; },
abstract = {Chemotherapy is an effective means to control infections caused by the protozoan parasite Leishmania. However, available treatments are limited, expensive, and associated with considerable toxicity. Genomic strategies have contributed to a better understanding of Leishmania's response to drugs and in the characterization of drug targets. Nonetheless, there is no knockdown system operational for Leishmania. In this study, we show that the CRISPR-Cas13 system can be an effective strategy to knockdown expression levels of both exogenous and endogenous transcripts. We succeeded in effectively knocking down the expression of the firefly luciferase gene integrated in the genome of L. infantum. This Cas13-mediated decrease in mRNA was paralleled with a significant reduction in both the luciferase protein level and its activity. Furthermore, we tested the effectiveness of the Cas13 system to target the endogenous miltefosine transporter (MT) and the aquaglyceroporin 1 (AQP1) genes. Knockdown was effective and parasites with less MT or AQP1 mRNA levels exhibited reduced susceptibility to miltefosine or antimonials, respectively. While further optimization is warranted, this knockdown system has the potential to facilitate numerous studies related to various aspects of Leishmania biology.},
}
@article {pmid41365534,
year = {2025},
author = {Skeate, JG and Lee, CJ and Stewart, C and Fischbach, MJ and Kar, B and Tsai, AK and Kenderian, SS and Stromnes, IM and Largaespada, DA and Moriarity, BS and Rogers, LM},
title = {Functional genomics for improving adoptive T-cell transfer therapies.},
journal = {Journal for immunotherapy of cancer},
volume = {13},
number = {12},
pages = {},
pmid = {41365534},
issn = {2051-1426},
support = {R37 CA276005/CA/NCI NIH HHS/United States ; },
mesh = {Humans ; *Genomics/methods ; *Immunotherapy, Adoptive/methods ; *T-Lymphocytes/immunology/transplantation ; Gene Editing/methods ; Animals ; *Neoplasms/therapy/immunology/genetics ; CRISPR-Cas Systems ; },
abstract = {Adoptive cell therapy (ACT) has shown remarkable success in the treatment of some malignancies, particularly leukemia. However, there are multiple factors that limit the durability of ACT in solid tumors, including dose-limiting toxicities, the immunosuppressive tumor microenvironment, and T-cell exhaustion. As the manufacture and preparation of adoptive T-cell therapies allows time and adequate conditions for ex vivo T-cell engineering, forward genetic screens can identify novel genetic targets that could improve their effectiveness. CRISPR is a commonly used functional genomics tool that has been successfully used to both enhance our understanding of mechanisms of resistance and to discover potential genetic edits to improve ACT. A complementary approach, Sleeping Beauty transposon mutagenesis provides additional opportunities to identify novel genetic edits without being constrained by the annotated human genome. Here, we summarize forward genetic screens and their tools to uncover strategies to enhance ACT. Complementary approaches can be combined and improved on to identify translatable genetic editing strategies through studies that accurately recapitulate disease-specific challenges.},
}
@article {pmid41365538,
year = {2025},
author = {Jung, SC and Oh, H and Eom, W and Jin, YS and Park, SH and Park, K and Koh, HG},
title = {Scarless Genetic Engineering of Saccharomyces cerevisiae for Enhanced Guanosine Monophosphate Production as a Natural Flavor Enhancer.},
journal = {Journal of microbiology and biotechnology},
volume = {35},
number = {},
pages = {e2508034},
pmid = {41365538},
issn = {1738-8872},
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *Metabolic Engineering/methods ; Saccharomyces cerevisiae Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Fermentation ; *Guanosine Monophosphate/biosynthesis/metabolism ; *Flavoring Agents/metabolism ; *Genetic Engineering/methods ; Promoter Regions, Genetic ; Inosine Monophosphate/metabolism ; Pentose Phosphate Pathway/genetics ; },
abstract = {Saccharomyces cerevisiae and Cyberlindnera jadinii are widely utilized in the natural food seasoning industry as sources of flavor enhancing nucleotides such as inosine monophosphate (IMP) and guanosine monophosphate (GMP), which contribute to umami taste and support sodium reduction in food. However, wild type yeast strains produce GMP at levels that are inadequate for industrial scale applications, necessitating metabolic engineering strategies to increase production efficiency. This study employed a CRISPR-Cas9-based scarless genome engineering approach to enhance GMP biosynthesis in S. cerevisiae via promoter replacement. The key genes IMD3 and GUA1, responsible for converting IMP to GMP, were overexpressed to redirect purine flux toward GMP production. To address precursor limitations, ZWF1 and RKI1, involved in the pentose phosphate pathway, were also overexpressed. In parallel, the expression of STB5 and RAP1 was increased to enhance NADPH regeneration and relieve transcriptional bottlenecks. As a result, the final engineered strain SCJ-7 demonstrated a 1.77-fold increase in GMP titer and a 1.40-fold increase in GMP content during flask fermentation compared to the wild-type. In fed-batch fermentation, GMP titer was further improved by 27.6%. These findings demonstrate that combining metabolic flux enhancement with transcriptional regulation provides an effective and scalable strategy for boosting GMP production in S. cerevisiae, offering strong potential for industrial application in the food industry.},
}
@article {pmid41365890,
year = {2025},
author = {Li, W and Liu, S and Fang, X and Zou, J and Jiang, Q and Min, X and Zhu, X and Cao, Y and Gao, X and Han, W and Azhar, M and Xing, X and Li, F and Zhang, Y and Liu, H and Cheng, L and Wang, C and Bao, J},
title = {Efficient high-precision transgene knock-in by Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 (RED-CRISPR).},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {538},
pmid = {41365890},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Animals ; *Gene Knock-In Techniques/methods ; *Transgenes/genetics ; Mice ; Humans ; Recombinational DNA Repair/genetics ; *Gene Editing/methods ; *Recombinases/metabolism/genetics ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {CRISPR-Cas9 tools have revolutionized genetic engineering, yet the efficient precise integration of DNA cargos, particularly for large DNA payloads (>1 kilobase, kb), remains a technical bottleneck. Herein, we develop a Recombinases (Redα/β)-enhanced DNA integration-CRISPR-Cas9 approach, referred to as RED-CRISPR, which offers a versatile yet robust homology-directed repair (HDR) strategy enabling efficient and precise kb-scale DNA insertion across various cell types, including immortalized and primary cells of variable origins. RED-CRISPR significantly enhances HDR efficiencies by 2- to 5-fold change across diverse loci and further elevates HDR rates by 1.5- to 2.5-fold when synergizing with other HDR-enhancing strategies. We achieved up to 45% knock-in efficiency for CAR-T cell manufacturing, and attained 43% knock-in rate for generation of genetically modified mice using an 8-kb DNA cargo. Through a head-to-head comparison, RED-CRISPR profoundly mitigates off-target mutational burden and chromosomal translocations. We envision RED-CRISPR as a powerful genome-editing tool with broad biomedical and therapeutic applications.},
}
@article {pmid41366133,
year = {2025},
author = {Bian, W and Mcquarrie, DWJ and Haussmann, IU and Arnold, R and Soller, M},
title = {Genetic evaluation of CRISPR-Cas9 off-target effects from deleterious mutations on Drosophila male single X chromosome.},
journal = {Functional &amp; integrative genomics},
volume = {25},
number = {1},
pages = {270},
pmid = {41366133},
issn = {1438-7948},
support = {P40 OD018537/OD/NIH HHS/United States ; },
mesh = {Animals ; Male ; *CRISPR-Cas Systems ; *X Chromosome/genetics ; *Mutation ; Gene Editing/methods ; *Drosophila/genetics ; *Drosophila melanogaster/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease protein 9 (Cas9) is a powerful tool used for genome engineering, but concerns remain about off-target effects. Here we evaluate potential deleterious effects of CRISPR-Cas9 by combining sequence analysis and the genetics of the male X chromosome in a Drosophila model. Since males have only one X chromosome deleterious mutations on the X chromosome will manifest in reducing viability or result in visible phenotypes and thus provide sensitive readouts of off-target activity. Our data do not support large scale off-target effects in Drosophila. To optimize sgRNA selection, we incorporated off-target evaluation into the PlatinumCRISPr sgRNA selection tool for a broad range of organisms.},
}
@article {pmid41366211,
year = {2025},
author = {Nuccio, SP and Cadoni, E and Nikoloudaki, R and Galli, S and Ler, AJ and Sanchez-Cabanillas, C and Maher, TE and Fan, E and Guneri, D and Flint, G and Zhu, M and Liu, LS and Fullenkamp, CR and Waller, Z and Magnani, L and Schneekloth, JS and Di Antonio, M},
title = {Chemically modified CRISPR-Cas9 enables targeting of individual G-quadruplex and i-motif structures, revealing ligand-dependent transcriptional perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {385},
pmid = {41366211},
issn = {2041-1723},
support = {Lister Prize 2022//Lister Institute of Preventive Medicine/ ; BB/R011605/1//RCUK | Biotechnology and Biological Sciences Research Council (BBSRC)/ ; },
mesh = {*G-Quadruplexes ; *CRISPR-Cas Systems/genetics ; Humans ; Ligands ; *Transcription, Genetic ; Promoter Regions, Genetic ; DNA/chemistry/genetics/metabolism ; Proto-Oncogene Proteins c-myc/genetics/metabolism ; Nucleotide Motifs ; CRISPR-Associated Protein 9/metabolism/genetics ; Gene Editing/methods ; },
abstract = {The development of selective ligands to target DNA G-quadruplexes (G4s) and i-motifs (iMs) has revealed their relevance in transcriptional regulation. However, most of these ligands are unable to target individual G4s or iMs in the genome, limiting their scope. Herein, we describe an Approach to Target Exact Nucleic Acid alternative structures (ATENA) that relies on the chemical conjugation of established G4 and iM ligands to a catalytically inactive Cas9 protein (dCas9), enabling their individual targeting in living cells. ATENA demonstrates that the selective targeting of the G4 present in the oncogene c-MYC leads to the suppression of transcripts regulated exclusively by one of its promoters (P1). Conversely, targeting the c-MYC iMs on the opposite strand leads to the selective increase of P1-driven transcripts. ATENA reveals that G4-mediated transcriptional responses are highly ligand-specific, with different ligands eliciting markedly different effects at the same G4 site. We further demonstrate that the basal expression levels of the gene targeted can be used to predict the transcriptional impact associated with G4-stabilization. Our study provides a platform for investigating G4- and iM-biology with high precision, unveiling the therapeutic relevance of individual DNA structures with selectivity.},
}
@article {pmid41366257,
year = {2025},
author = {Launspach, M and Macos, J and Afzal, S and Hohmann, J and Appis, ML and Pilgram, M and Beez, S and Ohlendorf, E and van der Ven, CFT and Lachiheb, C and Töws, K and Andersch, L and Jens, M and Zirngibl, F and Kath, J and Stecklum, M and Rodriguez-Fos, E and Anders, K and Wagner, DL and Henssen, AG and Kühn, R and Eggert, A and Künkele, A},
title = {Personalized CRISPR knock-in cytokine gene therapy to remodel the tumor microenvironment and enhance CAR T cell therapy in solid tumors.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {10987},
pmid = {41366257},
issn = {2041-1723},
mesh = {*Tumor Microenvironment/genetics/immunology ; Humans ; Animals ; Gene Knock-In Techniques ; Mice ; *CRISPR-Cas Systems ; *Immunotherapy, Adoptive/methods ; *Genetic Therapy/methods ; Chemokine CXCL10/genetics ; *Cytokines/genetics ; Cell Line, Tumor ; *Neuroblastoma/therapy/immunology/genetics ; Receptors, Chimeric Antigen/genetics ; T-Lymphocytes/immunology ; Chemokine CXCL11/genetics ; *Neoplasms/therapy/immunology/genetics ; Interferon-gamma/genetics ; Precision Medicine ; Clustered Regularly Interspaced Short Palindromic Repeats ; Female ; },
abstract = {The immunosuppressive tumour microenvironment (TME) remains a central barrier to effective immunotherapy in solid tumours. We present a gene-therapeutic strategy that enables localized remodelling of the TME via tumour-intrinsic cytokine expression. Central to this approach is CancerPAM, a multi-omics bioinformatics pipeline that identifies and ranks patient-specific, tumour-exclusive CRISPR-Cas9 knock-in sites with high specificity and integration efficiency. Using neuroblastoma as a model, CancerPAM analysis of tumour sequencing data identifies optimal knock-in sites for pro-inflammatory cytokines (CXCL10, CXCL11, IFNG), and CancerPAM rankings correlate strongly with target-site specificity and knock-in efficiency, validating its predictive performance. CRISPR-mediated CXCL10 knock-in enhances CAR T cell infiltration and antitumour efficacy in vitro and in vivo, including humanized CD34[+] HuNOG mice, where CXCL10-expressing tumours show stronger immune infiltration and prolonged tumour control within a reconstituted human immune microenvironment. Our findings establish a framework for safe and effective CRISPR-based cytokine delivery, integrating localized TME remodelling with cellular immunotherapies to enhance CAR T cells and other treatments in immune-refractory solid tumours.},
}
@article {pmid41367203,
year = {2025},
author = {Patel, J and Patel, D and Raval, A},
title = {Artificial Intelligence for Predictive Modeling in CRISPR/Cas9 Gene Editing: a Survey of Methods and Design Strategies.},
journal = {The journal of gene medicine},
volume = {27},
number = {12},
pages = {e70061},
doi = {10.1002/jgm.70061},
pmid = {41367203},
issn = {1521-2254},
mesh = {*CRISPR-Cas Systems ; *Artificial Intelligence ; *Gene Editing/methods ; Humans ; Machine Learning ; Algorithms ; },
abstract = {Ongoing developments in genome editing most notably the continued evolution of CRISPR-Cas systems and their orthogonal or modified counterparts have substantively altered both experimental and applied practices in biomedicine, agriculture, and therapeutic design. More recently, the systematic incorporation of artificial intelligence and machine learning methodologies has augmented the specificity, throughput, and explanatory capacity of genome-editing workflows, thereby refining the prediction of on-target efficiencies, the appraisal of off-target liabilities, and the tailoring of molecular therapeutic configurations. The present contribution offers an integrative survey of these computational developments, emphasizing (i) predictive algorithms, (ii) machine-learning and deep-learning frameworks, (iii) data-centric procedural strategies, and (iv) dedicated applications in oncology, neurology, rare-disease research, and precision-medicine contexts. Throughout, we evaluate architectural choices, sequence-encoding representations, and lingering dataset-related biases, while additionally addressing current constraints concerning model interpretability, ethical viability, and the procedural prerequisites for clinical translation. Moreover, we advance a structured taxonomy that organizes AI-mediated genome-editing approaches according to methodological lineage and functional scope, and we delineate extant research lacunae. By combining these elements, we supply a prospective assessment of the means by which artificial intelligence might be further leveraged to support secure, efficacious, and equitably accessible genome engineering outcomes.},
}
@article {pmid41367214,
year = {2025},
author = {Selokar, NL and Singh, P and Jose, B and Gautam, D and Patel, K and Verma, R and De, S and Singh, MK and Singh, D},
title = {A Myostatin (MSTN[-/-]) Knockout Buffalo Produced by CRISPR-Cas9 Mediated Genome Editing and Somatic Cell Nuclear Transfer.},
journal = {The CRISPR journal},
volume = {8},
number = {6},
pages = {436-442},
doi = {10.1177/25731599251401528},
pmid = {41367214},
issn = {2573-1602},
mesh = {Animals ; *Myostatin/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Buffaloes/genetics ; *Nuclear Transfer Techniques ; *Gene Knockout Techniques/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; Fibroblasts/metabolism ; Female ; Animals, Genetically Modified/genetics ; },
abstract = {CRISPR-Cas9 genome editing offers significant opportunities to improve livestock traits; however, its application in buffalo has been very limited, with no prior reports of live gene-edited animals. Here, we report the successful birth of a buffalo edited in the myostatin (MSTN) gene. To achieve this, five single-guide RNAs (sgRNAs) targeting the buffalo MSTN gene were designed and tested in skin-derived fibroblasts. Among these, sgRNA5 exhibited the highest editing efficiency, approaching ∼50%, as confirmed by T7 Endonuclease I assay, Tracking of Indels by Decomposition, and Inference of CRISPR Edits analyses. Single-cell cloning identified six edited fibroblast clonal populations, including one with a bi-allelic frameshift mutation predicted to severely truncate the MSTN protein. These bi-allelic clonal cells were subsequently used as nuclear donors to produce somatic cell nuclear transfer (SCNT) embryos, which were transferred into recipient buffaloes (n = 15). This effort established three pregnancies and resulted in the birth of one live MSTN knockout buffalo calf. Phenotypically, the calf displayed accelerated growth and increased muscle fiber number and size while maintaining normal meat composition. In conclusion, this study reports the world's first gene-edited buffalo generated through CRISPR-Cas9-mediated genome editing combined with SCNT. These findings provide a proof-of-concept for genome editing in buffalo and demonstrate that MSTN disruption can effectively enhance muscle growth and meat production traits.},
}
@article {pmid41367295,
year = {2026},
author = {Wyer, CAS and Amaro, IA and Pitcher, S and Ponlawat, A and Harrington, LC and Wolfner, MF and Hollis, B and Cator, LJ},
title = {Pickpocket315 affects male mating behavior in the yellow fever mosquito Aedes aegypti.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {2},
pages = {},
pmid = {41367295},
issn = {2160-1836},
support = {NE/S007415/1//Natural Environment Research Council/ ; R01-AI095491/NH/NIH HHS/United States ; //L.C.H and M.F.W and International Atomic Energy Association/ ; //Reproductive Biology of Male Aedes Mosquitoes for SIT Applications/ ; R01 AI095491/AI/NIAID NIH HHS/United States ; },
mesh = {Animals ; *Aedes/genetics/physiology ; Male ; *Sexual Behavior, Animal ; Female ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; RNA Interference ; Mosquito Vectors/genetics ; Copulation ; Yellow Fever/transmission ; },
abstract = {The molecular basis of mating behavior in the important disease vector mosquito, Aedes aegypti, remains poorly characterized. We investigated the functional role of a pickpocket gene, ppk315, in male mating behavior using both RNAi-mediated knockdown and CRISPR/Cas9 approaches. Behavioral assays revealed that RNAi-treated males (dsPPK315) made fewer mating attempts, were less responsive to female acoustic cues, and were less likely to achieve copulation, though their latency to initiate contact when attempts were made was comparable to controls. Males with a CRISPR/Cas9-induced disruption to ppk315 exhibited reduced success in inseminating multiple females, consistent with previous reports from RNAi knockdown males, ruling out off-target effects as the source of behavioral changes. In contrast to the results of behavioral assays with RNAi, ppk315 mutant males (ppk315-/-) attempted copulation as frequently as wild-type males (ppk315+/+) but were slower to contact females. Despite these impairments in one-on-one interactions, both dsPPK315 and ppk315-/- males displayed normal mating success under competitive swarm-like conditions, potentially due to the socially facilitated activation of mating behavior. Collectively, our findings support a role for ppk315 in the initiation of mating behaviors via sensory detection, with context-dependent consequences for reproductive success.},
}
@article {pmid41369349,
year = {2025},
author = {Cheng, X and Wang, D and Zhang, X and Li, L and Liu, Y and Cao, G and Zhang, Y},
title = {Regulation of the Homeostasis of Early Embryo Development in Dairy Cows by Targeted Editing of the PRLR Gene-Mediated Activation of the Anti-Heat Stress Pathway.},
journal = {Cells},
volume = {14},
number = {23},
pages = {},
pmid = {41369349},
issn = {2073-4409},
support = {No. 2023ZD04050//Guifang Cao/ ; },
mesh = {Animals ; Cattle ; *Gene Editing/methods ; *Heat-Shock Response/genetics ; *Homeostasis/genetics ; *Embryonic Development/genetics ; *Receptors, Prolactin/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Female ; Reactive Oxygen Species/metabolism ; Fibroblasts/metabolism ; Nuclear Transfer Techniques ; Oocytes/metabolism ; },
abstract = {The intensification of global climate warming exacerbates the issue of heat stress in dairy cows, making the SLICK mutation in the prolactin receptor (PRLR) gene a critical target for enhancing heat tolerance in these animals. This study aims to investigate the effects of CRISPR/Cas9-mediated editing of the PRLR gene on the biological characteristics of bovine fibroblasts and early embryonic development following somatic cell nuclear transfer (SCNT). Using the CRISPR/Cas9 system, we targeted and edited a 20 bp-150 bp region within exon nine of the PRLR gene. After conducting off-target predictions and activity screenings, we identified optimal guide RNA (sgRNA) sequences and established stable transgenic cell lines. Transcriptome sequencing was performed on edited cells to identify key genes and validate their expression profiles. Edited cells were utilized as donor cells for SCNT, during which we assessed oocyte levels of reactive oxygen species (ROS), glutathione (GSH), and mitochondrial function to analyze embryonic developmental performance. We constructed a cellular stress resistance network aimed at mitigating damage transmission while maintaining embryonic developmental homeostasis. This research provides technical support and theoretical reference for genetic editing breeding programs aimed at improving heat tolerance in dairy cattle.},
}
@article {pmid41369371,
year = {2025},
author = {Gardner-Kay, A and Le, L and Filla, M and Kibiryeva, N and O'Brien, JE and Bittel, DC},
title = {CRISPR Disruption of scaRNA1 Reduces Pseudouridylation in Spliceosomal RNA U2 at U89 and Perturbs the Transcriptome in HEK293T Cells.},
journal = {Cells},
volume = {14},
number = {23},
pages = {},
pmid = {41369371},
issn = {2073-4409},
support = {Intramural Faculty grant//Kansas City University/ ; },
mesh = {Humans ; HEK293 Cells ; *Spliceosomes/metabolism/genetics ; *Transcriptome/genetics ; *Pseudouridine/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA, Small Nuclear/metabolism/genetics ; RNA Splicing/genetics ; },
abstract = {Small Cajal body-associated RNAs (scaRNAs) are essential for biochemical modification of spliceosomal RNAs and spliceosome function. Changes in scaRNA expression level have been associated with developmental issues, including cancer and congenital heart defects (CHDs), although the mechanism remains unclear. Small Cajal body-associated RNA 1 (scaRNA1) guides pseudouridylation at uridine 89 (Ψ89) of the spliceosomal RNA U2, a highly conserved modification that may be critical for spliceosome function. To investigate the role of scaRNA1 in splicing regulation, CRISPR-Cas9 genome editing was used to introduce targeted deletions in the scaRNA1 locus in HEK293T cells. Edited clones were identified by T7 endonuclease I assay and confirmed by Sanger sequencing. Pseudouridylation at Ψ89 was quantified using CMC-based reverse transcription followed by quantitative PCR, and global mRNA splicing alterations were assessed by RNA sequencing. Clones harboring scaRNA1 disruptions exhibited a significant reduction in Ψ89 pseudouridylation, consistent with impaired scaRNA1 function. Transcriptome analysis (of mRNA from two clones) revealed >300 protein coding genes with significant changes in transcript isoform level, including >100 genes related to RNA-binding activity. These results indicate that scaRNA1 disruption alters spliceosomal function and leads to substantial changes in mRNA splicing. The dysregulated splicing of RNA-binding proteins may impair RNA processing and gene expression programs required for normal development, providing new insight into how noncoding RNA dysfunction may contribute to developmental pathogenesis.},
}
@article {pmid41369550,
year = {2026},
author = {Smith, DJ},
title = {Complementary human gene interaction maps from radiation hybrids and CRISPRi.},
journal = {Physiological genomics},
volume = {58},
number = {1},
pages = {42-57},
doi = {10.1152/physiolgenomics.00075.2025},
pmid = {41369550},
issn = {1531-2267},
support = {C25CR8562//University of California Cancer Research Coordinating Committee/ ; //Norton Simon Research Foundation/ ; },
mesh = {Humans ; Genome-Wide Association Study ; *Gene Regulatory Networks/genetics ; *CRISPR-Cas Systems/genetics ; *Radiation Hybrid Mapping/methods ; *Protein Interaction Maps/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Alleles ; },
abstract = {The only comprehensive human genetic interaction map was constructed using increased gene copy numbers in radiation hybrid (RH) cells. Recently, a second map restricted to essential genes was created using CRISPR interference (CRISPRi)-induced loss-of-function alleles. Here, the two maps are compared to understand their similarities and differences. Both maps showed significant overlap with protein-protein interaction databases and identified a shared set of interacting genes, although the specific gene pairs differed between approaches. Notably, the RH map exhibited strong overlap with genome-wide association study (GWAS) networks, whereas the CRISPRi map did not. These findings demonstrate how gain- and loss-of-function alleles reveal distinct yet complementary genetic interaction landscapes.NEW &amp; NOTEWORTHY This study compared two mammalian genetic interaction networks for cell growth: the radiation hybrid (RH) network used extra gene copies and the CRISPRi network used partial gene suppression. Both networks overlapped with protein-protein interaction data and identified common interacting genes, yet specific gene pair interactions differed dramatically. Only the RH network predicted genome-wide association study (GWAS) networks. As the first comparison of large-scale mammalian genetic interaction networks, this work reveals how gain- and loss-of-function variants capture diverse biological perspectives.},
}
@article {pmid41370122,
year = {2026},
author = {Pfisterer, L and Boyle, C and Cole, A and Mitchell, I and Flanagan, M and Gromley, Z and Gromley, A},
title = {Disruption of the centriolin/Cep110 gene (CNTRL) with CRISPR/Cas9 leads to cell cycle arrest and cell death of rhabdomyosarcoma cells in vitro.},
journal = {Molecular biology of the cell},
volume = {37},
number = {2},
pages = {br4},
pmid = {41370122},
issn = {1939-4586},
mesh = {CRISPR-Cas Systems/genetics ; Humans ; *Rhabdomyosarcoma/genetics/metabolism/pathology ; Cell Line, Tumor ; *Cell Cycle Checkpoints/genetics ; *Cell Cycle Proteins/genetics/metabolism ; Gene Editing/methods ; Centrosome/metabolism ; Cell Death/genetics ; *Microtubule-Associated Proteins/genetics/metabolism ; Cilia/metabolism ; Cell Survival/genetics ; Cell Proliferation/genetics ; Apoptosis/genetics ; },
abstract = {Rhabdomyosarcoma is the most common pediatric soft tissue cancer, thought to arise from primitive mesenchymal cells that differentiate into skeletal muscle. Previous studies suggest that primary cilia may play a role in the development of rhabdomyosarcoma. Primary cilia are cellular structures that arise from the centrosome and serve important functions in sensory signaling, cell migration, and developmental processes. However, most rhabdomyosarcoma cell lines do not have primary cilia. Because primary cilia are derived from centrosomes, the development of rhabdomyosarcoma may, in fact, be due to the function of centrosome proteins rather than the primary cilia itself. Therefore, this study sought to determine if the centrosomal protein centriolin/Cep110, which is localized to both centrosomes and primary cilia, plays a role in rhabdomyosarcoma biology. The gene editing tool CRISPR/Cas9 was used to disrupt the centriolin/Cep110 gene in the rhabdomyosarcoma cell line CCL-136, and the effects on cell viability and cell cycle progression were assayed. Our results show that loss of centriolin/Cep110 leads to cell cycle arrest and apoptotic cell death in rhabdomyosarcoma cells. These findings suggest that centriolin/Cep110 plays a key role in rhabdomyosarcoma cell proliferation and viability and that this centrosome protein may represent a potential target for future rhabdomyosarcoma therapies.},
}
@article {pmid41370200,
year = {2025},
author = {Shi, X and Hu, C and Jia, L and Lei, Z and Guo, B and Zhou, J and Wang, F},
title = {An SpC editor targeting pre-mRNA splicing for precise CRISPR control and enhanced antitumor efficacy.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41370200},
issn = {1362-4962},
support = {32271512//National Natural Science Foundation of China/ ; 82572281//National Natural Science Foundation of China/ ; 2022JC-56//Natural Science Basic Research Program of Shaanxi/ ; 2023-JC-ZD-43//Natural Science Basic Research Program of Shaanxi/ ; 2024JC-YBQN-0168//Natural Science Basic Research Program of Shaanxi/ ; 2023A1515110886//Guangdong Basic and Applied Basic Research Foundation/ ; },
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *RNA Precursors/genetics/metabolism ; *RNA Splicing/drug effects ; Animals ; Mice ; Cell Line, Tumor ; Spliceosomes/genetics/metabolism ; Macrolides/pharmacology ; Apoptosis/genetics ; Neoplasms/genetics/therapy ; RNA, Guide, CRISPR-Cas Systems/genetics ; Epoxy Compounds ; },
abstract = {The CRISPR/Cas9 system is a powerful genome editing tool that has the potential to be applied to a variety of biomedical applications. Despite the considerable potential of this gene editing technology, there are numerous safety concerns including the possibility of unpredictable off-target effects. The splicing process, which involves the removal of introns from pre-mRNA and the alignment of exons to produce mature transcripts, is a critical step in gene expression in most eukaryotes. In this study, we present a spliceosome-responsive CRISPR/Cas9 (SpC) editor that utilizes the splicing inhibitor pladienolide B (PB) to regulate pre-mRNA splicing and control the expression of the anti-CRISPR protein AcrIIA4, thereby modulating the activity of the Cas9 nuclease. This approach allows for precise regulation of the gene editing process, thereby effectively mitigating off-target effects. The reliability and robustness of the SpC editor were demonstrated through in vitro and in vivo bioluminescence imaging. Furthermore, a dual-target sgRNA was designed to target the diphtheria toxin A gene, resulting in apoptosis induction and growth inhibition of tumor cells across various types of cancer cells. Our results indicate that this SpC editor has the capacity to precisely regulate tumor cell growth, thus providing new insights and significant implications for cancer gene therapy.},
}
@article {pmid41370232,
year = {2026},
author = {Alok, A and Raman, V and D'Agostino, L and Kshetry, AO and Rai, KM and Wang, C and Gunapati, S and Stupar, RM and Patil, GB and Zhang, F},
title = {Developmental regulators enable rapid and efficient soybean transformation and CRISPR-mediated genome editing.},
journal = {Plant physiology},
volume = {200},
number = {3},
pages = {},
pmid = {41370232},
issn = {1532-2548},
support = {IOS-2040218//National Science Foundation/ ; IOS-2206920//National Science Foundation/ ; #2021-67013-34565//USDA NIFA/ ; //Texas Governor's University Research/ ; },
mesh = {*Glycine max/genetics/growth &amp; development ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Plants, Genetically Modified/genetics ; *Transformation, Genetic ; *Plant Growth Regulators/metabolism ; Gene Expression Regulation, Plant ; Plant Proteins/genetics/metabolism ; },
abstract = {Soybean (Glycine max) transformation remains challenging and has not kept pace with rapid advances in genetic engineering technologies due to low efficiency, lengthy timelines, and genotype dependency. Here, we developed a streamlined transformation method by leveraging developmental regulators (DRs) to promote de novo shoot regeneration directly from growing soybean plants. By evaluating multiple DR combinations, our results showed that co-expression of WUSCHEL2 (WUS2) and the gene encoding isopentenyltransferase (IPT) achieved higher transformation efficiencies (14.6% to 22.3%) in Williams 82 and Bert varieties than individual DRs without requiring exogenous hormones or selection agents. Moreover, this method produced heritable transgenic events within 9 to 11 weeks and successfully delivered CRISPR-Cas9 components, generating heritable mutations with 20% efficiency. The temporal transcriptomic and gene regulatory network analyses revealed that WUS2/IPT synergistically modulates stress responses and activates developmental pathways, orchestrating a transition from initial stress adaptation to regenerative programming. Our findings demonstrate that this DR-enabled approach significantly enhances soybean transformation frequency, reduces tissue culture requirements, and offers a promising genome-editing platform for soybean improvement.},
}
@article {pmid41370233,
year = {2026},
author = {Nomura, Y and Nomura, J and Tamada, K and Eguchi, N and Torigata, K and Tokumoto, S and Nemoto, A and Shirafuji, T and Yamamoto, K and Hishimoto, A and Nagase, H and Nishikawa, T and Takumi, T},
title = {Isogenic modeling of 1q21.1 reciprocal CNVs in human ES cells reveals divergent neurodevelopmental trajectories.},
journal = {Human molecular genetics},
volume = {35},
number = {2},
pages = {},
doi = {10.1093/hmg/ddaf184},
pmid = {41370233},
issn = {1460-2083},
support = {21&#xa0;K07820//KAKENHI/ ; 24&#xa0;K10078//KAKENHI/ ; 22&#xa0;K15750//KAKENHI/ ; 16H06316//KAKENHI/ ; 16H06463//KAKENHI/ ; 23KK0132//KAKENHI/ ; 24H00620//KAKENHI/ ; 24H01241//KAKENHI/ ; 23H04233//KAKENHI/ ; 24&#xa0;K22036//KAKENHI/ ; //Japan Society for the Promotion of Science and Ministry of Education, Culture, Sports, Science, and Technology/ ; JP21wm0425011//Japan Agency for Medical Research and Development/ ; JPMJPF2018//Japan Science and Technology Agency/ ; JPMJMS2299//Japan Science and Technology Agency/ ; JPMJMS229B//Japan Science and Technology Agency/ ; //Takeda Science Foundation/ ; //Smoking Research Foundation/ ; //Taiju Life Social Welfare Foundation/ ; },
mesh = {Humans ; *DNA Copy Number Variations/genetics ; *Chromosomes, Human, Pair 1/genetics ; Neurogenesis/genetics ; *Neurodevelopmental Disorders/genetics/pathology ; *Human Embryonic Stem Cells/metabolism ; Chromosome Deletion ; Neural Stem Cells/metabolism ; Cell Differentiation/genetics ; Neurons/metabolism ; Chromosome Duplication ; CRISPR-Cas Systems ; Intellectual Disability/genetics ; Cell Line ; Schizophrenia/genetics ; },
abstract = {Copy number variations (CNVs) in the distal 1q21.1 region, both deletion (1q del) and duplication (1q dup) are associated with various neurodevelopmental and neuropsychiatric disorders such as autism spectrum disorder, intellectual disability, epilepsy, and schizophrenia. Besides common phenotypes, 1q del and 1q dup manifest opposite clinical phenotypes, e.g. microcephaly in 1q del and macrocephaly in 1q dup. However, molecular and cellular mechanisms underlying these phenotypes are still elusive. Here, to identify molecular mechanisms associated with neurodevelopmental phenotypes from the viewpoint of neurogenesis and neurodevelopment, we generate isogenic human ES cell (hESC) lines with reciprocal 1q21.1 CNVs using CRISPR/Cas9 system and differentiate them into 2-dimensional (2-D) neurons and neural progenitor cell (NPC) spheroids. Our study recapitulates reciprocal brain size in the NPC spheroids and shows dosage-dependent differentiation changes i.e. more GABAergic components in 1q del and more proliferative state in 1q dup. These results demonstrate that 1q21.1 CNVs dramatically affect cell fate in the early neurodevelopmental periods. This is the first isogenic cell model of human 1q21.1 CNVs, and our findings provide new insights into the underlying mechanisms of neurodevelopmental disorders.},
}
@article {pmid41370671,
year = {2025},
author = {Gao, Z and Lin, K and Gong, Y and Zhao, Y and Zhang, S},
title = {Core-Shell Tripeptide-Lipid/PEI Nanocarriers Enable Efficient Plasmid-Based CRISPR/Cas9 Editing of VEGFR2.},
journal = {Langmuir : the ACS journal of surfaces and colloids},
volume = {41},
number = {50},
pages = {33716-33730},
doi = {10.1021/acs.langmuir.5c02069},
pmid = {41370671},
issn = {1520-5827},
mesh = {Humans ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Polyethyleneimine/chemistry ; *Plasmids/genetics/chemistry ; *Vascular Endothelial Growth Factor Receptor-2/genetics ; Animals ; *Lipids/chemistry ; *Nanoparticles/chemistry ; MCF-7 Cells ; Mice ; },
abstract = {The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a versatile genome editing technology that holds tremendous promise for the treatment of various diseases. Although several delivery technologies such as electroporation, viral vectors, and lipid nanoparticles have already shown promise in preclinical and clinical applications for hematological and neuromuscular genetic disorders, in vivo application is still restricted by the inefficient delivery of CRISPR/Cas9 components. Herein, by employing the tripeptide lipid N,N-ditetradecyloxyamidoethyl trimeric ornithine amide (CDO) and polyethylenimine (PEI), we constructed novel ternary systems (pDNA/PEI/CL) for the delivery of pDNA encoding Cas9 and single-guide RNAs (sgRNAs) targeting the VEGFR2 gene. The pDNA/PEI/CL delivery systems were fabricated by condensing pDNA with PEI, followed by coating with cationic liposomes composed of CDO. This system demonstrated high transfection efficiency, successfully delivering CRISPR/Cas9 to A549 and MCF-7 cells with efficiencies of up to 91.0% (n = 3, P < 0.001), while also exhibiting lower cytotoxicity. Notably, the sgRNA1/P1/C1 complex achieved higher genome editing efficiencies than sgRNA3/P1/C1, with 38.6% vs 31.0% in A549 cells (n = 3, P < 0.01) and 26.45% vs 20.18% in MCF-7 cells (n = 3, P < 0.01). Western blot analysis showed that VEGFR2 expression decreased by 48.1% in A549 and 44.3% in MCF-7 cells, while PI3K levels were reduced by 39.6% and 42.8%, respectively. This suppression of the PI3K/Akt signaling pathway led to cell cycle arrest, thereby inhibiting tumor cell proliferation and migration while promoting apoptosis. Furthermore, animal experiments validated the antitumor efficacy, highlighting the translational potential of this platform in cancer therapy. Collectively, these findings highlight the potential of the ternary complex system as a robust and biocompatible CRISPR/Cas9 delivery strategy, offering a promising avenue for gene therapy in cancer and other genetic diseases.},
}
@article {pmid41370881,
year = {2026},
author = {Li, SR and Li, Y and Yang, KB and Wang, SW and Sun, ML and Liu, Z and Zhang, XP and Zhong, Y and Yao, J},
title = {CRISPR/Cas12a coupled with MIRA: A specific and rapid assay for human DNA in challenging forensic matrices.},
journal = {Forensic science international. Genetics},
volume = {82},
number = {},
pages = {103393},
doi = {10.1016/j.fsigen.2025.103393},
pmid = {41370881},
issn = {1878-0326},
mesh = {Humans ; *CRISPR-Cas Systems ; Animals ; *Nucleic Acid Amplification Techniques/methods ; Cytochromes b/genetics ; Mice ; Swine ; Chickens ; *DNA Fingerprinting/methods ; DNA ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Human DNA detection is crucial in forensic medicine, particularly for trace, degraded, or mixed samples, which demand high sensitivity, specificity, and rapid processing. Traditional methods, such as immunological assays and PCR-based techniques, often suffer from operational complexity, limited sensitivity, or high equipment dependency. To address these challenges, we developed a novel detection system combining multienzyme isothermal rapid amplification (MIRA) with CRISPR-Cas12a for the rapid, specific, and portable human DNA identification. By targeting the human mitochondrial cytochrome b (CYTB) gene and sex-determining Region Y(SRY) gene, we designed MIRA primers and CRISPR-Cas12a crRNA to enable dual recognition and signal amplification. The method involves isothermal amplification at 37°C followed by CRISPR-Cas12a-mediated cleavage, producing detectable fluorescence or lateral flow chromatographic signals. Our system achieves ultra-sensitive detection and high specificity, distinguishing human DNA from non-human sources (e.g., pig, chicken, mouse), and also enables accurate gender identification, further enhancing its utility in forensic and genetic studies. Compared to traditional qPCR, this approach demonstrates superior sensitivity, faster turnaround (≤ 45 min), and minimal equipment requirements, making it ideal for forensic applications. Moreover, the blood, mixed, and degraded samples were used to confirm its robustness, with results interpretable via blue-light fluorescence or colloidal gold test strips. In summary, the MIRA-CRISPR/Cas12a system overcomes the limitations of conventional techniques, offering a rapid, cost-effective, and reliable solution for forensic human DNA identification, with potential extensions to wildlife conservation and food safety testing.},
}
@article {pmid41371153,
year = {2026},
author = {Longhi Cervantes, DS and Leal, GM and Fortirer, JDS and de Oliveira, LF and Navarro, BV and Buckeridge, MS},
title = {microRNAs and stress adaptation in grasses: A systematic review.},
journal = {Plant physiology and biochemistry : PPB},
volume = {230},
number = {},
pages = {110783},
doi = {10.1016/j.plaphy.2025.110783},
pmid = {41371153},
issn = {1873-2690},
mesh = {*MicroRNAs/genetics/metabolism ; *Poaceae/genetics/physiology/metabolism ; *Stress, Physiological/genetics ; *Adaptation, Physiological/genetics ; Gene Expression Regulation, Plant ; *RNA, Plant/genetics/metabolism ; },
abstract = {MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression after transcription, playing crucial roles in plant development and stress adaptation. In grasses, this regulation is vital under isolated biotic and abiotic stress conditions and combined stress scenarios, although many regulatory modules remain unexplored. This systematic review examined 60 studies out of 1823 publications indexed in Scopus, focusing on grass miRNAs with validated targets through Degradome-Seq and/or RACE approaches. Results indicate that miRNA-target modules were validated more often under abiotic stress than biotic or combined stress conditions. The most frequently studied miRNA families include miR156, miR159, miR164, miR169, and miR396, which are commonly linked to various types of stress, whether isolated or combined. Most research has concentrated on major crops such as rice and maize, with limited studies on other agriculturally important grasses. This review highlights advances in miRNA-phytohormone interactions, systemic signaling, and target validation strategies. It also underscores the potential of biotechnological tools such as RNAi, artificial miRNAs, target mimicry, and CRISPR/Cas for engineering more resilient grasses. Integrating multi-omics approaches and an increasing focus on combined stress responses suggest promising strategies for sustainable agriculture, food security, and bioenergy production amidst climate challenges. Together, these advances strengthen the potential of microRNA-based regulation as a key tool for enhancing crop resilience and adaptation.},
}
@article {pmid41371329,
year = {2026},
author = {Chen, B and Gao, J and Sun, H and Zhao, Y and Liu, Y and Qiu, X and Li, Y},
title = {Integrating CRISPR with SERS: Toward intelligent point-of-care diagnostics of the future.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {581},
number = {},
pages = {120782},
doi = {10.1016/j.cca.2025.120782},
pmid = {41371329},
issn = {1873-3492},
mesh = {*Spectrum Analysis, Raman/methods ; Humans ; *CRISPR-Cas Systems/genetics ; *Point-of-Care Systems ; Biosensing Techniques ; },
abstract = {In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas) system has emerged as a transformative genome-editing platform. Beyond its editing applications, the CRISPR/Cas system has attracted growing interest in molecular diagnostics particularly for nucleic acid detection due to its exceptional sensitivity and target specificity. Meanwhile, surface-enhanced Raman spectroscopy (SERS), which relies on plasmonic nanoparticles or nanostructures, has become a powerful biosensing technology known for its high sensitivity and distinct spectral fingerprinting capability. The integration of CRISPR/Cas-mediated molecular recognition with the ultrasensitive detection of SERS offers a rapid, low-volume, and direct strategy for identifying diverse nucleic acid targets. This synergistic combination has inspired the development of innovative biosensing platforms designed for ultrasensitive and precise molecular diagnostics. In this review, we first outline the fundamental principles of CRISPR/Cas and SERS, then summarize their hybrid applications in nucleic acid detection. Finally, we discuss the current progress, challenges, and future perspectives of CRISPR/Cas-integrated SERS biosensing.},
}
@article {pmid41372121,
year = {2025},
author = {Cheng, KW and Bhave, M and Markhard, AL and Peng, D and Bhatt, KD and Travisano, KA and Medicielo, JV and Anaya, A and Lembirik, S and Njoya, L and Anantpadma, M and Kuhn, JH and Puschnik, AS and Kistler, AL},
title = {Replicon-based genome-wide CRISPR knockout screening for the identification of host factors involved in viral replication.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11028},
pmid = {41372121},
issn = {2041-1723},
support = {HHSN272201800013C/OD/NIH HHS/United States ; },
mesh = {*Virus Replication/genetics ; Humans ; *Replicon/genetics ; *Dengue Virus/genetics/physiology ; Chikungunya virus/genetics/physiology ; *CRISPR-Cas Systems ; Gene Knockout Techniques/methods ; Ebolavirus/genetics/physiology ; Cell Line ; Animals ; *Host-Pathogen Interactions/genetics ; Membrane Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; HEK293 Cells ; Hexosyltransferases/genetics/metabolism ; },
abstract = {We describe a viral replicon-based CRISPR knockout (KO) screening approach to specifically identify host factors essential for viral replication which are often missed in live virus screens. We benchmark the replicon screening using a stable fluorescent dengue virus type 2 (DENV-2) replicon cell line and successfully identify host genes known to be required for viral DENV-2 replication (e.g., endoplasmic reticulum membrane complex and oligosaccharyltransferase complex components), along with additional genes that have not been reported in prior CRISPR KO screens with DENV-2. We extend this replicon screening approach to chikungunya virus (CHIKV), a positive-sense RNA virus, and Ebola virus (EBOV), a negative-sense RNA virus, and identify distinct sets of genes required for replication of each virus. Our findings indicate that viral replicon-based CRISPR screens are a useful approach to identify host factors essential for replication of diverse viruses and to elucidate potential novel targets for host-directed medical countermeasures.},
}
@article {pmid41372159,
year = {2025},
author = {Pan, R and Ren, J and Chen, X and Flores, LF and Gonzalez, RVL and Adonnino, AA and Lofts, B and Waldo, J and Halmai, J and Devinsky, O and Fink, K and Liu, XS},
title = {Editing DNA methylation in vivo.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {527},
pmid = {41372159},
issn = {2041-1723},
support = {R01 NS126185/NS/NINDS NIH HHS/United States ; R01 MH134519/MH/NIMH NIH HHS/United States ; R01NS126185//U.S. Department of Health &amp; Human Services | NIH | National Institute of Neurological Disorders and Stroke (NINDS)/ ; R01MH134519//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; F30 HD115371/HD/NICHD NIH HHS/United States ; },
mesh = {Animals ; *DNA Methylation/genetics ; *Gene Editing/methods ; Mice ; DNA Methyltransferase 3A ; Mice, Transgenic ; Promoter Regions, Genetic ; Methyl-CpG-Binding Protein 2/genetics/metabolism ; Proprotein Convertase 9/genetics/metabolism ; Liver/metabolism ; DNA (Cytosine-5-)-Methyltransferases/genetics/metabolism ; CRISPR-Cas Systems ; Proto-Oncogene Proteins/genetics/metabolism ; Male ; DNA-Binding Proteins/genetics/metabolism ; Epigenesis, Genetic ; Neurons/metabolism ; },
abstract = {DNA methylation is a crucial epigenetic mechanism that regulates gene expression. Precise editing of DNA methylation has emerged as a promising tool for dissecting its biological function. However, challenges in delivery have limited most applications of DNA methylation editing to in vitro systems. Here, we develop two transgenic mouse lines harboring an inducible dCas9-DNMT3A or dCas9-TET1 editor to enable tissue-specific DNA methylation editing in vivo. We demonstrate that targeted methylation of the Psck9 promoter in the liver of dCas9-DNMT3A mice results in decreased Pcsk9 expression and a subsequent reduction in serum low-density lipoprotein cholesterol level. Targeted demethylation of the Mecp2 promoter in dCas9-TET1 mice reactivates Mecp2 expression from the inactive X chromosome and rescues neuronal nuclear size in Mecp2[+/-] mice. Genome-wide sequencing analyses reveal minimal transcriptional off-targets, demonstrating the specificity of the system. These results demonstrate the feasibility and versatility of methylation editing, to functionally interrogate DNA methylation in vivo.},
}
@article {pmid41372199,
year = {2025},
author = {Nan, AX and Chickering, M and Bartolome, CL and Shadija, N and Li, D and Estes, BJG and Stetina, JV and Li, W and Andresen, J and Molugu, K and Amunugama, R and Fang, M and Bai, C and Wang, J and Norouzi, D and Cochrane, JC and Gatlin, JT and Dunyak, MT and Kumar, S and Chavez, L and Seth, A and Halperin, S and Finn, JD and Xie, J},
title = {Ligase-mediated programmable genomic integration (L-PGI).},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {563},
pmid = {41372199},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Mice ; Humans ; Genomics/methods ; *Ligases/metabolism/genetics ; HEK293 Cells ; },
abstract = {Since their discovery, CRISPR systems have been repurposed for programmable targeted genomic editing, leading to applications for gene disruption, single base editing, insertion, deletion, and manipulation of short genomic sequences. Pairing Cas9 nickase with reverse transcriptase allows applications for insertion, substitution, and deletion of short genomic sequences from an RNA template without generating double stranded breaks however this technology typically shows reduced efficacy in post mitotic cells, limiting its translatability in vivo. Here we present a novel, ligase-based method that addresses these limiations. We introduce edits through delivery and ligation of a synthetic DNA donor to genomic nicks created with Cas9 nickase and report editing activity in cell lines, primary cell cultures, and adult mice via nonviral delivery. With favorable on target outcomes compared to transcription-based editing in key cell types, good tolerability, and deliverability, ligation-mediated gene editing has the potential to further advance genomic medicine.},
}
@article {pmid41372233,
year = {2025},
author = {Macak, D and Kanis, P and Riesenberg, S},
title = {Repurposing clinically safe drugs for DNA repair pathway choice in CRISPR genome editing and synthetic lethality.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11077},
pmid = {41372233},
issn = {2041-1723},
mesh = {Humans ; *Gene Editing/methods ; *Synthetic Lethal Mutations/drug effects ; *Drug Repositioning/methods ; DNA End-Joining Repair/drug effects ; Induced Pluripotent Stem Cells/drug effects/metabolism ; *DNA Repair/drug effects ; Recombinational DNA Repair/drug effects ; *CRISPR-Cas Systems/genetics ; DNA Breaks, Double-Stranded/drug effects ; Tumor Suppressor p53-Binding Protein 1/metabolism/genetics ; },
abstract = {We evaluate the effect of most FDA-approved drugs (>7,000 conditions) on double-strand DNA break repair pathways by analyzing mutational outcomes in human induced pluripotent stem cells. We identify drugs that can be repurposed as inhibitors and enhancers of repair outcomes attributed to non-homologous and microhomology-mediated end joining (NHEJ, MMEJ), and homology-directed repair (HDR). We also identify functions of the proteins estrogen receptor 2 (ESR2) and aldehyde oxidase 1 (AOX1), affecting several key DNA repair proteins, such as ATM and 53BP1. Silencing of ESR2 can have a synergistic effect on increasing HDR when combined with NHEJ inhibition (mean 4.6-fold increase). We further identify drugs that induce synthetic lethality when NHEJ or HDR is blocked and may therefore be candidates for precision medicine. We anticipate that the ability to modulate the DNA repair outcomes with clinically safe drugs will help disease modeling, gene therapy, chimeric antigen receptor immunotherapy, and cancer treatment.},
}
@article {pmid41372414,
year = {2026},
author = {Habtewold, T and Lwetoijera, DW and Hoermann, A and Mashauri, R and Matwewe, F and Mwanga, R and Kweyamba, P and Maganga, G and Magani, BP and Mtama, R and Mahonje, MA and Tambwe, MM and Tarimo, F and Chennuri, PR and Cai, JA and Del Corsano, G and Capriotti, P and Sasse, P and Moore, J and Hudson, D and Manjurano, A and Tarimo, B and Vlachou, D and Moore, S and Windbichler, N and Christophides, GK},
title = {Gene-drive-capable mosquitoes suppress patient-derived malaria in Tanzania.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {442-448},
pmid = {41372414},
issn = {1476-4687},
support = {//Gates Foundation/ ; },
mesh = {Tanzania/epidemiology ; Animals ; *Plasmodium falciparum/isolation &amp; purification/genetics/drug effects/growth &amp; development ; *Anopheles/genetics/parasitology ; Humans ; *Malaria, Falciparum/prevention &amp; control/parasitology/transmission ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/parasitology ; Female ; Animals, Genetically Modified/genetics ; Male ; Child ; CRISPR-Cas Systems/genetics ; },
abstract = {Gene drive technology presents a transformative approach to combatting malaria by introducing genetic modifications into wild mosquito populations to reduce their vectorial capacity. Although effective modifications have been developed, these efforts have been confined to laboratories in the global north. We previously demonstrated that modifying Anopheles gambiae to express two exogenous antimicrobial peptides inhibits the sporogonic development of laboratory-cultured Plasmodium falciparum, with models predicting substantial contributions to malaria elimination in Africa when integrated with gene drive[1-3]. However, the effectiveness of this modification against genetically diverse, naturally circulating parasite isolates remained unknown. To address this critical gap, we adapted our technology for an African context by establishing infrastructural and research capacity in Tanzania, enabling the engineering of local A. gambiae under containment. Here we report the generation of a transgenic strain equipped with non-autonomous gene drive capabilities that robustly inhibits genetically diverse P. falciparum isolates obtained from naturally infected children. These genetic modifications were efficiently inherited by progeny when supplemented with Cas9 endonuclease provided by another locally engineered strain. Our work brings gene drive technology a critical step closer to application, providing a locally tailored and powerful tool for malaria eradication through the targeted dissemination of beneficial genetic traits in wild mosquito populations.},
}
@article {pmid41373007,
year = {2025},
author = {Song, J and Yang, D and Kong, L and Tsai, LK and Zhang, J and Chen, YE and Tsai, RY and Xu, J},
title = {Development of a high-yield Rabbit line for enhanced animal pharming.},
journal = {Biological research},
volume = {58},
number = {1},
pages = {73},
pmid = {41373007},
issn = {0717-6287},
support = {R41 GM110822/GM/NIGMS NIH HHS/United States ; R41GM110822/GM/NIGMS NIH HHS/United States ; },
mesh = {Animals ; Rabbits/genetics ; *Animals, Genetically Modified/genetics ; *Recombinant Proteins/biosynthesis/genetics ; *Milk/chemistry ; Gene Knock-In Techniques ; CRISPR-Cas Systems/genetics ; *Caseins/genetics ; Female ; Promoter Regions, Genetic/genetics ; },
abstract = {Animal pharming involves producing recombinant protein drugs using transgenic animals. The United States Food and Drug Administration (FDA) has approved certain drugs produced in the milk of transgenic Rabbits. Traditionally, these pharming Rabbits have been developed using conventional transgenic technology, which often results in an unpredictable success rate, uncontrollable transgene insertion sites, varying copy numbers, and generally low recombinant protein yields, typically 1-2 g/L or lower. We hypothesized that utilizing the promoter of a native major milk protein gene to drive transgene expression could significantly enhance yield. To test this, we developed a rabbit line that expresses tdTomato under the control of the CSN2 gene promoter, responsible for encoding β-casein, the most abundant protein in Rabbit milk. We successfully generated knock-in founder Rabbits using CRISPR/Cas9-mediated knock-in technology, augmented by the homology-directed repair (HDR)-promoting small molecule RS-1. These founder Rabbits were able to transmit the knock-in allele to their offspring, producing both heterozygous and homozygous tdTomato knock-in Rabbits. Remarkably, the recombinant protein yield reached 15-20 g/L in the milk of homozygous animals. Our work demonstrates a promising strategy to enhance recombinant protein production in Rabbit pharming.},
}
@article {pmid41373550,
year = {2025},
author = {Koller, F},
title = {The Potential of NGTs to Overcome Constraints in Plant Breeding and Their Regulatory Implications.},
journal = {International journal of molecular sciences},
volume = {26},
number = {23},
pages = {},
pmid = {41373550},
issn = {1422-0067},
support = {3522840500//Federal Agency for Nature Conservation/ ; },
mesh = {*Plant Breeding/methods ; CRISPR-Cas Systems ; Genome, Plant ; *Plants/genetics ; *Genomics/methods ; Gene Editing/methods ; },
abstract = {Conventional plant breeding relies on the occurrence of chromosomal crossover and spontaneous or non-targeted mutations in the genome induced by physical or chemical stressors. However, constraints exist concerning the number and variation of genotypes that can be achieved in this way, as the occurrence and combination of mutations are not equally distributed across the genome. The underlying mechanisms and causes of reproductive constraints can be considered the result of evolution to maintain the genomic stability of a species while at the same time allowing necessary adaptations. A continuous horizon scan was carried out to identify plants derived from new genomic techniques (NGTs), which show that CRISPR/Cas is able to circumvent at least some of these mechanisms and constraints. The reason for this is the specific mode of action: While physico-chemical mutagens such as radiation or chemicals merely cause a break in DNA, recombinant enzymatic mutagens (REMs), such as CRISPR/Cas, additionally interfere with cellular repair mechanisms. More recently developed REMs even expand the capabilities of NGTs to introduce new genetic variations within the target sequences. Thus, NGTs introduce genetic changes and combinations that are unknown in the current breeding pool and that are also unlikely to occur as a result of any previously used breeding methods. The resulting genotypes may need to be considered as 'new to the environment'. The technical potential of NGTs should also be taken into account in regulatory provisions. Previously unknown genotypes and phenotypes may negatively impact plant health, ecosystems, biodiversity, and plant breeding. It must further be acknowledged that the different outcomes of NGTs and conventional breeding are not always evident at first sight. As a starting point, within a process-oriented approval process, molecular characterization can inform the following steps in risk assessment and guide requests for further data.},
}
@article {pmid41373623,
year = {2025},
author = {Petrova, IO and Smirnikhina, SA},
title = {Ex Vivo Gene and Cell Therapy in Hematopoietic Stem Cells.},
journal = {International journal of molecular sciences},
volume = {26},
number = {23},
pages = {},
pmid = {41373623},
issn = {1422-0067},
support = {not applicable//Ministry of Education and Science of Russia/ ; },
mesh = {Humans ; *Genetic Therapy/methods ; *Hematopoietic Stem Cells/metabolism/cytology ; *Cell- and Tissue-Based Therapy/methods ; Animals ; *Hematopoietic Stem Cell Transplantation/methods ; Genetic Vectors/genetics ; Lentivirus/genetics ; },
abstract = {Ex vivo cell and gene therapy is a prospective approach to treatment of genetic diseases. To date, one of the most prevalent examples of genetically engineered cell therapies is hematopoietic stem/progenitor cells (HSPCs). This mini review is focused on HSPC therapy methods that have been approved for medical use. Most gene therapy methods rely on the lentiviral integration of the gene into the target cell genome, as lentiviruses are extremely effective, particularly in transduction of non-dividing cells. In this constantly evolving field, it is important to find the balance between safety concerns and efficiency. Analyzing cases of several diseases, for which ex vivo gene therapy was developed, we strive to understand which factors are crucial to success and what the potential drawbacks are. Although in general, viral gene integration demonstrates a considerable therapeutic effect, it has oncogenic potential. Development of self-inactivating vectors was a breakthrough in regard to safety, but the possibility of oncogenesis remains, and strict analysis of integration sites is required.},
}
@article {pmid41374373,
year = {2025},
author = {Sambo, CN and Skepu, A and Nxumalo, NP and Polori, KL},
title = {Diagnostic Advances and Public Health Challenges for Monkeypox Virus: Clade-Specific Insight and the Urgent Need for Rapid Testing in Africa.},
journal = {Diagnostics (Basel, Switzerland)},
volume = {15},
number = {23},
pages = {},
pmid = {41374373},
issn = {2075-4418},
support = {B1B0741B-9691-4333-B77E-3B8D42A7B5FF//Technology Innovation Agency/ ; },
abstract = {Background: Monkeypox (MPX), caused by the Monkeypox virus (MPOX) of the Orthopoxvirus genus, has re-emerged as a significant global health threat. Once confined to Central and West Africa, the 2022-2025 multi-country outbreaks, predominantly caused by Clade IIb, demonstrated sustained human-to-human transmission and global spread. Objective: This review summarizes current knowledge on MPX virology, epidemiology, clinical presentation, and diagnostic technologies, with a focus on innovations supporting rapid and field-deployable detection in resource-limited settings. Methods: The recent literature (2019-2025), including peer-reviewed studies, WHO and Africa CDC reports, and clinical guidelines, was critically reviewed. Data were synthesized to outline key developments in diagnostic methodologies and surveillance approaches. Results: MPX comprises two genetic clades: Clade I (Congo Basin) and Clade II (West African), which differ in virulence and transmission. Clade IIb is associated with sexual and close-contact transmission during recent outbreaks. Clinical manifestations have shifted from classic disseminated rash to localized anogenital lesions and atypical or subclinical infections. RT-PCR remains the diagnostic gold standard, while emerging assays such as loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and CRISPR/Cas-based platforms show promise for rapid point-of-care (POC) testing. Complementary serological tools, including ELISA and lateral flow assays, enhance surveillance and immune profiling. Conclusions: The resurgence of MPX highlights the urgent need for accessible, sensitive, and specific diagnostic platforms to strengthen surveillance and outbreak control, especially in endemic and resource-constrained regions.},
}
@article {pmid41375204,
year = {2025},
author = {Li, W and Shi, Y and Li, D and Wang, Y and Sun, Y and Li, H and Han, Y},
title = {A CRISPR Powered Immobilization-Free, Amplification-Free Carbon-Nanotube Field-Effect Transistor (FET) Sensor for Influenza A Virus (IAV).},
journal = {Molecules (Basel, Switzerland)},
volume = {30},
number = {23},
pages = {},
pmid = {41375204},
issn = {1420-3049},
support = {2023YFC2605101//National Key Research and Development Program of China/ ; },
mesh = {*Influenza A virus/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; Transistors, Electronic ; *Nanotubes, Carbon/chemistry ; Humans ; *RNA, Viral/genetics/analysis ; CRISPR-Cas Systems ; Limit of Detection ; *Influenza, Human/diagnosis/virology ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The epidemic of infectious diseases, such as influenza A, has imposed a severe health burden on the population. Early detection, diagnosis, reporting, isolation, and treatment are crucial for the prevention, control, and management of infectious diseases. Nucleic acid testing represents a vital approach for the rapid diagnosis of pathogenic microorganism types. However, current nucleic acid detection methods face notable bottlenecks: traditional CRISPR fluorescence assays require time-consuming pre-amplification of target nucleic acids, while existing carbon-nanotube field-effect transistor (FET)-based platforms, though amplification-free, often necessitate complex chip surface modification and probe immobilization, and suffer from non-reusable chips, all limiting their utility in point-of-care testing (POCT) and large-scale screening. This study reports a CRISPR-based amplification-free RNA detection platform (CRISPR-FET) for the rapid identification of influenza A virus. The CRISPR-FET platform described herein enables the detection of viral RNA without amplification within 20 min, with a limit of detection as low as 1 copy/μL. Secondly, a reporter RNA conjugated with gold particles is used to achieve signal amplification in FET detection; meanwhile, the method eliminates probe immobilization, thereby omitting this step and simplifying chip modification to reduce complex work-flows and pre-treatment costs. The chip's reusability further enhances cost-effectiveness. Additionally, streptavidin-modified magnetic bead adsorption minimizes background errors from excessive reporter RNA and non-target nucleic acids. Finally, validation with 24 clinical samples confirmed the platform's efficacy. By integrating rapidity, simplicity, and high sensitivity, alongside cost advantages from reusable chips, this CRISPR-FET platform meets the critical need for early influenza A diagnosis and holds promise for advancing POCT and large-scale epidemiological screening.},
}
@article {pmid41375334,
year = {2025},
author = {Lai, CM and Xiao, XS and Liu, LW and Lin, XD and Dou, DL and Cai, HY and Mei, ZF and Yang, F and Cheng, Y and Qin, Y},
title = {Nanotechnology Strategies in Plant Genetic Engineering: Intelligent Delivery and Precision Editing.},
journal = {Plants (Basel, Switzerland)},
volume = {14},
number = {23},
pages = {},
pmid = {41375334},
issn = {2223-7747},
support = {2024NZ029029//Major Science and Technology Project of Fujian Province/ ; },
abstract = {Plant genetic engineering is crucial for enhancing crop yield, quality, and resilience to both abiotic and biotic stresses, thereby promoting sustainable agriculture. Agrobacterium-mediated, biolistic bombardment, electroporation, and poly (ethylene glycol) (PEG)-mediated genetic transformation systems are widely applied in plant genetic engineering. However, these systems have limitations, including species dependency, destruction of plant tissues, low transformation efficiency, and high cost. Recently, gene-delivery methods based on nanotechnology have been developed for plant genetic transformation. This nanostrategy demonstrates remarkable transformation efficiency, excellent biocompatibility, effective protection of exogenous nucleic acids, and the potential for plant regeneration. However, the application of nanomaterial-mediated gene-delivery systems in plants is still in its early stages and faces numerous challenges for widespread adoption. Herein, the conventional genetic transformation techniques utilized in plants are succinctly examined. Subsequently, the advancements in nanomaterial-based gene-delivery systems are reviewed. The applications of CRISPR-Cas-mediated genome editing and its integration with plant nanotechnology are also examined. The innovations, methods, and practical applications of nanomaterial-mediated genetic transformation summarized herein are expected to facilitate the progress of plant genetic engineering in modern agriculture.},
}
@article {pmid41376155,
year = {2026},
author = {Jin, X and Wu, X and Song, J and Luo, M and Ye, Q and Ren, C and Song, L and Li, M and Hu, M and An, Y and Su, J and Fu, J and Xu, Q and Luo, M and Liu, F and Liu, M and Li, Q and Yao, S and Chen, L and Yang, Y},
title = {Comparative evaluation of liver-directed knockin strategies with viral and nonviral vectors in mouse inherited disease models.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1775-1793},
pmid = {41376155},
issn = {1525-0024},
mesh = {Animals ; *Genetic Vectors/genetics/administration &amp; dosage ; Mice ; Disease Models, Animal ; Dependovirus/genetics ; *Liver/metabolism ; *Gene Knock-In Techniques/methods ; Genetic Therapy/methods ; Gene Editing/methods ; Humans ; CRISPR-Cas Systems ; Recombinational DNA Repair ; *Hemophilia B/genetics/therapy ; Transgenes ; },
abstract = {CRISPR-Cas9-mediated gene knockin has emerged as a promising strategy for early-onset genetic disease intervention. However, the therapeutic efficacy and editing outcomes of different knockin strategies remain incompletely understood. Here, we systematically evaluated three major liver-directed knockin strategies, namely homology-directed repair (HDR), homology-independent targeted integration (HITI), and homology-mediated end joining (HMEJ), using neonatal mouse models of mucopolysaccharidosis type I and hemophilia B. Although all three approaches effectively rescued disease phenotypes, we observed distinct editing outcomes. Notably, the HMEJ approach, delivered via a combined adeno-associated virus-lipid nanoparticle (AAV-LNP) system, exhibited superior integration efficiency (5.8%-5.9%) and fidelity (97%-98%) compared with HDR and HITI. In contrast, whole-genome sequencing indicated that HITI induced a higher risk of random AAV donor integration than HDR or HMEJ. Furthermore, long-read sequencing analyses revealed that the frequencies of inverted terminal repeat (ITR)-mediated transgene integration differed between the 5' and 3' genomic junctions among the three strategies. Specifically, in HDR- and HMEJ-treated mice, ITR-mediated integration events were 7.7- to 19.7-fold more common at the 3' junctions than at the 5' junctions. These findings highlight the comprehensive advantages of the AAV-LNP-mediated HMEJ approach for liver-directed knockin therapy and suggest its strong potential for clinical translation.},
}
@article {pmid41376159,
year = {2026},
author = {Ha, AS and Kalter, N and Rosenberg, M and Acevedo, LA and Liang, B and Liu, W and Paruthiyil, S and Sinha, M and Vu, A and Nguyen, V and Qi, Z and Krishnappa, N and Shu, J and Yu, J and Catanzaro, J and Bluestone, JA and Tang, Q and Urnov, FD and Marson, A and Hendel, A and Herold, KC and Shy, BR and Esensten, JH},
title = {Gene-corrected regulatory T cell therapy for IL2RA deficiency.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {3},
pages = {1367-1381},
pmid = {41376159},
issn = {1525-0024},
mesh = {Humans ; *T-Lymphocytes, Regulatory/immunology/metabolism/transplantation ; *Interleukin-2 Receptor alpha Subunit/genetics/deficiency ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Gene Editing ; Male ; Female ; Interleukin-2 ; Mutation ; },
abstract = {Bi-allelic germline deficiency of IL2RA causes a rare autoimmune disease with impaired regulatory T cell (Treg) function and interleukin-2 (IL-2) signaling. Definitive treatment is currently limited to allogeneic hematopoietic stem cell transplantation, which carries significant morbidity and mortality risks. We previously identified a family with three siblings affected by compound heterozygous mutations in their IL2RA gene, resulting in dysfunctional Tregs. Here, we introduce a novel therapeutic approach involving ex vivo generation of gene-corrected autologous regulatory T cells (gcTregs). One of the two disease-causing mutations in patient-derived Tregs was corrected with CRISPR-Cas9-mediated homology-directed repair, restoring IL2RA expression. The resulting gcTregs demonstrated robust suppressive activity in vitro. Clinical-scale manufacturing from a patient with IL2RA deficiency showed efficient gene correction, restored IL2RA expression, and functional equivalence to healthy donor Tregs. This work establishes a Good Manufacturing Practice-compatible manufacturing process for personalized gcTreg therapies, potentially providing a safer treatment option for patients with IL2RA deficiency as well as a framework for treating other inborn errors of immunity.},
}
@article {pmid41377346,
year = {2025},
author = {Abedin, ZU and Waggan, AI and Khan, E and Suleman, MU and Tabassum, SN},
title = {Letter to the Editor: CRISPR-based gene editing for cardiac protection in Barth syndrome.},
journal = {Annals of medicine and surgery (2012)},
volume = {87},
number = {12},
pages = {9163-9164},
pmid = {41377346},
issn = {2049-0801},
abstract = {Barth syndrome is a rare X-linked mitochondrial disorder caused by mutations in the Tafazzin (TAZ) gene. These mutations make it hard for cardiolipin to remodel and mitochondria to work properly. This condition is characterized by growth retardation, neutropenia, skeletal myopathy, and dilated cardiomyopathy, frequently leading to significant morbidity and mortality, with numerous patients necessitating heart transplants. There are no treatments available at this time to fix the genetic problem. Recent progress in gene editing, especially CRISPR-based methods, holds great promise for fixing TAZ mutations. Research utilizing patient-derived cardiomyocytes has demonstrated that the rectification of TAZ mutations reinstates mitochondrial efficiency and enhances cellular functionality. Animal models, including TAZ-knockout mice, have exhibited substantial enhancements in cardiac function, survival rates, and diminished fibrosis subsequent to gene replacement therapy.},
}
@article {pmid41380236,
year = {2026},
author = {Yin, Z and Yin, H and Zhou, Y and Liu, H},
title = {CRISPR/Cas13a-mediated photoelectrochemical-colorimetric dual-mode biosensor for RNA N-acetyltransferase 10 detection based on Bi2O2S/Ag2S and laccase-like nanozyme.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118298},
doi = {10.1016/j.bios.2025.118298},
pmid = {41380236},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Laccase/chemistry ; Electrochemical Techniques/methods ; Colorimetry/methods ; *Acetyltransferases/isolation &amp; purification/analysis/chemistry ; Humans ; CRISPR-Cas Systems ; Limit of Detection ; Bismuth/chemistry ; },
abstract = {N-Acetyltransferase 10 (NAT10) is a crucial protein that catalyzes RNA acetylation modification and plays a significant role in biological activities. Accurate detection of NAT10 is of great importance in clinical testing and drug development. To achieve this goal, a novel biosensing platform was developed for NAT10 detection, relying on an RNA acetylation-inhibited CRISPR/Cas13a system, a Bi2O2S/Ag2S type-II heterojunction, and a laccase-mimetic nanocatalyst, using a photoelectric-colorimetric dual-mode strategy. Based on the catalysis effect of laccase-mimetic nanocatalyst, its substrates (hydroquinone and 2,4-dichlorophenol) were oxidized to form p-benzoquinone and 2,4-dichloroquinone, respectively. This oxidation process not only impaired the electron-donating ability of hydroquinone but also induced the coupling of 2,4-dichloroquinone with 4-antipyrine to generate a red product-enabling both photoelectrochemical and colorimetric detection of the NAT10 protein. The biosensor exhibited wide linear ranges of 0.1-1000 μg/L (photoelectrochemical mode) and 1-1000 μg/L (colorimetric mode), and the low detection limit of 0.056 and 0.348 μg/L for the two modes, respectively. Additionally, this biosensor was used to investigate the inhibitory effects of plasticizers and phosphorus-containing flame retardants on NAT10. Furthermore, molecular docking simulations were employed to explore the underlying inhibition mechanism. The developed biosensor not only provided a novel detection technique for NAT10, but also offers an alternative method for NAT10 inhibitor screening and a new tool for evaluating the ecotoxicological effects of organic pollutants.},
}
@article {pmid41380779,
year = {2026},
author = {Hill, AC and Schank, MB and Zhang, Y and Sun, N and Wang, L and Zhao, J and Banik, P and Pyburn, JS and Orfield, H and Lightner, JW and Leshaodo, TO and Wu, XY and Ning, S and El Gazzar, M and Moorman, JP and Guo, H and Yao, ZQ},
title = {Suppression of HBV replication and expression by CRISPR/Cas9 ribonucleoproteins.},
journal = {Antiviral research},
volume = {245},
number = {},
pages = {106326},
pmid = {41380779},
issn = {1872-9096},
support = {R01 AI177624/AI/NIAID NIH HHS/United States ; R21 AI157909/AI/NIAID NIH HHS/United States ; },
mesh = {*Hepatitis B virus/genetics/physiology/drug effects ; *Virus Replication/drug effects ; Humans ; *CRISPR-Cas Systems ; *Ribonucleoproteins/genetics/pharmacology/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Editing ; DNA, Viral/genetics ; Antiviral Agents/pharmacology ; Hep G2 Cells ; Genetic Therapy/methods ; Hepatitis B/therapy/virology ; Cell Line ; CRISPR-Associated Protein 9 ; },
abstract = {HBV infection is a global public health problem. The current treatment using nucleotide analogues (NA) can suppress viral replication but cannot eliminate HBV infection due to the persistence of covalently closed circular DNA (cccDNA), which sustains HBV replication and integration into the host cell genome and is refractory to NA treatment. CRISPR/Cas9 has been used to disrupt integrated HBV DNA and minichromosomal cccDNA for HBV suppression, but its expression and delivery require viral or non-viral vectors, which pose safety concerns for human application. We have previously reported the use of synthetic guide RNA (gRNA)/Cas9 ribonucleoprotein (RNP) as a non-viral formulation for HBV gene editing and viral suppression. To formulate highly effective CRISPR/Cas9 modalities for HBV gene therapy, here we designed additional gRNA/Cas9 RNPs and compared their antiviral efficacy in HBV-transfected as well as -infected cells. We found that two selected gRNA/Cas9 RNPs (gRNA5/Cas9, gRNA9/Cas9, and particularly their combinations) elicited the most potent antiviral efficacy, as evidenced by the significant inhibition of HBV DNA, RNA, and protein productions. DNA sequencing of the treated cells revealed moderate to high rates of insertion and deletion (indel) or knock-out (KO) mutations at the HBV target genes. Gene alignment analysis showed a high level of conservation for both gRNA5 and gRNA9 target sequences across major HBV genotypes, indicating that these CRISPR-based gene editing therapeutics have the potential to target different HBV strains worldwide. Thus, these synthetic gRNA/Cas9 RNPs represent promising novel therapeutics that can be developed and utilized for HBV gene disruption and viral eradication.},
}
@article {pmid41380995,
year = {2026},
author = {Roy, S and Nandy, S and Morita, D and Nandy, RK and Veeraraghavan, B and Walia, K and Das, S and Basu, S},
title = {Genomic analysis of a novel high-risk ST5217/ExoU+/O11 clone of carbapenem-resistant OXA-181- and VIM-2-producing Pseudomonas aeruginosa in India.},
journal = {Journal of global antimicrobial resistance},
volume = {46},
number = {},
pages = {158-161},
doi = {10.1016/j.jgar.2025.12.002},
pmid = {41380995},
issn = {2213-7173},
mesh = {*Pseudomonas aeruginosa/genetics/drug effects/isolation &amp; purification/enzymology/classification ; *beta-Lactamases/genetics/metabolism ; India ; Humans ; *Carbapenems/pharmacology ; Pseudomonas Infections/microbiology ; Phylogeny ; *Bacterial Proteins/genetics ; Genome, Bacterial ; Anti-Bacterial Agents/pharmacology ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Drug Resistance, Multiple, Bacterial/genetics ; Whole Genome Sequencing ; Genomics ; },
abstract = {OBJECTIVES: Studies of carbapenem-resistant Pseudomonas aeruginosa (CRPA)-harbouring OXA-48-like carbapenemases are rare. The study aimed to report the emergence and characterization of a novel high-risk clone of CRPA-harbouring OXA-48-like from India.<br><br>METHODS: Identification, AST, phenotypic detection of carbapenemases and WGS using Ion-Torrent-S5 platform were carried out. Analyses included ResFinder, VFDB, MLST, PAst, Phastest and CRISPR/Cas. SNP-based phylogenetic analysis with global OXA-48-like-harbouring CRPA genomes was carried out by CSI Phylogeny and iTOL for visualization.<br><br>RESULTS: The clinical MDR strain of CRPA AMRIR00655 belonged to a novel sequence type ST5217 and serotype O11. Phenotypic tests followed by WGS revealed the presence of dual carbapenemases, OXA-181 (serine-carbapenemase) and VIM-2 (zinc-carbapenemase), both located on chromosome. blaOXA-181 resides between chromosomal genes encoding RodZ and PAP2 in P. aeruginosa, confirming chromosomal integration.4,261 bp of blaOXA-181-bearing contig-DNA showed 100% homology to K. pneumoniae plasmid pKP3-A. ISEcp1 was present on upstream and on downstream, &#x25b3;lysR, &#x25b3;ereA and repA genes were detected. blaVIM-2 was located within class 1 integron along with aacC6-II, dfrB5, aac(3)-Id, tniC in surrounding regions and 13,242 bp showing 100% identity to P. aeruginosa chromosome. Presence of other ARGs (blaPAO, blaOXA-488,aph(3'')-Ib, aph(6)-Id, crpP, catB7, fosA, sul2) and efflux-pump genes might explain its MDR phenotype. Virulence factors including T3SS (PscF, PopB, PopD, PcrV) and its effectors (ExoT, ExoU, ExoY) indicated the pathogenic potential of ST5217. Core genome analysis showed that ST5217 was closest with other high-risk clones ST1339 and ST773-harbouring blaOXA-48-like.<br><br>CONCLUSIONS: To the best of our knowledge, this is the first report of blaOXA-181-harbouring novel high-risk clone of CRPA ST5217/ExoU+/O11 in India which emphasises the spread of OXA-181 among bacteria other than Enterobacteriaceae-family and warrant close monitoring.},
}
@article {pmid41381092,
year = {2025},
author = {Lane, KR and Jones, SE and Osborne, TH and Geller-McGrath, D and Nwaobi, BC and Chen, L and Thomas, BC and Hudson-Edwards, KA and Banfield, JF and Santini, JM},
title = {Bioleaching Microbial Community Metabolism and Composition Driven by Copper Sulphide Mineral Type.},
journal = {Environmental microbiology reports},
volume = {17},
number = {6},
pages = {e70261},
pmid = {41381092},
issn = {1758-2229},
support = {NE/L002485/1//Natural Environment Research Council/ ; BB/N012674/1/BB_/Biotechnology and Biological Sciences Research Council/United Kingdom ; //Hellenic Coppers Mines Ltd/ ; },
mesh = {*Copper/metabolism/chemistry ; *Bacteria/metabolism/genetics/classification/isolation &amp; purification ; *Archaea/metabolism/genetics/classification/isolation &amp; purification ; *Sulfides/metabolism/chemistry ; *Microbial Consortia ; Metagenomics ; *Minerals/metabolism/chemistry ; *Microbiota ; Plasmids/genetics ; },
abstract = {Copper bioleaching is a green technology for the recovery of copper from chalcopyrite (CuFeS2) and chalcocite (Cu2S) ores. Much remains to be learned about how mineral type and surface chemistry influence microbial community composition. Here, we established a microbial consortium from a copper bioleaching column in Cyprus on chalcopyrite and then sub-cultured it to chalcocite to investigate how the community composition shifts due to changes in mineral structure and the absence of mineral-derived Fe. The solution chemistry was determined and microbial communities characterised by genome-resolved metagenomics after 4 and 8 weeks of cultivation. Acidithiobacillus species and strains, a Rhodospirilales, Leptospirillum ferrodiazotrophum and Thermoplasmatales archaea dominated all enrichments, and trends in abundance patterns were observed with mineralogy and surface-attached versus planktonic conditions. Many bacteria had associated plasmids, some of which encoded metal resistance pathways, sulphur metabolic capacities and CRISPR-Cas loci. CRISPR spacers on an Acidithiobacillus plasmid targeted plasmid-borne conjugal transfer genes found in the same genus, likely belonging to another plasmid, evidence of intra-plasmid competition. We conclude that the structure and composition of metal sulphide minerals select for distinct consortia and associated mobile elements, some of which have the potential to impact microbial activity during sulphide ore dissolution.},
}
@article {pmid41381248,
year = {2026},
author = {Adamopoulos, PG and Athanasopoulou, K and Scorilas, A},
title = {A versatile type VI CRISPR-based approach for targeted m[6]A demethylation in mRNAs.},
journal = {Genome research},
volume = {36},
number = {1},
pages = {169-182},
pmid = {41381248},
issn = {1549-5469},
mesh = {Humans ; *RNA, Messenger/genetics/metabolism ; *Adenosine/analogs &amp; derivatives/metabolism ; HeLa Cells ; AlkB Homolog 5, RNA Demethylase/genetics/metabolism ; Demethylation ; *CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; },
abstract = {Epitranscriptomics, a rapidly evolving field mainly driven by massive parallel sequencing technologies, explores post-transcriptional RNA modifications. N [6]-methyladenosine (m[6]A) has emerged as the most prominent and dynamically regulated modification in human mRNAs, being implicated in the regulation of diverse biological processes, including spermatogenesis, heat shock response, ultraviolet-induced DNA damage response and maternal mRNA clearance. Despite the recognized significance of m[6]A in mRNA regulation, limited studies have focused on the targeted and efficient manipulation of this modification in mRNAs. Here, we present Dem6A-Vec, an "all-in-one" plasmid vector designed for site-specific m[6]A demethylation in human mRNAs. Dem6A-Vec integrates the expression of a catalytically inactive RfxCas13d fused to the m[6]A demethylase ALKBH5 and a U6-driven customizable guide RNA in a single construct, simplifying experimental workflows and enhancing targeting efficiency. Using nanopore direct RNA sequencing, we identify high-confident m[6]A sites in HeLa cells, which serve as targets for Dem6A-Vec. We validate the targeted demethylation of m[6]A sites in the EEF2 and RRAGA genes using the established SELECT-qPCR method, confirming the impacts on mRNA stability and highlighting the tool's precision and versatility. The presented approach is implemented in multiple mRNA sites with diverse methylation stoichiometries, underscoring its adaptability to various transcriptomic contexts. This study provides a robust and scalable method for investigating the functional roles of m[6]A modifications, offering a transformative platform for advancing epitranscriptomic research and potential therapeutic applications.},
}
@article {pmid41381416,
year = {2025},
author = {Cheng, AS and Li, LX and Zhou, JX and Harris, PC and Calvet, JP and Li, X},
title = {In vivo base editing rescues ADPKD in a humanized mouse model.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11212},
pmid = {41381416},
issn = {2041-1723},
support = {DK126662//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; R01 DK126662/DK/NIDDK NIH HHS/United States ; K01 DK107729/DK/NIDDK NIH HHS/United States ; DK058816//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; DK129241//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; DK059597//U.S. Department of Health &amp; Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (National Institute of Diabetes &amp; Digestive &amp; Kidney Diseases)/ ; R01 DK059597/DK/NIDDK NIH HHS/United States ; PR221810//U.S. Department of Defense (United States Department of Defense)/ ; R01 DK129241/DK/NIDDK NIH HHS/United States ; R01 DK058816/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; *Polycystic Kidney, Autosomal Dominant/genetics/therapy/pathology ; *Gene Editing/methods ; Disease Models, Animal ; Humans ; Mice ; Dependovirus/genetics ; TRPP Cation Channels/genetics ; Kidney/pathology/metabolism ; CRISPR-Cas Systems/genetics ; Mutation ; *Genetic Therapy/methods ; Liver/pathology/metabolism ; Male ; Promoter Regions, Genetic ; Female ; },
abstract = {Autosomal dominant polycystic kidney disease (ADPKD) is a genetic kidney disease, caused by mutations of the PKD1 and PKD2 genes, characterized by the development of renal cysts and extrarenal complications, such as cardiac hypertrophy. Recently, a revolutionary approach, adeno-associated virus (AAV) delivered CRISPR-Cas9 gene editing, has been developed to treat inherited diseases. However, the use of this technology in kidney diseases in vivo is challenged. In this study, we adapt one of the gene editing systems, adenine base editor (ABE9), to develop a broadly expressed and a kidney-specific promoter mediated base editors, and test the effects of these two systems delivered by AAV9 on preventing disease in humanized Pkd1[RC/RC] mice carrying an arginine (R) to cystine (C) mutation that mimics a mutation in ADPKD patients. We show that one dose of the broadly expressed dual ABE9-AAV9 treatment corrects the pathogenic variant in kidneys, hearts and livers, and result in delaying cyst growth, decrease heart hypertrophy and improve liver function. To confirm the specificity of the base editor system in kidneys, we show that one dose of the kidney specific promoter mediated dual-ABE9-AAV9 treatment corrects the Pkd1 gene mutation in the kidney, and not in the heart, resulting in delaying cyst growth in Pkd1[RC/RC] kidneys, supporting a promising strategy of using base editor to target specific organs. Treatment with ABE9 base editors mediated by either the broadly expressed or kidney specific promoter increased the survival rate of Pkd1[RC/null] mice. These preclinical studies support a potential that single-dose genetic therapies may be through the correction of pathogenic variants to prevent ADPKD development in the clinic.},
}
@article {pmid41381501,
year = {2025},
author = {Padilla, R and Shipman, GA and Horth, C and Gravel, M and Bareke, E and Majewski, J},
title = {H3K36 Methylation as a Guardian of Epigenome Integrity.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11371},
pmid = {41381501},
issn = {2041-1723},
support = {P01 CA196539/CA/NCI NIH HHS/United States ; PJT-183939//Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Sant&#xe9; du Canada)/ ; P01-CA196539//U.S. Department of Health &amp; Human Services | NIH | NCI | Division of Cancer Epidemiology and Genetics, National Cancer Institute (National Cancer Institute Division of Cancer Epidemiology and Genetics)/ ; },
mesh = {Animals ; *Histones/metabolism/genetics ; Humans ; Mice ; Methylation ; *Epigenome/genetics ; Heterochromatin/metabolism/genetics ; Mesenchymal Stem Cells/metabolism ; Methyltransferases/metabolism/genetics ; Histone-Lysine N-Methyltransferase/genetics/metabolism ; *Epigenesis, Genetic ; Repressor Proteins/metabolism/genetics ; Mice, Knockout ; Chromatin/metabolism ; Euchromatin/metabolism ; CRISPR-Cas Systems ; Cell Line, Tumor ; },
abstract = {H3K36 methylation is a key epigenetic mark with critical roles in development and disease. Here, we systematically dissect its functions using CRISPR-engineered mouse mesenchymal stem cells lacking combinations of the five H3K36 methyltransferases, culminating in quintuple knockout cells devoid of H3K36me2/3. We show that H3K36me2 influences enhancer activity, supports the expression of their target genes, and safeguards active genes from encroachment of the repressive marks, H3K27me2/3. In addition, we find that the loss of H3K36me triggers redistribution of large heterochromatic H3K9me3 domains into euchromatin, in part mediated by SUV39H1, leading to global epigenomic remodelling, constitutive heterochromatin erosion, and a collapse of 3D genome organization. Parallel analyses in human HNSCC cells overexpressing the H3K36M oncohistone reveal conserved disruptions to the epigenome and chromatin architecture. Together, these results establish H3K36 methylation as a pivotal regulator of chromatin state and genomic structure.},
}
@article {pmid41381532,
year = {2025},
author = {Zaada, DSY and Toren, O and Krsticevic, F and Haber, DA and Gildman, D and Galpaz, N and Häcker, I and Schetelig, MF and Marois, E and Arien, Y and Papathanos, PA},
title = {Mosquito sex separation using complementation of selectable traits and engineered neo-sex chromosomes.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11175},
pmid = {41381532},
issn = {2041-1723},
support = {3-1679//Ministry of Science, Technology and Space/ ; 1833/7-1//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; 101059523//EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)/ ; ANR-23-CE35-0003//Agence Nationale de la Recherche (French National Research Agency)/ ; },
mesh = {Animals ; Female ; Male ; *Aedes/genetics/physiology ; *Sex Chromosomes/genetics ; CRISPR-Cas Systems ; Genetic Engineering ; *Sex Determination Processes/genetics ; Animals, Genetically Modified ; Pigmentation/genetics ; Phenotype ; Insect Proteins/genetics ; },
abstract = {Effective sex separation remains a critical challenge for mosquito genetic control. Genetic sexing strains (GSS) address this by linking maleness with selectable traits, enabling efficient female removal. Here, we present a versatile platform for GSS development in the invasive Aedes albopictus mosquito that integrates CRISPR-engineered selectable phenotypes with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow pigmentation gene and restore its function in males using nix-containing transgenes, producing a stable strain with yellow females and dark males. Beyond serving as a vivid marker, yellow confers added advantages: GSS females pupate later than wild females, enhancing protandry-based sorting, and lay desiccation-sensitive eggs, lowering accidental female release risk. The strain is compatible with size-based separation, improving sexing accuracy through the integration of natural and engineered dimorphisms. To our knowledge, this represents the first engineered sex-linked selectable trait in mosquitoes based on endogenous genes, establishing a foundation for scalable GSS development.},
}
@article {pmid41381927,
year = {2025},
author = {Yousuf, F and Solanki, M and Singh, SS and Ch, SR and Neeraja, CN and Sundaram, RM and Mangrauthia, SK},
title = {Tissue culture optimization and genome editing for yield improvement of an Indian rice landrace Chittimuthyalu.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {54},
pmid = {41381927},
issn = {1573-9368},
support = {ICAR-EFC Sub-Scheme 10: Enhancing climate resilience and ensuring food security with genome editing tools//Indian Council of Agricultural Research/ ; },
mesh = {*Oryza/genetics/growth &amp; development/drug effects ; *Gene Editing/methods ; *Tissue Culture Techniques/methods ; Plant Breeding ; India ; *Plants, Genetically Modified/genetics/growth &amp; development ; Purines/pharmacology ; CRISPR-Cas Systems ; Phenylurea Compounds ; Thiadiazoles ; },
abstract = {Chittimuthyalu, a rice landrace from Southern India, is known for its pleasant aroma, rich nutritive value, and excellent cooking qualities. However, it has a poor plant type (tall and weak stem prone to lodging) and is low yielding. The efforts to improve such valuable rice accessions with existing cross-breeding or random mutagenesis often result in undesirable traits due to linkage drag or untargeted mutations in large numbers. Genome editing, the most precise breeding tool, offers a viable solution to address such issues. In this study, we developed an efficient tissue culture protocol for callus induction, transformation, and regeneration of Chittimuthyalu. The highest callus induction frequency was achieved on L3 basal media enriched with 2.5 mg/l 2,4-Dichlorophenoxyacetic acid (2,4-D) and 600 mg/l of both proline and glutamine. For regeneration, a combination of Thidiazuron (TDZ), 6-Benzylaminopurine (BAP), and kinetin yielded an optimal regeneration frequency. The optimized tissue culture protocol was utilized to transform a multiplex gene editing construct developed by combining the four guide RNAs designed from yield and disease resistance-associated genes OsDEP1, OsTB1, OsCKX2, and OsSWEET14. The OsDEP1genome-edited rice plants exhibit thicker culm, enhanced grain size, ~ 100% increase in the thousand-grain weight, and ~ 50% increase in total grain yield per plant. The optimized tissue culture protocol and development of further edits in the remaining genes will pave the way for improving the agronomic traits of Chittimuthyalu. This study also highlights much-needed efforts to develop efficient tissue culture and genome editing methods for wild rice species and landraces, which will help bring these hardy, climate-resilient, and nutrient-rich accessions into mainstream cultivation.},
}
@article {pmid41382257,
year = {2025},
author = {Chen, W and Wu, P and Champer, J},
title = {Strategies to improve the efficiency of homing gene drives with multiplexed gRNAs.},
journal = {BMC biology},
volume = {24},
number = {1},
pages = {12},
pmid = {41382257},
issn = {1741-7007},
support = {32270672//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *Gene Drive Technology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems ; },
abstract = {BACKGROUND: CRISPR homing gene drive holds great potential for pest control, but its success is challenged by the generation of resistance alleles through end-joining repair. Using multiple gRNAs to target adjacent sites within a conserved gene can prevent functional resistance by allowing repeated cleavage events, but poor homology during DNA repair may compromise efficiency.<br><br>RESULTS: We first assessed the efficiency of single gRNA drives with truncated homology arms in Drosophila melanogaster mimicking a multiplexed system in which only one site is cleaved. Integrating results into a detailed gRNA multiplexing model, we found that efficiency loss was greater than expected. To mitigate this, we evaluated two new strategies: (1) extended homology arms to span all target sites (with mutations in the PAMs to prevent self-cleavage) and (2) a population-level gRNA multiplexing system involving two or more drives, each carrying two gRNAs. Extended homology arms did not result in notable improvement in conversion efficiency, and the extended region could be lost during drive conversion. The population-level multiplexing gRNAs strategy was more promising, though the intentionally mutated PAM also could not be consistently inherited. Simulations of homing suppression drives applying population-level multiplexed gRNAs increased the success rate of population elimination and reduced the time required for suppression.<br><br>CONCLUSIONS: Future drive designs requiring a larger number of gRNAs could potentially be improved. The design relying on extended homology arms may not represent an optimal strategy. However, population-level multiplexing gRNAs could serve as a promising alternative, enhancing efficiency while maintaining tolerance to functional resistance.},
}
@article {pmid41384994,
year = {2025},
author = {Zhang, J and Liu, J and Bayani, A},
title = {Phage therapy and the microbiome in hematologic malignancies: opportunities, mechanisms, and early evidence.},
journal = {Journal of cancer research and clinical oncology},
volume = {152},
number = {1},
pages = {8},
pmid = {41384994},
issn = {1432-1335},
mesh = {Humans ; *Hematologic Neoplasms/therapy/microbiology/immunology ; *Phage Therapy/methods ; Animals ; *Bacteriophages ; *Gastrointestinal Microbiome ; *Microbiota ; },
abstract = {Hematologic malignancies remain among the most difficult cancers to treat, challenged by profound heterogeneity, treatment-induced immune dysfunction, and the frequent emergence of drug resistance. Beyond tumor-intrinsic mechanisms, dysbiosis of the gut microbiome is increasingly recognized as a critical determinant of therapeutic outcomes, shaping hematopoiesis, immune responses, and drug metabolism. Bacteriophage (phage) therapy has re-emerged as a precision tool capable of selectively eradicating pathogenic taxa while preserving commensal short-chain fatty acid-producing communities. Preclinical and early human studies demonstrate that phages can recalibrate microbial ecosystems, disrupt antibiotic-tolerant biofilms, and enrich metabolites such as butyrate that support mucosal integrity and immune balance. Mechanistically, phage DNA enriched with CpG motifs engages Toll-like receptor 9, activating dendritic cells and enhancing cytotoxic T lymphocyte responses, suggesting dual benefits in infection control and anti-tumor immunity. Emerging applications extend further, with engineered phages serving as vectors for CRISPR-Cas gene editing, targeted cytokine delivery, and nanocarrier platforms for leukemia therapy. Despite translational promise, major hurdles persist, including immunogenicity, horizontal gene transfer, resistance evolution, and regulatory uncertainty. Addressing these challenges through GMP-compliant manufacturing, metagenomics-guided personalization, and AI-optimized cocktail design could establish phage therapy as a microbiome-informed adjunct to overcome drug resistance in blood cancers. However, direct clinical evidence of phage therapy efficacy in hematologic malignancies remains limited, and current data are largely derived from preclinical and compassionate-use contexts.},
}
@article {pmid41385323,
year = {2025},
author = {Zahm, AM and Cranney, CW and Gormick, AN and Rondem, KE and Schmitz, B and Himes, SR and English, JG},
title = {ConSeqUMI, an error-free nanopore sequencing pipeline to identify and extract individual nucleic acid molecules from heterogeneous samples.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41385323},
issn = {1362-4962},
support = {DP2 GM146247/GM/NIGMS NIH HHS/United States ; //NIH/ ; 1DP2GM146247-01/GM/NIGMS NIH HHS/United States ; },
mesh = {*Nanopore Sequencing/methods ; Humans ; SARS-CoV-2/genetics ; Spike Glycoprotein, Coronavirus/genetics ; COVID-19/virology ; High-Throughput Nucleotide Sequencing/methods ; CRISPR-Cas Systems ; *Sequence Analysis, DNA/methods ; Dependovirus/genetics ; *Nucleic Acids/genetics/isolation &amp; purification ; Genome, Viral ; },
abstract = {Nanopore sequencing has revolutionized genetic analysis by offering linkage information across megabase-scale genomes. However, the high intrinsic error rate of nanopore sequencing impedes the analysis of complex heterogeneous samples, such as viruses, bacteria, complex libraries, and edited cell lines. Achieving high accuracy in single-molecule sequence identification would significantly advance the study of diverse genomic populations, where clonal isolation is traditionally employed for complete genomic frequency analysis. Here, we introduce ConSeqUMI, an innovative experimental and analytical pipeline designed to address long-read sequencing error rates using unique molecular indices for precise consensus sequence determination. ConSeqUMI processes nanopore sequencing data without the need for reference sequences, enabling accurate assembly of individual molecular sequences from complex mixtures. We establish robust benchmarking criteria for this platform's performance and demonstrate its utility across diverse experimental contexts, including mixed plasmid pools, recombinant adeno-associated virus genome integrity, and CRISPR/Cas9-induced genomic alterations. Furthermore, ConSeqUMI enables detailed profiling of human pathogenic infections, as shown by our analysis of severe acute respiratory syndrome coronavirus 2 spike protein variants, revealing substantial intra-patient genetic heterogeneity. Lastly, we demonstrate how individual clonal isolates can be extracted directly from sequencing libraries at low cost, allowing for post-sequencing identification and validation of observed variants. Our findings highlight the robustness of ConSeqUMI in processing sequencing data from UMI-labeled molecules, offering a critical tool for advancing genomic research.},
}
@article {pmid41385544,
year = {2025},
author = {Fu, YZ and Luo, FF and Yang, L and Zhang, YX and Li, JY and Wang, SY and Zhang, Y and Wang, YY},
title = {SPNS1 is an essential cellular factor for EV-A71 by acting as a transporter of viral pocket factor.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {50},
pages = {e2510020122},
pmid = {41385544},
issn = {1091-6490},
support = {2023YFC2306100//The National Key R&amp;D Program China/ ; 82372230//The National Natural Science Foundation of China/ ; U23A20168//The National Natural Science Foundation of China/ ; No.2022338//The Youth Innovation Promotion Association CAS/ ; },
mesh = {Humans ; Animals ; *Enterovirus A, Human/physiology/metabolism/genetics ; Virus Replication ; *Enterovirus Infections/virology/metabolism/genetics ; Lysosomes/metabolism ; Mice ; Capsid Proteins/metabolism ; CRISPR-Cas Systems ; HEK293 Cells ; Endosomes/metabolism ; Receptors, Scavenger ; Lysosomal Membrane Proteins ; },
abstract = {Human enterovirus A71 (EV-A71) is a major cause of hand, foot and mouth disease. Cellular factors critical for EV-A71 infection remain enigmatic. Here, we performed CRISPR/Cas9 screens and identified sphingolipid transporter 1 (SPNS1) as an essential factor for EV-A71. SPNS1 deficiency inhibits infection of EV-A71 and 9 of 11 examined enteroviruses. Mechanistically, the endo/lysosomal localization of SPNS1 and the acidification of the endo/lysosomes are essential for SPNS1 to support EV-A71 infection. SPNS1 deficiency inhibits EV-A71 genomic RNA replication, but barely affects replication of EV-A71 RNA directly transfected into the cytoplasm. SPNS1 interacts with the EV-A71 capsid protein VP1 and entry receptor SCARB2 in the endo/lysosomes, where it acts as a transporter to release the viral pocket factor into the cytosol, leading to uncoating. Animal experiments show that SPNS1 deficiency results in reduced viral loads, pathological effects, and lethality following EV-A71 infection. Our findings collectively identified SPNS1 as a transporter of the EV-A71 viral pocket factor.},
}
@article {pmid41385899,
year = {2026},
author = {Fathy, K and Bharti, J and Khan Sony, S and Nehra, M and Kaul, R and Rawat, B and Sopory, SK and Agrawal, PK and Prakash, A and Kaul, T},
title = {Triumphing over hidden hunger: Redesigning rice (Oryza sativa L.) for enhanced nutraceutical grain composition utilizing multiplexed genome editing.},
journal = {Journal of plant physiology},
volume = {316},
number = {},
pages = {154667},
doi = {10.1016/j.jplph.2025.154667},
pmid = {41385899},
issn = {1618-1328},
mesh = {*Oryza/genetics/metabolism/chemistry ; *Gene Editing/methods ; *Edible Grain/chemistry/genetics ; Cadmium/metabolism/analysis ; *Dietary Supplements ; Plants, Genetically Modified/genetics ; CRISPR-Cas Systems ; Plant Breeding ; Zinc/metabolism ; Iron/metabolism ; },
abstract = {Rice, a staple food crop, is consumed by most of the world's population. Micronutrient malnutrition is a severe health issue, leading to diseases such as cancer, anemia, diabetes, heart disease, and disorders in physical and psychological development. We aimed to create rice with low cadmium in the grain but having high cadmium in shoots, safe biofortified protein, high iron, and zinc using CRISPR/Cas9 and breeding technologies instead of adding drugs. The triple gene Knockout rice lines for two iron sensors and one negative regulator gene for cadmium were created to offer high Fe/Zn and low Cd content for breeders. Multiplexed gene editing mediated biolistic transformation of rice callus, and genotyping was used to check the genetic stability of the edited rice lines. Rice lines were found to have enhanced iron, zinc, and protein content, with concentrations varying based on growth conditions. These lines can be used as phytoremediators for cadmium by storing Cd on plant shoots. The rice-edited plants possessed excellent agro-morphological traits, photosynthetic, and physiological performance. The developed edited indica rice lines have crucial agronomic traits with more nutritional value. Compared to the other lines and the wild wildtype, the genome-edited free Cas9 line 2 showed better traits: 13.48 μg/g (iron), 22.9 μg/g (zinc), and a high protein content, which depends on how bioavailable metals and nutrients are in the soil. The line also had 20.60 g of seeds per 1000 g of plant, a total plant yield of 102.76 g, and 101 days of 50 % flowering. This work offers efficient and precise multiple gene-editing in rice with an effective, sustainable strategy for multi-trait enhancement. The developed lines could be used in breeding programs for sustainable solutions for malnutrition worldwide. The experimental results can provide reference and support for the safe use of edited crops as a diet.},
}
@article {pmid41385994,
year = {2026},
author = {Luo, Y and Wang, X and Yang, F and Zhao, Y and Hu, S and Liu, S and Li, S and Luo, G and Sun, Q},
title = {Construction and validation of a rapid semen identification system based on SHERLOCK technology.},
journal = {Forensic science international. Genetics},
volume = {82},
number = {},
pages = {103410},
doi = {10.1016/j.fsigen.2025.103410},
pmid = {41385994},
issn = {1878-0326},
mesh = {Humans ; *Semen/chemistry ; Male ; *CRISPR-Cas Systems ; *RNA, Messenger/genetics ; *Forensic Genetics/methods ; DNA Primers ; Sensitivity and Specificity ; },
abstract = {This study developed a rapid detection system for semen-specific mRNA based on CRISPR/Cas13a system to meet the timeliness requirements of forensic on-site body fluid identification. Specific primers and CRISPR RNA (crRNA) short fragments on semen specific mRNA genes were designed and screened, to establish a SHERLOCK detection method based on technology principles of CRISPR/Cas. Furthermore, nucleic acid rapid release agents for treating samples were screend to construct a new detection method in combination with SHERLOCK, and the specificity and sensitivity of the method were tested. The method can rapidly detect the presence of semen from unknown body fluid samples, and the relative fluorescence unit (RFU) value of the semen sample is significantly higher than those of non-semen samples (P < 0.0001), with a sample detection sensitivity of down to 0.25 μL. The construction of the rapid semen detection method using rapid extraction and SHERLOCK reduces operation time, significantly reduces instrument dependence, and provides an innovative solution for forensic on-site rapid body fluid identification.},
}
@article {pmid41386334,
year = {2026},
author = {Svane, N and Kurosawa, T and Schmid, B and Saaby, L and Kristensen, M and Tabata, H and Kubo, Y and Terasaki, T and Brodin, B and Deguchi, Y},
title = {Elucidating the roles of TM7SF3 and LHFPL6 in the putative H[+]/OC antiporter function in the human brain capillary endothelial cell line, hCMEC/D3.},
journal = {European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences},
volume = {217},
number = {},
pages = {107409},
doi = {10.1016/j.ejps.2025.107409},
pmid = {41386334},
issn = {1879-0720},
mesh = {Humans ; *Endothelial Cells/metabolism ; Blood-Brain Barrier/metabolism ; *Brain/metabolism/blood supply ; Cell Line ; *Antiporters/metabolism/genetics ; Biological Transport ; *Membrane Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: The putative proton/organic cation (H[+]/OC) antiporter has been shown to mediate transport of CNS drug compounds like oxycodone and pyrilamine across the blood-brain barrier (BBB). This transporter has a broad substrate profile and is able to transport substrates against their concentration gradient, making it an interesting target for brain drug delivery. However, the molecular identity of this transporter remains unknown. Recent studies have indicated that the two proteins TM7SF3 and LHFPL6 might be components of this transporter. The present study aimed to investigate the roles of TM7SF3 and LHFPL6 in the H[+]/OC antiporter function to advance understanding of its molecular identity and potential in CNS drug delivery.<br><br>METHODS: CRISPR-Cas9 gene-editing was used to generate three hCMEC/D3 knockout (KO) cell lines: TM7SF3 KO (TM-KO), LHFPL6 KO (LH-KO), and a double KO of TM7SF3 and LHFPL6 (TMLH-KO). The uptake of pyrilamine analogue (EDMPG) and [[3]H]-pyrilamine was assessed in wild type (WT) and KO lines. Quantitative Realtime Polymerase Chain Reaction (qRT-PCR) confirmed successful gene knockouts. Passive diffusion properties and the expression and functionality of known BBB transporters, including LAT1 (SLC7A5), GLUT1 (SLC2A1), and MCT1 (SLC16A1), were also examined.<br><br>RESULTS: The EDMPG uptake was significantly reduced in TM-, LH-, and TMLH-KO cells, suggesting that TM7SF3 and LHFPL6 contribute to the H[+]/OC antiporter function. However, [[3]H]-pyrilamine uptake remained unchanged across all KOs, indicating a TM7SF3- and LHFPL6-independent transport mechanism. This was further supported by the persistent inhibition of [[3]H]-pyrilamine uptake in the presence of known H[+]/OC antiporter substrates. While passive diffusion and GLUT1- and MCT1-mediated transport were unaffected, LAT1-mediated uptake of [[3]H]L-leucine and gabapentin (Neurontin) was significantly reduced in LH- and TMLH-KO cells, correlating with decreased LAT1 mRNA expression in these cells.<br><br>CONCLUSIONS: This study suggests that the H+/OC antiporter operates via two distinct mechanisms: a high-capacity, TM7SF3- and LHFPL6-independent pathway and a low-capacity, TM7SF3- and LHFPL6-dependent pathway. These findings underscore the complexity of the H[+]/OC antiporter molecular composition and highlight the need for further research to fully elucidate its identity.},
}
@article {pmid41387334,
year = {2025},
author = {Song, N and Tian, G and Li, H and Zhang, L and Wang, Y and Zhao, W and Yao, C and Yang, D},
title = {DNA Nanoflowers Efficiently Encapsulate Photodynamic Agents and CRISPR/Cas9 for Synergistic Pancreatic Cancer Therapy.},
journal = {Nano letters},
volume = {25},
number = {51},
pages = {17693-17701},
doi = {10.1021/acs.nanolett.5c04676},
pmid = {41387334},
issn = {1530-6992},
support = {//National Natural Science Foundation of China/ ; //Tianjin Key Medical Discipline Construction Project/ ; },
mesh = {*Pancreatic Neoplasms/drug therapy/pathology/genetics/therapy/metabolism ; *Photochemotherapy/methods ; Humans ; Animals ; *CRISPR-Cas Systems ; Mice ; NF-E2-Related Factor 2/genetics/metabolism ; Cell Line, Tumor ; *Photosensitizing Agents/chemistry/pharmacology/administration &amp; dosage/therapeutic use ; Chlorophyllides ; *DNA/chemistry ; Reactive Oxygen Species/metabolism ; Porphyrins/chemistry/pharmacology/administration &amp; dosage ; Hemin/chemistry ; Apoptosis/drug effects ; Gene Editing ; Ribonucleoproteins/genetics ; G-Quadruplexes ; },
abstract = {Photodynamic therapy (PDT) holds significant promise for treating pancreatic cancer by utilizing photosensitizers to generate reactive oxygen species (ROS) that induce tumor cell death. However, the therapeutic efficacy of PDT is hindered by inadequate ROS accumulation. Herein, we develop a DNA nanoflower that enables the controlled codelivery of Cas9 ribonucleoprotein (RNP), hemin, and chlorin e6 for synergistic PDT. The Cas9 RNP selectively knocks out the antioxidant regulator nuclear factor E2-related factor 2 (Nrf2), thereby increasing cancer cells' sensitivity to ROS. Simultaneously, the G-quadruplex/hemin complex catalyzes the conversion of endogenous H2O2 into O2, alleviating tumor hypoxia and supplying additional oxygen for PDT. This synergistic approach substantially amplifies ROS accumulation by attenuating ROS elimination and enhancing ROS generation, demonstrating high gene editing efficiency, significant Nrf2 down-regulation, elevated apoptosis, and remarkable antitumor efficacy in pancreatic cancer cells and a mouse model, underscoring the potential for precision medicine.},
}
@article {pmid41387457,
year = {2025},
author = {Cheng, F and Soleimani Samarkhazan, H and Khazaei, Y},
title = {CRISPR-engineered microbiome: living therapeutics revolutionize blood cancer immunotherapy.},
journal = {NPJ biofilms and microbiomes},
volume = {12},
number = {1},
pages = {17},
pmid = {41387457},
issn = {2055-5008},
mesh = {Humans ; *Immunotherapy/methods ; Animals ; *Hematologic Neoplasms/therapy/immunology/microbiology ; *CRISPR-Cas Systems ; *Gastrointestinal Microbiome/genetics ; *Microbiota ; },
abstract = {Blood cancers such as leukemia, lymphoma, and myeloma remain refractory in many patients due to immune escape, antigen heterogeneity, and therapy‑related toxicities. To address these challenges, we review recent strategies that harness CRISPR‑engineered gut commensals as precision "living therapeutics" to modulate host immunity and directly target malignant clones. We frame this review around three principal themes: (1) mechanistic strategies whereby CRISPR-engineered commensals modulate host immunity and directly antagonize malignant clones; (2) the enabling technologies and delivery/containment platforms, CRISPR variants, phage/LNP delivery, genetic circuits and biocontainment, that make living therapeutics feasible; and (3) translational progress, outstanding technical and safety barriers, and ethical/regulatory challenges that must be addressed for clinical deployment. To illustrate these themes, we discuss three concrete therapeutic modalities: engineered microbial secretion of immunomodulators, targeted delivery of tumor-lytic payloads, and engineered production of anticancer metabolites, and how these are enabled by contemporary CRISPR and synthetic-biology toolkits. Selected preclinical models report substantial antitumor effects, often >60% tumor reduction in rodent studies, and restoration of CAR-T cell function in controlled settings; however, effect sizes vary across models, and human translation remains unproven. We also analyze key technical barriers, strain stability, biocontainment, off‑target effects, and propose solutions, including auxotrophic kill-switches and AI‑guided strain optimization. Finally, we outline future directions, from in situ phage delivery to multi‑omics-driven patient stratification. CRISPR‑microbiome editing represents a paradigm shift in hematologic oncology, offering localized, sustained therapy with reduced systemic toxicity.},
}
@article {pmid41387726,
year = {2025},
author = {Srinivasan, R and Sun, T and Sandles, A and Wu, D and Wang, L and Patel, H and Pabalate, R and Bader, M and Heidersbach, A and Ho, C and Xie, S and Ng, A and Haley, B},
title = {Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {504},
pmid = {41387726},
issn = {2041-1723},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; HEK293 Cells ; CRISPR-Associated Protein 9/genetics/metabolism ; },
abstract = {CRISPR/Cas9 technologies provide unique capabilities for modeling disease and understanding gene-to-phenotype connections. In cultured cells, chemical-mediated control of Cas9 activity can limit off-target effects and enable mechanistic study of essential genes. However, widely-used Tet-On systems often show leaky Cas9 expression, leading to unintended edits, as well as weak activity upon induction. Leakiness can be problematic in the context of Cas9 nuclease activity, which may result in cumulative DNA damage and degradation of the target cell genome over time. To overcome these deficiencies, we have established transgenic platforms that minimize Cas9 functionality in the OFF-state along with maximized and uncompromised ON-state gene editing efficiency. By combining conditional destabilization and inhibition of Cas9, we have developed an all-in-one (one or multiple guide RNAs and Cas9) ultra-tight, Tet-inducible system with exceptional dynamic range (ON vs. OFF-state) across various cell lines and targets. As an alternative to Tet-mediated induction, we have created a Branaplam-regulated splice switch module for low-baseline and robust Cas9 activity control. Lastly, for circumstances where DNA damage needs to be avoided, we have constructed a dual-control, Tet-inducible CRISPRi module for tight and potent transcriptional silencing. This upgraded suite of inducible CRISPR systems has broad applications for numerous cell types and experimental conditions.},
}
@article {pmid41387738,
year = {2025},
author = {Zhang, X and Zhu, T and Zhang, W and Zhang, Y and Zhang, J and Yang, J and Xia, C and Zhao, H and Yu, Y and Wen, C},
title = {Ethylene promotes branch formation but inhibits tendril development in cucumber.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {745},
pmid = {41387738},
issn = {2041-1723},
support = {JKZX202207//Beijing Academy of Agricultural and Forestry Sciences (BAAFS)/ ; },
mesh = {*Ethylenes/pharmacology/metabolism ; *Cucumis sativus/growth &amp; development/genetics/metabolism/drug effects ; Gene Expression Regulation, Plant/drug effects ; Plant Proteins/genetics/metabolism ; Signal Transduction/drug effects ; CRISPR-Cas Systems ; *Plant Growth Regulators/pharmacology/metabolism ; Mutation ; Promoter Regions, Genetic ; },
abstract = {Ethylene coordinates numerous plant growth processes, particularly in cucurbit crops, yet its role in vegetative growth regulation remains largely unexplored. Here, we report the function of ethylene in controlling branch and tendril development in cucumber. We find that ethylene promotes branches formation but inhibits tendrils development in a dose-dependent manner. CRISPR-Cas9-generated gene-edited Csein2 and Csein3/Cseil1 mutants exhibit few branches and more tendrils. Exogenous ethylene can recover the branch/tendril defective phenotypes of the Csein3 and Cseil1 mutants but not those of the Csein2 mutant or the Csein3/Cseil1 double mutant. Transcriptomic and metabolic analyses reveal that CsCYP707A4 and CsTL are the key downstream targets of ethylene signaling. We show that CsEIN3 can bind to its promoters to activate the expression of CsCYP707A4 but inhibit the expression of CsTL, which leads to the opposite effect on branch and tendril development. The study sets the foundation for designing ideal plant architecture to increase production efficiency.},
}
@article {pmid41387770,
year = {2025},
author = {Ge, L and Li, W and Dou, Y and Ma, Y and Sun, M and Chen, X and Feng, X and Li, Y and Yu, Q},
title = {Callus and endosperm green fluorescence reporter-assisted selection system in maize CRISPR/Cas9 gene editing.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {92},
pmid = {41387770},
issn = {1471-2229},
support = {2022SZX13//Science&amp;Technology Specific Projects in Agricultural High-tech Industrial Demonstration Area of the Yellow River Delta/ ; ZR2024MC067//Natural Science Foundation of Shandong Province/ ; },
mesh = {*Zea mays/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Endosperm/genetics/metabolism ; *Green Fluorescent Proteins/genetics/metabolism ; Plants, Genetically Modified/genetics ; Genes, Reporter ; },
abstract = {BACKGROUND: Genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) has emerged as a promising approach for functional gene analysis and genetic improvement. Since stable transformation remains the primary method for implementing this system, the ultimate goal in crop breeding programs would require the selection of transgene-free plants with the CRISPR/Cas expression cassette removed.<br><br>RESULTS: In this study, we developed an endosperm-specific fluorescence reporter-assisted selection system for CRISPR/Cas9 gene editing (pAZS22-eGFP/CRISPR/Cas9) in maize (Zea mays L.), utilizing enhanced green fluorescent protein (eGFP) expressed specifically in the endosperm to facilitate the easy identification of transgenic and transgene-free plants from the T1 generation on. In addition, the 22&#xa0;kDa alpha zein (z1C1_10) promoter from maize, employed in this system, has been shown to be active in both callus and endosperm, thereby being able to enhance the accuracy of transformant identification during the tissue culture process by reducing false positives compared to the traditional selective media methods. Our studies targeting the ZmSnRK2.1 or Dwarf1 (D1) genes demonstrated a reasonable editing efficiency, with rates ranging from 56.3% for T0 plants targeting ZmSnRK2.1, to 87.5% and 100% for T1 plants targeting D1 and ZmSnRK2.1, respectively. In addition, we successfully identified 1 transgene-free homozygous d1 mutant in the T1 generation and 7 transgene-free homozygous snrk2.1 mutants in the T2 generation.<br><br>CONCLUSIONS: The pAZS22-eGFP/CRISPR/Cas9 system provides an efficient tool for gene editing, transformant selection and transgene status identification in maize breeding.},
}
@article {pmid41388295,
year = {2025},
author = {Ji, T and Zhang, Y and Wang, Y and Yuan, K and Wang, M and Ye, J and Zhang, H and Zhang, N and Zhang, H},
title = {AND logic-gated CRISPR/Cas9 and hybridization chain reaction system for precise ctDNA detection.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {43},
pmid = {41388295},
issn = {1477-3155},
support = {2022YFB3808200//National Key Research and Development Program of China/ ; },
mesh = {*Circulating Tumor DNA/genetics/blood/analysis ; Humans ; *CRISPR-Cas Systems/genetics ; Polymorphism, Single Nucleotide ; Nucleic Acid Amplification Techniques/methods ; Biomarkers, Tumor/genetics/blood ; Nucleic Acid Hybridization ; Mutation ; Proto-Oncogene Proteins p21(ras)/genetics ; Limit of Detection ; },
abstract = {Circulating tumor DNA (ctDNA) is a critical biomarker for liquid biopsies, enabling the non-invasive acquisition of cancer-related information from blood samples. Precise detection of ctDNA, particularly the identification of single-nucleotide variations (SNVs), is crucial for early cancer diagnosis, therapeutic monitoring, and prognostic evaluation. However, current ctDNA detection methods often encounter challenges such as complex procedures, difficult data analysis, and false-positive signals during pre-amplification. In this study, we introduce a novel detection method based on AND logic-gated integration of interspaced short palindromic repeats and associated proteins (CRISPR/Cas9) system with hybridization chain reaction (HCR) isothermal amplification. This strategy enhances the specific and sensitive detection of ctDNA. The incorporation of the AND logic gate effectively minimizes the off-target effects of Cas9 and enables the differentiation of single-nucleotide mutations, such as KRAS G12D, even in complex serum environments. Our system exhibits high sensitivity and specificity, achieving a limit of detection as low as 1 fM and capable of identifying SNVs mutations with allele fractions as low as 0.1% among wild-type sequences. Furthermore, we validated the specificity of our approach by successfully detecting various mutations, including KRAS G12C, KRAS G12D, EGFR T790M and TP53 R273H, in simulated clinical samples. These findings highlight a reliable method for precise ctDNA detection, offering high specificity, selectivity, and accuracy, thus paving the way for potential cancer diagnostic application.},
}
@article {pmid41389042,
year = {2025},
author = {Nie, YG and Zhang, HS and Su, M and Zha, CJ and Yang, K and Ying, ZM},
title = {Proximity-Inducible CRISPR/Cas12a Activity by Scaffold RNA Assembly for Sensing Applications.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {28088-28097},
doi = {10.1021/acs.analchem.5c06530},
pmid = {41389042},
issn = {1520-6882},
mesh = {Humans ; *MicroRNAs/analysis/genetics/blood ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Breast Neoplasms/diagnosis ; *RNA/chemistry/genetics/metabolism ; Adenosine Triphosphate/analysis ; *CRISPR-Associated Proteins/metabolism ; *Endodeoxyribonucleases/metabolism/genetics ; Female ; *Bacterial Proteins/metabolism ; },
abstract = {The programmability and flexibility of the RNA-directed CRISPR/Cas12a system underpin its utility as a potent tool for diagnostic applications. However, existing engineered crRNA strategies are still limited by a narrow target range, inadequate specificity, and operational complexity. To overcome these challenges, a proximity-assembly and activate (PAA) strategy was developed, employing split dumbbell activators with terminally modified target-binding modules that reassemble on scaffold RNA to reconstruct functional crRNA and activate Cas12a trans-cleavage activity. The design allows universal detection of both nucleic acid and non-nucleic acid targets. Notably, owing to its strict target dependency, the assembled crRNA biosensor significantly reduces background signal and suppresses nonspecific leakage. We demonstrated that the PAA system facilitates rapid and highly specific detection of miRNA-21, ATP, and anti-Dig antibody in complex matrices, enabling single-base discrimination among miRNA variants. Moreover, the platform successfully detected endogenous miRNA-21 in serum and cellular samples from breast cancer patients, clearly distinguishing them from healthy controls. This work presents a modular, plug-and-play, and versatile platform for molecular diagnostics, holding considerable potential for advancing clinical diagnostics and precision medicine.},
}
@article {pmid41389205,
year = {2025},
author = {Zobel, M and Damaggio, G and Mignogna, ML and Besusso, D and Scalzo, D and Cossu, A and Trovesi, C and Crosti, M and Cortina, F and Campus, I and Formenti, G and Mazzara, S and Gregoretti, F and Antonelli, L and Oliva, G and Zuccato, C and Colonna, V and Conforti, P and Cereda, M and Rossi, RL and Maestri, S and Scolz, A and Iennaco, R and Cattaneo, E},
title = {A human CAGinSTEM platform for decoding HTT repeats' somatic instability links CAG interruption to HD pathology in neurons.},
journal = {Cell reports},
volume = {44},
number = {12},
pages = {116685},
pmid = {41389205},
issn = {2211-1247},
mesh = {Humans ; *Huntington Disease/genetics/pathology ; *Huntingtin Protein/genetics/metabolism ; *Neurons/metabolism/pathology ; *Genomic Instability ; *Trinucleotide Repeats/genetics ; Trinucleotide Repeat Expansion ; CRISPR-Cas Systems/genetics ; },
abstract = {Somatic CAG instability in the mutant Huntingtin (HTT) gene is increasingly recognized as a key hallmark of Huntington's disease (HD). Using our novel human CAGinSTEM platform, we manipulated cis genetic elements influencing instability in human HD neurons, monitoring repeat length. Quality-controlled CRISPR-engineered stem cells with increasing CAG lengths and clinical haplotypes were analyzed using third-generation sequencing. Our findings link interruptions in the CAG repeat, especially the loss or duplication of the penultimate CAA of canonical alleles, to significant instability modulation. Notably, four internal CAA interruptions completely abolish CAG instability, reversing HD phenotypes such as altered striatal fate acquisition and nuclear disorganization. This platform highlights the role of cis modifiers, emphasizing the direct influence of HTT DNA repeat composition on CAG instability and providing a robust framework for modeling HTT repeat instability in vitro.},
}
@article {pmid41389602,
year = {2026},
author = {Kumari, P and Gupta, V and Chhikara, A and Dalal, J},
title = {Revolutionizing forensic DNA analysis: The potential of CRISPR-Cas9 technology in genetic investigations.},
journal = {Journal of forensic and legal medicine},
volume = {117},
number = {},
pages = {103047},
doi = {10.1016/j.jflm.2025.103047},
pmid = {41389602},
issn = {1878-7487},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Fingerprinting/methods ; *Forensic Genetics/methods ; *Gene Editing ; },
abstract = {The newest achievements in the field of molecular biology and gene-editing technologies have transformed the paradigm of forensic DNA analysis. However, there are still great difficulties in interpreting degraded, low-template, mixed genetic samples. The review critically evaluates the transformative potential of Clustered Regularly Interspaced Short Palindromic Repeats and an associated protein 9 (CRISPR-Cas9) as an accurate, effective, and cost-efficient system of genome-editing in the field of forensic science. Based on the evidence of the current literature, the paper critically analyzes the mechanisms of CRISPR-Cas9 activity, its RNA-guided specificity, dual-strand cleavage, and high-fidelity targeting, and compares its functionality with other standard methods like the STR and SNP profiling. The review also discusses more complex CRISPR-based diagnostic systems, such as SHERLOCK, DETECTR, and HOLMES that allow the analysis of DNA rapidly, without amplification, and in a portable format. Among major discoveries, there is the ability of CRISPR to increase the accuracy of DNA profiling, resolve mixture, recapitulate damaged genetic material, and reduce the possibility of contamination. In addition to genetic analysis, it has applications in forensic epigenetics, prediction of phenotypes, microbial forensics and environmental trace analysis. The review also covers the ethical, legal and governance implications of implementing CRISPR-based evidence in the judicial process especially in as far as data privacy; admissibility and equity of access are concerned. In general, CRISPR-Cas9 is a paradigm shift in forensic genomics, the one that has the potential to transform personal identification, reconstruction of the crime scene, and the interpretation of molecular evidence. Future efforts should focus on method validation, standardization, and ethical governance to ensure the responsible and sustainable implementation of this technology in forensic practice.},
}
@article {pmid41389662,
year = {2026},
author = {Ma, J and Zhao, CF and Liu, X},
title = {Advances in targeted therapeutics and smart delivery systems based on precision nano-oncology.},
journal = {International immunopharmacology},
volume = {169},
number = {},
pages = {115946},
doi = {10.1016/j.intimp.2025.115946},
pmid = {41389662},
issn = {1878-1705},
mesh = {Humans ; *Neoplasms/therapy ; *Precision Medicine/methods ; Animals ; *Drug Delivery Systems/methods ; *Nanomedicine/methods ; Immunotherapy/methods ; *Antineoplastic Agents/administration &amp; dosage/therapeutic use ; Nanoparticles ; Drug Carriers ; },
abstract = {The convergence of nanotechnology and precision oncology is revolutionizing cancer treatment by enabling highly specific, minimally invasive, and personalized therapeutic strategies. This review explores recent breakthroughs in nano-therapeutics and their pivotal role in overcoming the limitations of conventional cancer therapies. Emphasis is placed on the design and function of nanocarriers that facilitate targeted drug delivery via both passive (EPR effect) and active ligand-mediated mechanisms. Special attention is given to stimuli-responsive systems that release therapeutic agents in response to pH, enzymes, temperature, or redox environments, enhancing spatiotemporal control. The article further discusses the integration of nanotechnology with emerging modalities including immunotherapy, photothermal and photodynamic therapies, gene editing tools (e.g., CRISPR/Cas systems), and multifunctional theranostic platforms. While these innovations offer transformative potential, the review also addresses persistent challenges such as tumor heterogeneity, immune clearance, off-target effects, large-scale manufacturing, and regulatory complexity. By highlighting both promise and hurdles, this article provides a comprehensive lens into the future of precision cancer nanomedicine.},
}
@article {pmid41390353,
year = {2025},
author = {Howe, LJ and Aulchenko, YS and Davey Smith, G and Davies, NM and Esparza-Gordillo, J and Johnson, T and Liu, JZ and Richardson, TG and Sanseau, P and Scott, RA and Seaton, DD and Sharma, A and Cortes, A},
title = {Evaluating transportability of in vitro cellular models to in vivo human phenotypes using gene perturbation data.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {513},
pmid = {41390353},
issn = {2041-1723},
mesh = {Humans ; Phenotype ; Lysosomes/metabolism ; *Models, Biological ; Cholesterol, LDL/blood/metabolism ; CRISPR-Cas Systems ; Cholesterol/metabolism ; },
abstract = {Gene perturbation screens (e.g. CRISPR-Cas9) assess the impact of gene disruption on in-vitro cellular phenotypes (e.g., proliferation, anti-viral response). In-vitro experiments can be useful models for in-vivo (organismal) phenotypes (e.g., immune cell anti-viral response and infectious diseases). However, assessing whether an in-vitro cellular model effectively captures in-vivo biology is challenging. An in-vitro model is 'transportable' to an in-vivo phenotype if perturbations impacting the in-vitro phenotype also impact the in-vivo phenotype with mechanism-consistent directionality and effect sizes. We propose a framework; Gene Perturbation Analysis for Transportability (GPAT), to assess model transportability using gene perturbation effect estimates from perturbation screens (in-vitro) and loss-of-function burden tests (in-vivo). In hypothesis-driven analyses, GPAT provides evidence for model transportability of higher lysosomal cholesterol accumulation in-vitro to lower human plasma LDL-cholesterol (P = 0.0006), consistent with the known role of lysosomes in lipid biosynthesis. In contrast, there was limited evidence for other putative in-vitro models. In hypothesis-free analyses, we find evidence for transportability of cancer cell line proliferation to in-vivo human plasma cellular phenotypes (e.g. erythroleukemia proliferation and plasma lymphocyte percentage). Here we show that perturbation data can be used to evaluate transportability of in-vitro cellular models, informing assay prioritisation and supporting novel hypothesis generation.},
}
@article {pmid41390487,
year = {2025},
author = {Carruthers, DN and Kinnunen, PC and Li, Y and Chen, Y and Gin, JW and Yunus, IS and Galliard, WR and Tan, S and Radivojevic, T and Adams, PD and Singh, AK and Sustarich, J and Petzold, CJ and Mukhopadhyay, A and Garcia Martin, H and Lee, TS},
title = {Automation and machine learning drive rapid optimization of isoprenol production in Pseudomonas putida.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11489},
pmid = {41390487},
issn = {2041-1723},
support = {DE-AC0205CH11231//U.S. Department of Energy (DOE)/ ; },
mesh = {*Pseudomonas putida/metabolism/genetics ; *Machine Learning ; *Metabolic Engineering/methods ; *Hemiterpenes/metabolism ; CRISPR-Cas Systems ; Proteomics ; Metabolic Networks and Pathways/genetics ; Automation ; },
abstract = {Advances in genome engineering have improved our ability to perturb microbial metabolic networks, yet bioproduction campaigns often struggle with parsing complex metabolic datasets to efficiently enhance product titers. We address this challenge by coupling laboratory automation with machine learning to systematically optimize the production of isoprenol, a sustainable aviation fuel precursor, in Pseudomonas putida. The simultaneous downregulation through CRISPR interference of combinations of up to four gene targets, guided by machine learning, permitted us to increase isoprenol titer 5-fold in six consecutive design-build-test-learn cycles. Moreover, machine learning enabled us to swiftly explore a vast experimental design space of 800,000 possible combinations by strategically recommending approximately 400 priority constructs. High-throughput proteomics allowed us to validate CRISPRi downregulation and identify biological mechanisms driving production increases. Our work demonstrates that ML-driven automated design-build-test-learn cycles, when combined with rigorous data validation, can rapidly enhance titers without specific biological knowledge, suggesting that it can be applied to any host, product, or pathway.},
}
@article {pmid41390513,
year = {2025},
author = {Vermeulen, M and Craig, AW and Babak, T},
title = {Challenges and opportunities for oncology drug repurposing informed by synthetic lethality.},
journal = {NPJ systems biology and applications},
volume = {11},
number = {1},
pages = {143},
pmid = {41390513},
issn = {2056-7189},
support = {PJT 178214//Canadian Institutes of Health Research Project Grant/ ; },
mesh = {Humans ; *Drug Repositioning/methods ; *Synthetic Lethal Mutations/genetics ; Cell Line, Tumor ; CRISPR-Cas Systems/genetics ; *Antineoplastic Agents/pharmacology/therapeutic use ; *Neoplasms/genetics/drug therapy ; Gene Knockout Techniques ; Mutation ; },
abstract = {Although two-thirds of cancers arise from loss-of-function mutations in tumor suppressor genes, there are few approved targeted therapies linked to these alterations. Synthetic lethality offers a promising strategy to treat such cancers by targeting vulnerabilities unique to cancer cells with these mutations. To identify clinically relevant synthetic lethal interactions, we analyzed genome-wide CRISPR/Cas9 knock-out (KO) viability screens from the Cancer Dependency Map and evaluated their clinical relevance in patient tumors through mutual exclusivity, a pattern indicative of synthetic lethality. Indeed, we found significant enrichment of mutual exclusivity for interactions involving cancer driver genes compared to non-driver mutations. To identify therapeutic opportunities, we integrated drug sensitivity data to identify inhibitors that mimic the effects of CRISPR-mediated KO. This approach revealed potential drug repurposing opportunities, including BRD2 inhibitors for bladder cancers with ARID1A mutations and SIN3A-mutated cell lines showing sensitivity to nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. However, we discovered that pharmacological inhibitors often fail to phenocopy KO of matched drug targets, with only a small fraction of drugs inducing similar effects. This discrepancy reveals fundamental differences between pharmacological and genetic perturbations, emphasizing the need for approaches that directly assess the interplay of loss-of-function mutations and drug activity in cancer models.},
}
@article {pmid41390669,
year = {2025},
author = {Teske, M and Wertheimer, T and Butz, S and Zwicky, P and Mallona, I and Nopper, SL and Münz, C and Elling, U and Lancrin, C and Becher, B and Grosso, AR and Baubec, T and Schmolka, N},
title = {Targeted CRISPR-Cas9 screening identifies core transcription factors controlling murine haemato-endothelial fate commitment.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11412},
pmid = {41390669},
issn = {2041-1723},
support = {186012//Schweizerischer Nationalfonds zur F&#xf6;rderung der Wissenschaftlichen Forschung (Swiss National Science Foundation)/ ; FAN//Universit&#xe4;t Z&#xfc;rich (University of Zurich)/ ; },
mesh = {Animals ; Mice ; *CRISPR-Cas Systems/genetics ; *Transcription Factors/genetics/metabolism ; Mesoderm/cytology/metabolism ; Cell Differentiation/genetics ; Cell Lineage/genetics ; *Hematopoietic Stem Cells/cytology/metabolism ; Gene Expression Regulation, Developmental ; Mouse Embryonic Stem Cells/cytology/metabolism ; *Endothelial Cells/cytology/metabolism ; Hematopoiesis/genetics ; },
abstract = {During development, blood generation begins in the yolk sac with the differentiation of haemato-endothelial mesoderm forming haematopoietic progenitors. This study aims to identify the crucial molecular regulators of haemato-endothelial mesoderm formation and to extend our knowledge of the process in an unbiased way. We employ a murine embryonic stem cell model that recapitulates embryonic blood development, and perform targeted CRISPR-Cas9 knock out screens focusing on transcription factors and chromatin regulators. We identify the transcription factors ETV2, LDB1, SMAD1, SIX4 and ZBTB7b as regulators of haemato-endothelial mesoderm commitment. Embryonic stem cells lacking these regulators give rise to mesodermal subsets with a defined lineage differentiation bias, while transcriptome analysis of these cells uncovers the precise impact of each factor on gene expression in the developing mesoderm. Our study reveals molecular pathways governing mesodermal development crucial to allow endothelial and haematopoietic lineage specification and paves the way for future advances in haematopoietic stem cell applications.},
}
@article {pmid41390734,
year = {2025},
author = {Ren, J and Yao, J and Cao, Q and Li, Y and Li, Y and Zhang, Z and Ge, X and Wang, S and Zhang, Y and Wang, X and Zhang, X},
title = {Protein-nucleic acid language model-assisted design of precise and compact adenine base editor.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11207},
pmid = {41390734},
issn = {2041-1723},
mesh = {Animals ; *Gene Editing/methods ; Humans ; HEK293 Cells ; *Adenine/metabolism/chemistry ; Mice ; Genetic Therapy/methods ; Nanoparticles/chemistry ; CRISPR-Cas Systems/genetics ; *Nucleic Acids/genetics/chemistry ; Proprotein Convertase 9/genetics/metabolism ; Mutation ; },
abstract = {Adenine base editors (ABEs) are powerful tools for gene therapy. However, efficient version of ABEs (e.g. ABE8e) always induce excessive bystander and off-target editing events and are large in size, hindering their potential in clinical disease treatment. Here, we develop a pre-trained Protein-Nucleic Acid Constrained Language Model to design ABE8e with high activity, reduced editing window and decreased size. By further engineering, the smallest ABE8e- PNLM-pcABE- with a 27% size reduction, exhibits high activity, precise 3-nt editing window, and reduced off-target events near background level in HEK293T cells. Compared to ABE8e, PNLM-pcABE has up to 133.5-fold precision improvement in pathogenic mutation correction. By PNLM-pcABE, the albino mouse model carrying desired base mutation is nearly 100% obtained via zygotes microinjection and the expression of PCSK9 substantially decreases in mice receiving in vivo delivery with lipid nanoparticle (LNP), indicating their great potential in gene therapy and disease modeling.},
}
@article {pmid41390996,
year = {2026},
author = {Wang, AJ and Du, C and Liu, H and Wang, HL},
title = {Effect of the sdc4 gene knockdown on muscle development in zebrafish.},
journal = {Comparative biochemistry and physiology. Part B, Biochemistry &amp; molecular biology},
volume = {281},
number = {},
pages = {111171},
doi = {10.1016/j.cbpb.2025.111171},
pmid = {41390996},
issn = {1879-1107},
mesh = {Animals ; *Zebrafish/genetics/growth &amp; development/metabolism ; *Syndecan-4/genetics/metabolism ; *Muscle Development/genetics ; *Zebrafish Proteins/genetics/metabolism ; *Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; MicroRNAs/genetics/metabolism ; *Muscle, Skeletal/metabolism/growth &amp; development ; Wnt Signaling Pathway ; CRISPR-Cas Systems ; },
abstract = {The growth and regeneration of skeletal muscle are closely related to syndecan-4 (Sdc4), which is a type I transmembrane heparan sulfate proteoglycan belonging to the syndecan family. However, it remains unclear how the sdc4 gene affects fish muscle development. Therefore, an sdc4 knockdown zebrafish line (sdc4[-/-]) was generated by CRISPR/Cas9 technology in this study, and its phenotypes were analyzed. The results revealed that sdc4[-/-] zebrafish exhibited reduced body length and weight compared to the wild-type (WT) at 90 days post fertilization (dpf). Furthemore, sdc4[-/-] zebrafish also showed a significantly larger number of muscle fibers, and significantly reduced individual muscle fiber cross-sectional area. The mRNA expression levels of genes associated with myogenic regulatory factors (MRFs) and the wnt/β-catenin pathway were all significantly downregulated. Based on dual luciferase reporter assays, sdc4 gene expression was regulated by the transcription factor myocyte enhancer factor 2aa (Mef2aa) and miR-141-3p, which bind to its promoter and 3' untranslated region (UTR), respectively. Additionally, the reduced average swimming speed and distance observed in sdc4[-/-] zebrafish at 90 dpf were concomitant with a significant downregulation of mitochondrial respiratory chain complex-related genes and a reduction in ATP concentration. This research aids understanding of sdc4 function in fish and may provide a new perspective for studying the molecular mechanisms of muscle growth and development.},
}
@article {pmid41391580,
year = {2026},
author = {Zarei, S and Hosseiniara, SM and Zijoud, SSH and Hosseiniara, R},
title = {Electrochemical MicroRNA biosensors for kidney Cancer: From biomarker discovery to point-of-care diagnostics.},
journal = {Clinica chimica acta; international journal of clinical chemistry},
volume = {582},
number = {},
pages = {120786},
doi = {10.1016/j.cca.2025.120786},
pmid = {41391580},
issn = {1873-3492},
mesh = {Humans ; *MicroRNAs/analysis/genetics ; *Biosensing Techniques ; *Biomarkers, Tumor/analysis/genetics ; *Kidney Neoplasms/diagnosis/genetics ; *Electrochemical Techniques ; *Point-of-Care Systems ; },
abstract = {Kidney cancer, particularly clear cell renal cell carcinoma (ccRCC), presents a significant clinical burden due to late-stage detection and limited effectiveness of current diagnostic modalities. Minimally invasive strategies, such as liquid biopsy, have emerged as promising alternatives, with microRNAs (miRNAs) gaining attention as stable, disease-specific biomarkers detectable in biofluids. miRNAs function as oncogenes or tumor suppressors, offering advantages over conventional protein biomarkers in early cancer detection and prognostic assessment. Electrochemical biosensors provide a highly sensitive, rapid, and cost-effective platform for miRNA detection, enabling potential point-of-care applications. Recent advances include the integration of nanomaterials, enzymatic and isothermal amplification methods, and CRISPR-Cas systems to enhance specificity and signal sensitivity. Prototype sensors targeting RCC-relevant miRNAs, multiplexed detection for biomarker panels, and smartphone-compatible platforms demonstrate the feasibility of translating these technologies into clinical practice. Despite challenges in assay standardization, pre-analytical variability, and regulatory pathways, electrochemical miRNA biosensors hold transformative potential for non-invasive RCC diagnostics, treatment monitoring, and precision oncology. Continued innovation and clinical validation may establish these platforms as integral tools for personalized patient management.},
}
@article {pmid41391726,
year = {2026},
author = {Liao, XR and Han, D and Qi, LJ and Huang, QY and Gao, QY and He, XY and Guo, T and Lei, JJ and Cheng, SX},
title = {Aptamer-functionalized nanoparticles for CRISPR-Cas9 delivery to circulating malignant cells for therapeutic efficacy evaluation.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {390},
number = {},
pages = {114542},
doi = {10.1016/j.jconrel.2025.114542},
pmid = {41391726},
issn = {1873-4995},
mesh = {Humans ; *CRISPR-Cas Systems ; *Nanoparticles/chemistry/administration &amp; dosage ; *Aptamers, Nucleotide/administration &amp; dosage/chemistry ; Cell Line, Tumor ; Hyaluronic Acid/chemistry ; *Neoplastic Cells, Circulating/metabolism ; Gene Editing/methods ; *Neoplasms/therapy/genetics ; Hyaluronan Receptors/metabolism ; Epithelial Cell Adhesion Molecule/genetics ; Proto-Oncogene Proteins c-met/genetics ; *Gene Transfer Techniques ; ErbB Receptors/genetics ; Plasmids ; },
abstract = {Genome editing therapies targeting oncogenic pathways represent a promising alternative to small-molecule inhibitors, enabling durable therapeutic responses without inducing drug resistance. However, their success hinges on overcoming tumor heterogeneity, as malignant cells of cancer patients exhibit significant phenotypic variability. To advance personalized research on genome editing efficacy, tailored delivery systems capable of precisely targeting heterogeneous cancer cell populations are essential. Herein, we developed a facile modification strategy to construct a multiplexed surface-functionalized gene delivery system targeting heterogeneous cancer cells for personalized therapeutic studies. The system integrates the EGFR-targeting TuTu22 aptamer with SYL3C-conjugated hyaluronic acid (SYL3C-HA) for EpCAM and CD44 recognition. This triple-targeting platform enables efficient delivery of genome editing plasmid for c-Met knockout in both cancer cell lines and circulating malignant cells (CMCs) from cancer patients. The c-Met knockout not only reduces tumor malignancy but also reverses immune suppression, evidenced by PD-L1 downregulation and restored immune surveillance. By combining gene delivery with an ex vivo patient-derived evaluation platform, this system provides a robust tool for personalized research on the therapeutic strategies for tumor progression inhibition and immunity restoration.},
}
@article {pmid41392542,
year = {2025},
author = {Qiao, Z and Choi, S and Chen, Z and Rodriguez, RM and Wang, Q and Yang, Z and Theuerkauf, SA and Nabhan, JF and Hensch, TK and Buchholz, CJ and Lu, Q},
title = {Targeted Intracellular Delivery via Precision Programming of ARRDC1-Mediated Microvesicles.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {12},
pages = {e70199},
pmid = {41392542},
issn = {2001-3078},
support = {//Vesigen Therapeutics/ ; P42ES030990/NH/NIH HHS/United States ; R01ES029097/NH/NIH HHS/United States ; R01HL139496/NH/NIH HHS/United States ; },
mesh = {Animals ; Mice ; CD8-Positive T-Lymphocytes/metabolism ; Humans ; *Cell-Derived Microparticles/metabolism ; Neurons/metabolism ; *Drug Delivery Systems/methods ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; },
abstract = {Efficient and cell-specific delivery remains a major barrier to realising the full therapeutic potential of modalities such as mRNA and CRISPR-based gene editors. Here, we report a versatile delivery platform based on engineered ARRDC1-mediated microvesicles (ARMMs) capable of delivering cargo to defined cell populations. By decorating ARMMs with engineered Nipah virus (NiV)-derived fusion and attachment proteins conjugated to cell-specific ligands, we enable selective binding and membrane fusion-mediated cargo release. ARMMs functionalized with anti-CD8 single-chain variable fragment (scFv) delivered protein, mRNA, or CRISPR-Cas9 base editor selectively to CD8[+] T cells. Similarly, ARMMs displaying a designed ankyrin repeat protein (DARPin) targeting the GluA4 receptor enabled delivery to parvalbumin-positive (PV[+]) neurons. In vivo, administration of targeted ARMMs resulted in functional delivery to CD8[+] splenocytes and PV[+] cortical neurons in mice. These findings establish surface-engineered ARMMs as a programmable and modular system for precision delivery of therapeutic macromolecules, with broad applicability in gene and RNA-based medicine.},
}
@article {pmid41394966,
year = {2025},
author = {Punde, A and Dey, S and Pandire, R and Bhattacharjee, A and Patra, C},
title = {Expanding the CRISPR/Cas toolkit: applications in proteomics and theranostics.},
journal = {Frontiers in bioengineering and biotechnology},
volume = {13},
number = {},
pages = {1713700},
pmid = {41394966},
issn = {2296-4185},
abstract = {Conventional methods available for genome editing have proven non-specific, labour-intensive, and time-consuming. In this context, CRISPR/Cas technology represents a significant breakthrough. It is derived from a sophisticated microbial defence system consisting of clustered regularly interspaced short palindromic repeats, or CRISPR, and the RNA-guided DNA endonuclease Cas. Beyond its original role in genome editing, CRISPR continues to play a major role in the field of proteomics, functional genomics, and molecular therapy. Animal models, including mice, Drosophila, zebrafish, etc., have substantially benefited from CRISPR in uncovering protein function through reverse genetics approaches, including knock-in, knockout, CRISPRi, and indel mutation strategies. On the clinical front, CRISPR gene therapy has also seen successes, including applications in sickle cell disease, hypercholesterolemia, and cancer immunotherapy. However, notable challenges remain, including in vivo packaging and delivery efficiency, toxicity, and genomic off-target effects. Ongoing efforts to overcome these include the development of novel delivery formulations (e.g., nanoparticles, exosomes), artificial intelligence-guided experimental design, and miniaturization of Cas proteins. This review focuses on CRISPR/Cas gene editing mechanisms and explores its state-of-the-art applications in the field of proteomics and theranostics.},
}
@article {pmid41395238,
year = {2025},
author = {Zou, Y and Yao, ZW and Xiao, T and Ma, YR and He, J and Chen, LM and Chen, XQ and Chen, N},
title = {Emerging Trichomonad Infections in Companion Animals: Rapid Visual Detection of Pentatrichomonas hominis and Tritrichomonas foetus Using an RPA-CRISPR/Cas12a Assay.},
journal = {Transboundary and emerging diseases},
volume = {2025},
number = {},
pages = {9995679},
pmid = {41395238},
issn = {1865-1682},
mesh = {Animals ; Cats ; *Protozoan Infections, Animal/diagnosis/parasitology ; *Tritrichomonas foetus/isolation &amp; purification/genetics ; Dogs ; *Trichomonadida/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *Cat Diseases/parasitology/diagnosis ; CRISPR-Cas Systems ; Sensitivity and Specificity ; Pets ; *Dog Diseases/diagnosis/parasitology ; },
abstract = {Pentatrichomonas hominis (P. hominis) and Tritrichomonas foetus (T. foetus) are prevalent intestinal protozoa. P. hominis is associated with chronic diarrhea in humans and animals, whereas T. foetus causes gastrointestinal disease in companion animals and reproductive-tract infection in cattle. Rapid and accurate identification of these infections at the point-of-care (POC) is crucial for the diagnosis and effective management of zoonotic diseases. In this study, we developed two novel recombinase polymerase amplification (RPA) assays coupled with CRISPR/Cas12a detection. The dual-species assay, using a lateral-flow format, targeted species-specific regions of the 18S rRNA gene of P. hominis and T. foetus, and under ideal conditions, delivered visual results within 40 min for a single sample at 37°C. P. hominis-specific assay: To differentiate P. hominis in mixed infections with T. foetus, a second assay targeted the highly conserved Spo11-1 gene of P. hominis. Optimal crRNA-412 and RPA primers were selected for maximal Cas12a cleavage efficiency. Analytical sensitivity and specificity were compared with conventional nested polymerase chain reaction (PCR) and Sanger sequencing. The results showed that The dual-species assay detected as few as 50 DNA copies/µL of either parasite with no cross-reactivity to Giardia lamblia, Cystoisospora canis, Cryptosporidium spp., Toxoplasma gondii, Toxocara canis, and Toxascaris leonina. Among 70 fecal samples of companion animal (48 dogs and 22 cats), 14 (29.2%) dogs tested positive for P. hominis, and eight cats (36.4%) tested positive for T. foetus by nested PCR. Due to financial and logistical constraints, we selected a smaller subset for subsequent analysis with the RPA-CRISPR/Cas12a lateral-flow strip (LFS) assay, which showed 100% diagnostic concordance with PCR. The Spo11-1 assay achieved a limit of detection of 20 DNA copies/µL and specifically recognized P. hominis among a panel that included seven non-target protozoa and helminths. Validation on 10 additional canine and feline samples (four positives and six negatives) showed complete agreement with nested-PCR results. In conclusion, this CRISPR-based diagnostic approach significantly enhances the efficiency and accuracy of Trichomonads detection, offering a practical, cost-effective solution particularly suitable for veterinary and potentially human healthcare diagnostics in resource-limited settings.},
}
@article {pmid41395656,
year = {2025},
author = {Lin, Z and Pu, Z and Wu, J and Zeng, J and Dou, Q and Mao, M and Zhang, Y},
title = {A Versatile CRISPR/Cas12a Autocatalytic Cascade System via Structure-Switching V-Type Split Probe for Highly Sensitive DNA Diagnostics.},
journal = {Analytical chemistry},
volume = {97},
number = {50},
pages = {28079-28087},
doi = {10.1021/acs.analchem.5c06488},
pmid = {41395656},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *DNA Probes/chemistry/genetics ; *DNA, Viral/analysis/genetics ; Humans ; *Endodeoxyribonucleases/metabolism/genetics ; Limit of Detection ; Nucleic Acid Amplification Techniques ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {The rapid detection of pathogen nucleic acids is critical for controlling infectious disease outbreaks and providing timely treatment. However, current molecular diagnostic applications, including sensitive CRISPR/Cas-based detection systems, rely on target preamplification, which often requires expensive equipment and strict adherence to sometimes complex workflows. Here, we describe a rapid, simple, and amplification-free CRISPR/Cas-based diagnostic system that employs a structure-switching V-shaped DNA probe with a Cas12a recognition sequence split by an ssDNA loop to establish a positive feedback loop and a signal amplification cascade. This approach exhibited an ultralow background signal, rapid production of an exponential signal, and atto-molar sensitivity. It was incorporated into microfluidic and lateral flow assay applications for multiplex detection of distinct papillomavirus strains and point-of-care detection of monkeypox virus infections, respectively. The approach thus has significant potential for rapid and sensitive detection of specific pathogen-derived DNA targets in both clinical laboratory and point-of-care applications.},
}
@article {pmid41396047,
year = {2026},
author = {Matsuoka, T and Kano, S},
title = {Impact of patent-granting differences between Japan and the United States on patent protection for medical methods: insights from genome editing patents.},
journal = {Expert opinion on therapeutic patents},
volume = {36},
number = {2},
pages = {133-144},
doi = {10.1080/13543776.2025.2605318},
pmid = {41396047},
issn = {1744-7674},
mesh = {*Patents as Topic/legislation &amp; jurisprudence ; Japan ; United States ; Humans ; *Gene Editing/legislation &amp; jurisprudence ; CRISPR-Cas Systems ; },
abstract = {INTRODUCTION: Securing patents in multiple countries has become essential for the development of global medical products. However, differences in national patent systems result in varying patentability standards. Although global claim construction strategies have been applied in practice, these approaches have not yet been systematically organized.<br><br>AREA COVERED: This study examines how the patent scope for patent families of international applications related to genome editing technologies filed in 2013, differs between Japan, where medical method patents are prohibited, and the United States, where such patents are permitted.<br><br>EXPERT OPINION: For CRISPR-Cas system patents, claim structures varied significantly, even among the corresponding family patents. To navigate these differences, the following strategies were proposed for filing patents in countries that prohibit medical method patents such as Japan: Convert medical method claims in the U.S. into composition claims that include product inventions, as this process ensures that such claims allow for the enforcement of rights against the suppliers of infringing products.Clearly define the scope of the claimed use-inventions when specifying the characteristics of the product based on its effects.Explicitly describe cells produced by a specific manufacturing method within the claimed rights.},
}
@article {pmid41396964,
year = {2026},
author = {Jiang, C and Liu, Y and Han, W and Zou, D and Chen, K and Jiang, X and Ma, A and Wei, X},
title = {Regulation of Single and Multiple Genes in Bacillus amyloliquefaciens by an Evolution System In Vivo.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {88-98},
doi = {10.1021/acssynbio.5c00480},
pmid = {41396964},
issn = {2161-5063},
mesh = {*Bacillus amyloliquefaciens/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Editing/methods ; *Directed Molecular Evolution/methods ; Bacterial Proteins/genetics/metabolism ; Gene Expression Regulation, Bacterial ; Cytidine Deaminase/genetics/metabolism ; Mutation ; },
abstract = {With the development of synthetic biology, an evolution system in vivo has been applied to accelerate the construction of cell factories. In this study, an efficient in vivo evolution system was developed for regulation of single and multiple genes in Bacillus amyloliquefaciens. First, the CRISPR/Cas9n-AID base editor was constructed through integration expression of the fused Cas9n protein and activation-induced cytidine deaminase (AID), and the base conversion efficiency from C to T was as high as 90% in single-gene editing. Subsequently, the evolution template (XP43) with an editable RBS sequence (GGGGGGGG) was designed for in vivo evolution through two strategies. By next-generation sequencing of RBS mutation libraries, the extended sgRNA strategy was confirmed to be the optimal evolution scheme. Using the alkaline protease gene (aprE) as the single gene target, the evolution program was initiated to successfully obtain a series of mutant strains with gradient AprE activities. Furthermore, multiple key genes (dhemA, SAM2, and hemEHY) were evolved simultaneously to balance the heme metabolic network, and the optimal mutant strain (HZHA-C2) produced 14.02 mg/L heme, 93% higher than the control strain. Finally, the overexpression of the hemH gene further increased the heme titer by 49%. By a fed-batch fermentation strategy, the heme titer of the optimal engineered strain (HZHA2/pHY-hemH) was improved by 64%, achieving 32.61 mg/L.},
}
@article {pmid41397585,
year = {2026},
author = {Hsu, CY and Polatova, D and Hamad, RH and Patel, PN and Akram, M and Singh, G and Arora, V and Nayak, PP and Kadhem, M and Hamzah, HF},
title = {Phage therapy in cancer treatment: Mechanisms, emerging innovations, and translational progress.},
journal = {Critical reviews in oncology/hematology},
volume = {218},
number = {},
pages = {105085},
doi = {10.1016/j.critrevonc.2025.105085},
pmid = {41397585},
issn = {1879-0461},
mesh = {Humans ; *Phage Therapy/methods ; *Neoplasms/therapy ; *Bacteriophages ; Animals ; Translational Research, Biomedical ; },
abstract = {Bacteriophage therapy has re-emerged as a rapidly advancing field in oncology, bridging antimicrobial precision with tumor-targeted biotherapy. Beyond infection control, phages are now recognized as programmable biological systems capable of eradicating multidrug-resistant (MDR) pathogens, modulating tumor-associated microbiota, activating immune responses, and delivering therapeutic genes or drugs. Preclinical evidence shows that phages can selectively eliminate Fusobacterium nucleatum in oral squamous cell carcinoma, restore microbial balance in colorectal cancer, and enhance immune infiltration via cytokine or antigen display. Engineered constructs including GM-CSF-expressing and MAGE-A1-displaying phages, λ-phage ASPH vaccines, and PEGylated nanocarriers delivering MEG3 or TRAIL have demonstrated strong anti-tumor efficacy across melanoma, hepatocellular, and colorectal cancer models. Additionally, CRISPR-Cas-armed phages precisely remove resistance genes such as bla-CTX-M and mecA, while AI-driven selection pipelines enable data-guided design of personalized phage cocktails. These advances represent a paradigm shift from empirical antibacterial use toward mechanistically engineered, multifunctional phage platforms that integrate microbiome modulation, immune activation, and nanocarrier-mediated gene delivery. Although challenges such as immune clearance, bacterial resistance, and regulatory complexity remain, the convergence of AI, CRISPR, and synthetic biology is accelerating the evolution of phage therapy into a clinically viable precision-oncology strategy. In this context, bacteriophages emerge not merely as antibacterial agents but as intelligent, patient-specific nanomedicines poised to redefine therapeutic boundaries in cancer treatment.},
}
@article {pmid41397613,
year = {2026},
author = {Yin, C and Chen, B and Zheng, X and Wang, N and Wang, J and Li, R and Li, J and Yao, S and Zhai, Y and Song, X},
title = {Portable visual platform integrates polymerase spiral amplification and CRISPR/Cas12a for foodborne bacteria point-of-care testing.},
journal = {Journal of dairy science},
volume = {109},
number = {2},
pages = {1036-1051},
doi = {10.3168/jds.2025-27493},
pmid = {41397613},
issn = {1525-3198},
mesh = {*Staphylococcus aureus/isolation &amp; purification/genetics ; *Point-of-Care Testing ; CRISPR-Cas Systems ; Animals ; Nucleic Acid Amplification Techniques ; Food Microbiology ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Staphylococcus aureus, a prominent global foodborne pathogen, frequently triggers epidemics with severe public health impacts. Timely and reliable detection of S. aureus is crucial for mitigating the disease burden in low- and middle-income countries. However, conventional laboratory-based detection methods remain impractical in resource-limited settings, highlighting the urgent need for accessible point-of-care solutions. Here, we present an inner-outer-tube (IOT) assay that synergistically integrates the polymerase spiral amplification (PSR) technology for enhanced sensitivity with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 12a (Cas12a) system for sequence-specific identification. Additionally, we have created a portable all-in-one mobile detection (PAMD) device that combines all the steps needed for testing in the field, allowing for quick visual detection of S. aureus in just 60 min. The PSR-CRISPR/Cas12a-IOT method implemented with the PAMD device achieves a detection limit of 10 cfu/mL without needing extra preparation or costly equipment. The detection platform developed in this work has advantages of ease of operation, manageable costs, and robust performance, making it highly ideal for low-resource contexts and on-site detection scenarios. Furthermore, the PSR-CRISPR/Cas12a-IOT-PAMD detection platform provides global versatility through the interchangeable use of primer sets, hence broadening its applicability to various infections.},
}
@article {pmid41397982,
year = {2025},
author = {Okuwa, T and Himeda, T and Kobayashi, K and Nomura, N and Utani, K and Koike, S and Nakamura, A and Higuchi, M},
title = {Saffold virus exploits integrin αvβ8 and sulfated glycosaminoglycans as cooperative attachment receptors for infection.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {534},
pmid = {41397982},
issn = {2041-1723},
support = {25K10386//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 21K07045//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; 24K10234//MEXT | Japan Society for the Promotion of Science (JSPS)/ ; JP25fk0108716//Japan Agency for Medical Research and Development (AMED)/ ; S2023-4//Kanazawa Medical University/ ; K2024-3//Kanazawa Medical University/ ; },
mesh = {Humans ; *Integrins/metabolism/genetics ; *Glycosaminoglycans/metabolism ; HeLa Cells ; Virus Internalization ; CRISPR-Cas Systems ; *Receptors, Virus/metabolism ; Virus Attachment ; Host-Pathogen Interactions ; Gene Knockout Techniques ; },
abstract = {Saffold virus (SAFV), a member of the species Cardiovirus saffoldi within the Picornaviridae family, causes acute respiratory and gastrointestinal illnesses as well as hand, foot, and mouth disease. It is also suspected to be associated with neuronal disorders, such as encephalitis and meningitis, in severe cases. Despite its clinical significance, the virus-host interactions underlying SAFV pathogenicity remain largely unknown. Using a genome-wide CRISPR-Cas9 knockout screen, we identify the following receptors for SAFV infection: sulfated glycosaminoglycans (GAGs) and integrin αVβ8. Single knockouts of SLC35B2, an essential gene for sulfated GAG synthesis, or the integrin genes ITGAV or ITGB8 partially reduce SAFV-3 and SAFV-2 susceptibility in HeLa cells, and a double knockout confers complete resistance. Furthermore, we demonstrate that SAFV-3 virions bind directly to sulfated GAGs and integrin αVβ8. Based on these findings, we propose a model of SAFV infection in which sulfated GAGs and integrin αVβ8 act through dual and cooperative pathways to facilitate viral entry.},
}
@article {pmid41397984,
year = {2025},
author = {Chen, J and Hu, L and Vernuccio, R and Shi, N and Tian, J and Zhang, Y and Tian, S and Cao, X and Ha, Z and Lu, J and Battini, L and Raynal, B and Haouz, A and Xue, J and Cai, Q and Zhao, Y and Lu, Y and Smith, GL and Xie, Y and Lu, H and Guardado-Calvo, P and Zhang, P and Zhang, R},
title = {Development of a replication-defective mpox virus platform for fundamental and therapeutic research.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {798},
pmid = {41397984},
issn = {2041-1723},
mesh = {*Virus Replication/drug effects/genetics ; Humans ; *Antiviral Agents/pharmacology/chemistry ; Animals ; CRISPR-Cas Systems ; Chromosomes, Artificial, Bacterial/genetics ; Genome, Viral ; *Orthopoxvirus/genetics/drug effects/physiology ; Virion/genetics/drug effects ; *Defective Viruses/genetics/drug effects ; Virus Assembly/drug effects/genetics ; },
abstract = {The recent global outbreaks of mpox highlight the urgent need for both fundamental research and antiviral development. However, studying the mpox virus (MPXV), with its large and complex genome, remains challenging due to the requirement for high-containment facilities. Here, we describe a strategy for de novo assembly of MPXV clade IIb genomes in bacterial artificial chromosomes using transformation-associated recombination cloning. Leveraging CRISPR-Cas9 and Lambda Red recombination, we engineer replication-defective MPXV particles with dual deletions of OPG96 (M2R) and OPG158 (A32.5 L)-genes essential for virion assembly, that are capable of recapitulating key stages of the viral life cycle. We apply this system to screen a compound library and identify G243-1720, a potent anti-poxvirus inhibitor with broad activity in vitro and in vivo. G243-1720 blocks the formation of extracellular enveloped virions and cell-cell spread. Resistance mutation selection, crystallographic analysis, analytical ultracentrifugation, and mass photometry reveal that, despite its distinct chemical structure, G243-1720 shares a mode of action with tecovirimat, both functioning by affecting dimerization of protein OPG57 (F13). Our findings underscore the potential of G243-1720 as a promising broad-spectrum anti-poxvirus lead compound and demonstrate the utility of replication-defective MPXV particles as a reliable platform for viral biology studies and antiviral development.},
}
@article {pmid41398311,
year = {2025},
author = {Kang, YJ and Ha, HJ and Jin, HB and Lee, SY and Park, HH},
title = {Structural basis of dimerization and cascade formation by Cas5.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2976},
pmid = {41398311},
issn = {2045-2322},
support = {2025//Chung-Ang University/ ; RS-2025-02316334//National Research Foundation of Korea/ ; },
mesh = {*Protein Multimerization ; *CRISPR-Cas Systems ; Crystallography, X-Ray ; Models, Molecular ; *CRISPR-Associated Proteins/chemistry/metabolism/genetics ; *Bacterial Proteins/chemistry/metabolism/genetics ; Protein Conformation ; },
abstract = {CRISPR-Cas systems are essential for prokaryotic adaptive immune mechanisms; however, the structural details of many subtype-specific components remain unclear. Herein, we report the crystal structure and biophysical characterization of Cas5 from Moraxella bovoculi (MboCas5), a component of the type I-C CRISPR-Cas system. We found that M. bovoculi encodes both type I-C and type III-B systems, and that MboCas5 forms a dimer that is stabilized by key interactions, including a salt bridge between R72 and D167. Structural comparisons with other Cas5 homologs and AlphaFold 3 predictions further validated the unique dimer configuration, suggesting that it is conserved across species. Additionally, structural comparison revealed a highly flexible loop region, which likely undergoes conformational changes upon Cascade assembly and might mediate interactions with Cas8 and crRNA. Overall, the findings provided structural and mechanistic insights into Cas5 function and could potentially contribute to our understanding of the assembly of type I-C Cascade complexes.},
}
@article {pmid41398410,
year = {2025},
author = {Chi, H and Hoikkala, V and McMahon, S and Graham, S and Gloster, T and White, MF},
title = {Structure and mechanism of the broad spectrum CRISPR-associated ring nuclease Crn4.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {889},
pmid = {41398410},
issn = {2041-1723},
support = {101018608//EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council)/ ; },
mesh = {Crystallography, X-Ray ; *CRISPR-Cas Systems ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/chemistry/genetics ; Models, Molecular ; *Endonucleases/metabolism/chemistry/genetics ; Bacteriophages/genetics ; Nucleotides, Cyclic/metabolism ; Oligoribonucleotides/metabolism ; },
abstract = {Type III CRISPR systems detect the presence of RNA from mobile genetic elements (MGE) in prokaryotes, providing antiviral immunity. On activation, the catalytic Cas10 subunit conjugates ATP to form cyclic oligoadenylate (cOA) signalling molecules that activate ancillary effectors, providing an immune response. Cellular ring nucleases degrade cOA to reset the system. Here, we describe the structure and mechanism of a new family of ring nucleases, Crn4, associated with type III-D CRISPR systems. The crystal structure of Crn4 reveals a small homodimeric protein with a fold unrelated to any known ring nuclease or, indeed, any known protein structure. Crn4 degrades a wide range of cOA species to linear oligoadenylates in vitro and ameliorates type III CRISPR immunity in vivo. Phage and plasmids also encode Crn4 orthologues that may function as anti-CRISPRs. These observations expand our understanding of ring nucleases and reveal a new protein fold for cyclic nucleotide recognition.},
}
@article {pmid41399197,
year = {2026},
author = {Singh, K and Sharma, S and Kalia, A and Manchanda, P},
title = {Advancement in Mushroom Transformation: From Conventional Techniques to Modern Genetic Engineering.},
journal = {Journal of basic microbiology},
volume = {66},
number = {1},
pages = {e70132},
doi = {10.1002/jobm.70132},
pmid = {41399197},
issn = {1521-4028},
mesh = {*Genetic Engineering/methods ; *Agaricales/genetics/growth &amp; development ; *Transformation, Genetic ; CRISPR-Cas Systems ; Gene Editing ; Agrobacterium/genetics ; },
abstract = {Mushrooms have long been valued for their nutritional, pharmaceutical, and culinary benefits. Recent studies showcased mushrooms as bio-factories for protein production, and as a source of value-added products by employing genetic manipulation and molecular transformation techniques. Advancements in molecular tools and transformation methods have enhanced the efficiency of genetic improvements in mushrooms by both conventional and modern genetic engineering techniques, paving the way for their use in various industrial applications. Genetic transformation in mushrooms involves transferring genes within and across species to understand gene functions and improve mushroom qualities. The techniques involved in transformation includes Agrobacterium-mediated transformation, hybridization, mutation breeding, particle bombardment, protoplast fusion, and CRISPR/Cas9. This review outlines the life cycle of mushrooms, major difficulties in mushroom transformation, various transformation techniques, their history, efficiency, and success rate. It also highlights the potential of genetic engineering to revolutionize mushroom cultivation and their applications.},
}
@article {pmid41399500,
year = {2025},
author = {Swartjes, T and Bouzetos, E and Adiego-Pérez, B and Pool, VD and Staals, RHJ and van der Oost, J and Wu, WY},
title = {Base editing both DNA strands in distinct editing windows with small CRISPR-associated effector Cas12f1.},
journal = {iScience},
volume = {28},
number = {12},
pages = {114033},
pmid = {41399500},
issn = {2589-0042},
abstract = {CRISPR-associated base editors have been established as genome editing tools that enable base conversions in targeted DNA sequences, without generating double-strand breaks. Here, we describe the development of new base editors based on CRISPR-Cas12f1, a miniature Cas protein of only 422 amino acids. Chimeric constructs have been generated by fusing a catalytically inactive dCas12f1, to either a cytosine deaminase or an adenine deaminase. Using these synthetic fusion proteins, systematic analyses have been performed on base editing of a target sequence on a plasmid in Escherichia coli. Interestingly, apart from the previously described base editing of the displaced non-target DNA strand, we also observed efficient editing of the target DNA strand. This effect was not observed for Un1Cas12f1 BEs. In addition to the small size of AsCas12f1 base editors, its unique editing profile makes it a valuable addition to the CRISPR-Cas toolbox.},
}
@article {pmid41401738,
year = {2026},
author = {Feng, J and Lin, X and Kang, L and Duan, M and Duan, N and Wang, Z and Wu, S},
title = {Integrating a microfluidic chip@LFA biosensor enabled by Pt3Sn@MGO nanocomposites for RAA/CRISPR-Cas12b mediated food adulteration monitoring.},
journal = {Biosensors &amp; bioelectronics},
volume = {296},
number = {},
pages = {118315},
doi = {10.1016/j.bios.2025.118315},
pmid = {41401738},
issn = {1873-4235},
mesh = {*Biosensing Techniques/instrumentation ; Animals ; *Food Contamination/analysis ; *Nanocomposites/chemistry ; Salmon/genetics ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Lab-On-A-Chip Devices ; Nucleic Acid Amplification Techniques ; Food Analysis ; Recombinases/chemistry ; Myoglobin/genetics ; },
abstract = {Traditional methods for food species genetic authentication typically involve time-consuming laboratory procedures and inconsistent operations, easily resulting in gene damage and inaccurate diagnostics. Here, by fully integrating reagent flow and reactions through micromachining technology, a standardized and lab-free operational PMMA-based microfluidic chip@ lateral flow assay (LFA) biosensor was developed for salmon adulteration detection. The primers recognizing the myoglobin nuclear gene of salmon were designed and optimized, enabling efficient recombinase-aided amplification (RAA) of target gene and subsequent activation of the CRISPR-Cas12b system. A multifunctional Pt3Sn@MGO nanocomposite was synthesized with enhanced FRET efficiency and photothermal properties, then employed as a signal probe in LFA test strip, achieving fluorescent, photothermal, and colorimetric quantitative detection of salmon contents in mixed samples, with detection limits of 0.007 %, 0.092 %, and 0.153 %, respectively, salmon contents in commercially products were evaluated to verify the practicality. This work presents an integrated, portable, and automation-enabled platform for standardized genetic authentication of food adulteration, which would be utilized as a universal lab-free method for on-site adulteration monitoring and species gene diagnostics by matching conserved genes and primer designs.},
}
@article {pmid41402279,
year = {2025},
author = {Wang, D and Ritz, C and Luo, Y and Suresh, A and Pierce, A and Veo, B and Brunt, B and Dahl, N and Serkova, N and Venkataraman, S and Danis, E and Kus, K and Mazan, M and Rzymski, T and Vibhakar, R},
title = {Transcriptional regulation of protein synthesis by mediator kinase represents a therapeutic vulnerability in MYC-driven medulloblastoma.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11152},
pmid = {41402279},
issn = {2041-1723},
support = {P30 CA046934/CA/NCI NIH HHS/United States ; },
mesh = {*Medulloblastoma/genetics/metabolism/pathology/drug therapy ; Humans ; *Proto-Oncogene Proteins c-myc/metabolism/genetics ; *Cyclin-Dependent Kinase 8/metabolism/genetics/antagonists &amp; inhibitors ; *Protein Biosynthesis/genetics ; Animals ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Mice ; *Cerebellar Neoplasms/genetics/metabolism/pathology/drug therapy ; Transcription, Genetic ; TOR Serine-Threonine Kinases/metabolism/antagonists &amp; inhibitors ; RNA Polymerase II/metabolism ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; },
abstract = {MYC-driven medulloblastoma (MB) is a highly aggressive brain tumor with poor prognosis and limited treatment options. Through CRISPR-Cas9 screening, we identify the Mediator-associated kinase CDK8 as a critical regulator of MYC-driven MB. Both genetic loss and pharmacological inhibition of CDK8 impair MB tumor growth. Moreover, we find that CDK8 cooperates with MYC to sustain the MYC-mediated translational program, as CDK8 depletion induces pronounced transcriptional changes in translation-associated gene sets, reduces ribosome biogenesis, and impairs protein synthesis. Mechanistically, CDK8 regulates the occupancy of RNA polymerase II at specific chromatin loci, facilitating epigenetic alterations that promote the transcription of ribosomal genes. Furthermore, combined inhibition of CDK8 and mTOR synergistically enhances therapeutic efficacy in vivo, leading to more pronounced tumor growth suppression. Overall, our findings establish a functional link between CDK8-mediated transcriptional regulation and mRNA translation, suggesting a promising therapeutic approach targeting protein synthesis for MYC-driven MB.},
}
@article {pmid41402283,
year = {2025},
author = {Sivanandan, S and Leitmann, B and Lubeck, E and Sultan, MM and Stanitsas, P and Ranu, N and Ewer, A and Mancuso, JE and Phillips, ZF and Kim, A and Bisognano, JW and Cesarek, J and Ruggiu, F and Feldman, D and Koller, D and Sharon, E and Kaykas, A and Salick, MR and Chu, C},
title = {A pooled Cell Painting CRISPR screening platform enables de novo inference of gene function by self-supervised deep learning.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {77},
pmid = {41402283},
issn = {2041-1723},
mesh = {*Deep Learning ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Regulatory Networks ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Pooled CRISPR screening enables large-scale interrogation of gene functions but typically measures simple phenotypes such as fitness. High-content methods like Perturb-seq extend dimensionality to transcriptomics but are costly and limited in scope. Optical pooled screening (OPS) combines pooled CRISPR screening with imaging to yield scalable, information-rich readouts, yet existing implementations remain pathway-specific. Here we describe an OPS-compatible Cell Painting platform that enables hypothesis-free reverse genetic screening through multiplexed morphological profiling. We validate this technique using a well-defined morphological gene set, compare classical image analysis to self-supervised learning methods using a mechanism-of-action library, and perform discovery screening with a druggable genome library. By combining rich morphological data with deep learning, gene networks emerge without the need for target-specific biomarkers, leading to unbiased discovery of gene functions.},
}
@article {pmid41402624,
year = {2025},
author = {Sinkunas, T and Tamulaitiene, G},
title = {A DNA mimic jams the Cas9 scissors.},
journal = {The FEBS journal},
volume = {},
number = {},
pages = {},
doi = {10.1111/febs.70374},
pmid = {41402624},
issn = {1742-4658},
support = {S-MIP-20-39//Lietuvos Mokslo Taryba/ ; },
abstract = {Anti-CRISPR (Acr) proteins are small protein inhibitors that block the RNA-guided nucleic acid (DNA or RNA) targeting activity of CRISPR-Cas enzymes. Despite their shared function, Acr proteins display minimal sequence or structural similarity and employ diverse mechanisms to block nuclease activity. Lee and Park characterized the previously undescribed AcrIIA13b protein, which inhibits Cas9 protein. Structural, biochemical, and mutational analyses revealed that AcrIIA13b acts as a DNA mimic, thereby disabling the Cas9 complex from binding to the DNA target.},
}
@article {pmid41402634,
year = {2025},
author = {Oliynyk, RT and Mahas, A and Karpinski, E and Church, GM},
title = {Plasmid2MC: efficient cell-free generation of high-purity minicircle DNA for genome editing in mammalian cells.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1778},
pmid = {41402634},
issn = {2399-3642},
mesh = {*Gene Editing/methods ; *Plasmids/genetics ; Humans ; HEK293 Cells ; Animals ; *DNA, Circular/genetics ; Mice ; CRISPR-Cas Systems ; Cell-Free System ; Mouse Embryonic Stem Cells/metabolism ; },
abstract = {DNA plasmids are widely used for delivering proteins and RNA in genome editing. However, their bacterial components can lead to inactivation, cell toxicity, and reduced efficiency compared to minicircle DNA (mcDNA), which lacks such bacterial sequences. Existing commercial kits that recombine plasmids into mcDNA within proprietary bacterial strains are labor-intensive, yield inconsistent results, and often produce endotoxin-contaminated low-quality mcDNA. To address this challenge, we developed Plasmid2MC, a novel cell-free method utilizing ΦC31 integrase-mediated recombination to efficiently excise the bacterial backbone from conventionally prepared plasmids, followed by digestion of the bacterial backbone and all other DNA contaminants, resulting in highly pure and virtually endotoxin-free mcDNA. We demonstrated the application of mcDNA to express CRISPR-dCas9 for base editing in HEK293T cells and mouse embryonic stem cells, as well as for homology-independent targeted insertion (HITI) genome editing. The method's ease of preparation, high efficiency, and the high purity of the resulting mcDNA make Plasmid2MC a valuable tool for applications requiring bacterial backbone-free circular DNA.},
}
@article {pmid41402770,
year = {2025},
author = {Jiang, M and Zhang, K and Wang, Z and Gao, M and Su, S and He, J and Xu, H and Bo, Z and Jiang, Z and Zhang, C and Hui, JH and Wei, R},
title = {Nanomaterials in gene therapy and genome editing: challenges and emerging directions.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {56},
pmid = {41402770},
issn = {1477-3155},
mesh = {*Gene Editing/methods ; Humans ; *Genetic Therapy/methods ; *Nanostructures/chemistry/therapeutic use ; Animals ; CRISPR-Cas Systems ; Nanoparticles/chemistry ; Lipids/chemistry ; },
abstract = {Nanomaterials are redefining the landscape of gene and genome editing, yet their translation to clinical reality remains constrained by multiple unresolved challenges. While they provide structural and functional advantages for delivering nucleic acids and CRISPR/Cas systems across biological barriers, their behavior within living systems is often unpredictable, leading to issues such as off-target editing, immune activation, and inconsistent biodistribution. The design of nanocarriers, whether lipid-based, polymeric, inorganic, must therefore balance efficiency with safety, integrating physicochemical precision with biological adaptability. Recent advances in ionizable lipid nanoparticles demonstrate how fine-tuning charge, surface chemistry, and degradation kinetics can enhance endosomal escape and target specificity, but reproducibility and large-scale manufacturing continue to limit broader application. Moreover, polymeric and exosome-inspired systems promise modularity and targeted reuse, yet they demand clearer understanding of long-term biocompatibility and regulatory acceptance. The future of nanomaterial-enabled genome engineering depends not only on optimizing delivery vehicles but also on establishing predictive models of nano-bio interactions, harmonizing ethical oversight, and developing standardized evaluation pipelines that link nanoscale design to therapeutic outcomes.},
}
@article {pmid41403193,
year = {2025},
author = {Su, S and Zuo, Y and Ma, B and Zhao, Z and Wang, X and Zhang, X and Ignatus, AD and Piñero, JC and Peng, X and Li, F and Chen, M},
title = {Functional Validation of GmGSTs2 in the Resistance to Abamectin in the Oriental Fruit Moth, Grapholita molesta (Lepidoptera: Tortricidae).},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {52},
pages = {33033-33045},
doi = {10.1021/acs.jafc.5c12427},
pmid = {41403193},
issn = {1520-5118},
mesh = {Animals ; *Ivermectin/analogs &amp; derivatives/pharmacology ; *Moths/genetics/drug effects/enzymology/growth &amp; development ; *Insecticides/pharmacology ; *Glutathione Transferase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Insecticide Resistance ; CRISPR-Cas Systems ; Larva/drug effects/genetics/growth &amp; development/enzymology ; },
abstract = {Abamectin has been used for decades as an insecticide and acaricide in arthropod pest management. However, there is no direct evidence from CRISPR/Cas9 studies confirming the involvement of GSTs in insect resistance to abamectin. The oriental fruit moth, Grapholita molesta, is a destructive pest of fruit trees worldwide. The role of GSTs in the oriental fruit moth remains unclear. In this study, an abamectin-resistant strain (AB-R) was derived from a susceptible laboratory strain (AB-S) of G. molesta. Synergist bioassays showed that the GST inhibitor diethyl maleate (DEM) significantly increased abamectin toxicity in AB-R. Biochemical assays indicated that glutathione S-transferase (GST) activity in AB-R was 1.63-fold higher than in AB-S. Among 25 GST genes examined, GmGSTs2 showed the largest expression difference between AB-R and AB-S and was expressed across developmental stages and body parts. Recombinant GmGSTs2 significantly reduced the effective quantity of abamectin in vitro. CRISPR/Cas9 knockout of GmGSTs2 in both genetic backgrounds increased susceptibility to abamectin and significantly affected the development and survival of G. molesta. The transgenic Drosophila melanogaster strain expressing GmGSTs2 showed an LC50 of 74.12 mg L[-1] (34.59-126.63) versus 25.48 mg L[-1] (12.28-39.82) in W[1118] controls, indicating a 2.91-fold difference. Together, synergism assays, enzyme activity measurements, in vitro metabolism, CRISPR knockout in both resistant and susceptible backgrounds, and a heterologous in vivo assay identify GmGSTs2 as a key metabolic driver of abamectin resistance in G. molesta, providing a practical target for resistance management.},
}
@article {pmid41403730,
year = {2025},
author = {Khari, M and Jain, N and Kaul, S and Pandey, M and Sharma, N},
title = {siRNA and mRNA-Based Preventive and Therapeutic Strategies for HPV-Induced Cervical Cancer.},
journal = {Advanced pharmaceutical bulletin},
volume = {15},
number = {3},
pages = {552-573},
pmid = {41403730},
issn = {2228-5881},
abstract = {Human papillomavirus (HPV), specifically types 16 and 18, is the main cause of cervical cancer and a significant cause of death among women. Specifically, HPV E6 and E7 oncogenes hinder the normal cell cycle regulation, resulting in uncontrolled cell growth and cervical cancer. The available therapy options include surgery, radiotherapy, and chemotherapy, which show success but also demonstrate notable complications. SiRNA (small interfering RNA) and mRNA (messenger RNA) therapies have emerged as precise and effective tools to silence the HPV E6 and E7 oncogenes and stimulate the immune system to fight against HPV infection, respectively, presenting a targeted therapy approach and overcoming the available therapy challenges. Nanoparticles and Pegylated liposomes are the delivery systems that increase the efficacy and safety of siRNA and mRNA therapies. This review critically appreciates the effective targeting of siRNA and mRNA-based therapies by highlighting their key advantages and limitations. Despite being a target-specific and effective approach, there are certain challenges like scale-up, cost-effectiveness, and developing stable delivery systems, which are required to be discussed. In addition, other precision medicine approaches, such as CRISPR/CAS-9, antisense oligonucleotides, or immunotherapy, have also been included as compared to siRNA/mRNA therapies. Their preclinical, patent, and clinical translations have also been discussed exhaustively.},
}
@article {pmid41404141,
year = {2025},
author = {Shilpha, J and Kang, WH},
title = {Molecular and genomic insights into viral resistance in Capsicum spp.: pathogenesis, defense mechanisms, and breeding innovations.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1716114},
pmid = {41404141},
issn = {1664-462X},
abstract = {Plant viruses represent a major challenge to agricultural systems, threatening global food security amid a rising population. Specifically, pepper cultivation (Capsicum annuum L.) is often hindered by various viral diseases, with more than 60 viruses identified as affecting pepper plants. The most efficient strategy for controlling viral diseases is the development of resistant cultivars of peppers. A comprehensive understanding of complex interactions between plant defense mechanisms and the strategies employed by viruses to evade these defenses, coupled with host factors that facilitate viral replication and movement, is essential for developing resistant cultivars. Natural antiviral defense mechanisms in plants are well characterized and include resistance genes, RNA silencing, autophagy-mediated degradation, translational repression, and resistance to viral movement. Recent advances in next-generation sequencing (NGS), genome-wide association studies (GWAS), high-density genotyping platforms and gene-editing tools such as CRISPR/Cas have accelerated the identification of resistance loci and key host factors involved in viral pathogenesis. This review summarizes current molecular and genomic insights into virus-host interactions in Capsicum spp., highlighting their role in advancing marker-assisted selection (MAS) and genomic-assisted breeding. The integration of molecular markers and genome editing into breeding pipelines offers new opportunities for developing durable, broad-spectrum viral resistance in peppers, ultimately supporting sustainable crop production and agricultural resilience.},
}
@article {pmid41404500,
year = {2025},
author = {Hundal, T and Luo, Y and Qie, Y and Gadd, ME and Brim, AD and Vazquez-Rosario, I and Guo, S and Kharfan-Dabaja, MA and Qin, H},
title = {Novel allogeneic CAR T-cell platform involving microhomology-mediated end joining repair and low off-targeting potential.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102778},
pmid = {41404500},
issn = {2162-2531},
abstract = {Several allogeneic chimeric antigen receptor (CAR) T-cell therapies in clinical trials rely on CRISPR-Cas genome editing, but the enzyme's random repair mechanism increases the risk of undesired off-target effects, challenging safe CAR T-cell generation. To address this, we developed a novel CRISPR RNA (crRNA) targeting the T-cell receptor beta constant (TRBC) gene. Combined with AsCas12a Ultra, this crRNA edits primary human T-cells via a predictable microhomology-mediated end joining (MMEJ) DNA repair pathway, significantly lowering off-target risks. During evaluation, we sequestered a unique T-cell subset with disrupted T-cell receptor (TCR), retained CD3 expression, and no in vivo alloreactivity. Termed CD3-retained, allogeneically functioning T-cells (CRAFT-cells), these cells exhibited growth kinetics comparable to unedited T-cells. When engineered with CD19- or BAFF-R-targeted CARs, CRAFT CAR T-cells showed strong antigen-specific cytotoxicity and significant ex vivo expansion compared to conventional CD3-disrupted CAR T-cells. Moreover, CRAFT CAR T-cells effectively served as effector cells for bispecific T-cell engagers (BiTEs), enabling CD3-dependent tumor cell killing. Our CRAFT crRNA platform offers a novel strategy to generate safer allogeneic CAR T-cells. The distinct properties of CRAFT CAR T-cells, combined with BiTE therapy, represent a promising and potentially more durable approach for next-generation allogeneic CAR T-cell therapies in clinical applications.},
}
@article {pmid41404502,
year = {2025},
author = {Gao, H and Gao, S and Kan, G and Valentovich, LN and An, Y},
title = {Research progress of base editing and prime editing tools based on the CRISPR/Cas system.},
journal = {Molecular therapy. Nucleic acids},
volume = {36},
number = {4},
pages = {102771},
pmid = {41404502},
issn = {2162-2531},
abstract = {The base editor (BE) and prime editing guide RNA (pegRNA)-based prime editor (PE) technologies relying on the CRISPR/Cas system are very efficient gene editors that have been developed in recent years. The BEs include cytosine base editors (CBEs) that mediate the conversion of C to T, adenine base editors (ABEs) that mediate the conversion of A to G, glycosylase base editors (GBEs) that mediate the conversion of C to G, and the dual-base editors (DBEs) that mediate the simultaneous conversion of C to T and A to G. The BEs and PEs have been successfully applied for genome editing of various animals, plants, and microorganisms due to their advantages of high efficiency and independence of DNA double-strand breaks or donor DNA. The development process and characteristics of various BEs and PEs and their effectiveness of application are systematically introduced to provide a reference for selecting appropriate genome editing technologies. Moreover, the urgent issues that need to be addressed for more efficient and precise editing are summarized and prospected.},
}
@article {pmid41404798,
year = {2025},
author = {Yang, F and Xu, C and Li, C and Xiang, X and Zhao, Y and Hu, C and Rong, H and He, Y and Li, J and Wang, Y and Tang, C and Liu, X and Li, R and Deng, F and Xiang, T},
title = {Amplification-free cancer diagnosis based on inhibition of Cas12a activity by site-specific 5mC-modified cfDNA.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41404798},
issn = {1362-4962},
support = {82172619//National Natural Science Foundation of China/ ; YXQN202421//Outstanding Young Talents Program of Chongqing/ ; 2023nlts007//Enhancing Scientific Research Capabilities of Chongqing University Cancer Hospital/ ; 2024MSXM136//Chongqing Municipal Science and Health Joint Medical Research/ ; },
mesh = {Humans ; *DNA Methylation ; *CRISPR-Cas Systems/genetics ; *Cell-Free Nucleic Acids/genetics/blood/chemistry ; *Neoplasms/diagnosis/genetics/blood ; *CRISPR-Associated Proteins/metabolism/antagonists &amp; inhibitors/genetics ; *Endodeoxyribonucleases/metabolism/genetics/chemistry ; Bacterial Proteins ; },
abstract = {DNA methylation detection holds significant value for cancer diagnosis and recurrence monitoring. However, current methods are often time-consuming, costly, and necessitate specialized techniques. The CRISPR-Cas system, particularly Cas12a, presents a precise and user-friendly platform for disease diagnosis. We developed the CRISPR-Methylated DNA Detection Test (CRISPR-MeDNA Test), a Cas12a-based method for detecting methylation in plasma cell-free DNA (cfDNA). The results reveal that 5mC-modified DNA significantly suppresses the trans-cleavage activity of Cas12a, depending on the methylation site, number, and interval spacing. Simultaneously, methylation of the non-target strand (NTS) suppresses Cas12a activity more strongly than methylation of the target strand (TS), as the NTS plays a critical role in R-loop formation, which is essential for Cas12a cleavage target DNA. Mechanistically, 5mC-modified DNA was found to trigger conformational rearrangements in the Cas12a complex, as predicted by AlphaFold3 modeling and corroborated by FRET assays. Notably, the combination of Cas12a with multiplexed guide RNAs enables effective discrimination between cfDNA from healthy donors and cancer patients without the need for pre-amplification, based on the inhibitory effects of methylated DNA on the Cas12a trans-cleavage activity. This work provides a Cas12a-based detection for a rapid, cost-effective, low-complexity method for 5mC-modified cfDNA in liquid biopsies.},
}
@article {pmid41405812,
year = {2025},
author = {Wang, L and Ren, S and Behan, AA and Buzdar, JA and Arain, MA and Li, Y},
title = {Promising Future of Engineered Probiotics for Antimicrobial Peptides and Protein Production: Prospects and Industrial Challenges.},
journal = {Probiotics and antimicrobial proteins},
volume = {},
number = {},
pages = {},
pmid = {41405812},
issn = {1867-1314},
}
@article {pmid41406514,
year = {2026},
author = {Ghodrat, R and Ramachandran, H and Hildebrandt, B and Binder, S and Rossi, A and Reichert, AS},
title = {CRISPR/Cpf1-mediated editing of PINK1 in induced pluripotent stem cells.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103887},
doi = {10.1016/j.scr.2025.103887},
pmid = {41406514},
issn = {1876-7753},
mesh = {*Induced Pluripotent Stem Cells/metabolism/cytology ; Humans ; *Protein Kinases/genetics/metabolism ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Cell Differentiation ; Cell Line ; },
abstract = {The PTEN induced kinase 1 (PINK1) gene is crucial for mitophagy and mitochondrial quality control. Mutations in the PINK1 gene are associated with several neurological disorders. To decipher the role of PINK1-mediated mitophagy in human induced pluripotent stem cells (hiPSCs) and in their differentiated counterparts, we used CRISPR/Cpf1 and generated a human iPSC line with homozygous out-of-frame deletions by targeting exon 6 of the PINK1 gene. The generated homozygous PINK1 mutant cell line showed normal cell morphology, genomic stability, and expression of classical stem cell markers. Furthermore, the cells can be differentiated efficiently into the three germ layers.},
}
@article {pmid41406662,
year = {2026},
author = {Schalper, KT and Yang, R and Guan, X and Zhang, J and Schreiber, D and Moon, J and Liu, C},
title = {Programmable CRISPR-mediated gold nanoparticle adhesion for visual colorimetric detection.},
journal = {Biosensors &amp; bioelectronics},
volume = {295},
number = {},
pages = {118319},
pmid = {41406662},
issn = {1873-4235},
support = {U01 CA269147/CA/NCI NIH HHS/United States ; },
mesh = {*Colorimetry/methods ; *Gold/chemistry ; *Biosensing Techniques/methods ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/diagnosis/virology ; *DNA, Viral/isolation &amp; purification/genetics/analysis ; *Papillomaviridae/isolation &amp; purification/genetics ; Female ; Limit of Detection ; DNA, Single-Stranded/chemistry ; },
abstract = {While the aggregation behavior of gold nanoparticles (AuNPs) has been extensively studied in biosensing, catalysis, and nanomedicine, their potential for programmable surface adhesion via tunable surface chemistry remains largely untapped. Here, a programmable CRISPR-mediated hydrophobic adhesion phenomenon using streptavidin-coated AuNPs functionalized with Cy5-ssDNA-biotin probes is introduced. Hydrophobic Cy5 moieties on the AuNP surface induce localized aggregation and strong adhesion to hydrophobic surfaces. This unique behavior was leveraged by coupling CRISPR-Cas12a-mediated ssDNA cleavage with Cy5-labeled ssDNA-coated AuNPs to develop a simple, visual-readout colorimetric assay for nucleic acid detection. When combined with recombinase polymerase amplification, the method achieved ultrasensitive detection of human papillomavirus (HPV) DNA down to 10 aM, without the need for complex instrumentation. The platform's clinical utility was validated by detecting HPV DNA in cervical swab samples, highlighting its promise for low-cost, sensitive, and accessible point-of-care diagnostics in resource-limited settings. Unlike conventional aggregation-dispersion systems, this platform introduces a fundamentally distinct signal transduction mechanism based on surface adhesion, defining a new modality within CRISPR-based colorimetric diagnostics and offering a simple, low-cost solution for point-of-care testing.},
}
@article {pmid41406894,
year = {2026},
author = {Heinemann, JA and Hiscox, TC and Zanatta, CB and Kurenbach, B and Walker, S and McCabe, AW and Hoepers, AM and Agapito-Tenfen, SZ},
title = {Genome editing outside of controlled facilities: A review of plausible futures and risks.},
journal = {Ecotoxicology and environmental safety},
volume = {309},
number = {},
pages = {119565},
doi = {10.1016/j.ecoenv.2025.119565},
pmid = {41406894},
issn = {1090-2414},
mesh = {Animals ; Humans ; *Gene Editing/methods/trends ; Risk Assessment ; },
abstract = {Vectors for delivering proteins and/or nucleic acids into the cells of whole organisms, from single to multicellular, are rapidly advancing. Common cargos are nucleic acids needed to express the components of a genome editing reaction, or ribonucleoproteins (RNP) that can act immediately upon delivery. In only 20 years, improvements in associated formulation technologies have decreased the dependence of genome editing on the need for a laboratory or trained personnel, allowing for genome editing outside of controlled facilities. As this happens, both target and non-target organisms may be exposed to active biological agents, necessitating a new framework for risk assessment. Some scientists deny developments for gene editing in uncontrolled environments, leading to scientifically unjustified dismissals of risk.},
}
@article {pmid41407085,
year = {2026},
author = {Qi, H and Yang, Y and Hou, X and Chen, Y and Gong, S},
title = {Sensitivity-improving CRISPR-Cas strategies for non-nucleic acid targets detection.},
journal = {Methods (San Diego, Calif.)},
volume = {246},
number = {},
pages = {151-173},
doi = {10.1016/j.ymeth.2025.12.004},
pmid = {41407085},
issn = {1095-9130},
mesh = {*CRISPR-Cas Systems/genetics ; *Nucleic Acid Amplification Techniques/methods ; Humans ; *Gene Editing/methods ; Nucleic Acids/genetics ; },
abstract = {CRISPR-Cas systems have revolutionized non-nucleic acid targets detection across diverse applications. Nevertheless, the relatively low enzymatic turnover rate of activated Cas nucleases during substrate cleavage remains a critical bottleneck, limiting the sensitivity of such detection methods. To address this challenge, numerous innovative strategies have been proposed to enhance the sensitivity of CRISPR-Cas systems, enabling high-sensitive non-nucleic acid targets detection. This review systematically summarizes the sensitivity-enhancing methodologies for non-nucleic acid targets detection using CRISPR-Cas technologies. We first delineate the working mechanisms of various CRISPR-Cas systems and the signal transduction pathways specific to non-nucleic acid targets. Subsequently, we detail diverse sensitivity-improving approaches, including nucleic acid amplification-facilitated strategies, multimolecular labeling techniques, dual-enzyme cascade methods, and multiplex amplification methodologies. Additionally, the current challenges and future perspectives in this field are discussed, aiming to inspire researchers to develop more ingenious solutions and facilitate real-world applications of CRISPR-Cas system for non-nucleic acid targets detection.},
}
@article {pmid41407557,
year = {2025},
author = {Tyagi, E and Sachan, A and Bhuyan, R and Kumari, P and Prakash, A},
title = {Next-Gen Biofilm Control: Gene Editing and Computational Approaches.},
journal = {APMIS : acta pathologica, microbiologica, et immunologica Scandinavica},
volume = {133},
number = {12},
pages = {e70122},
doi = {10.1111/apm.70122},
pmid = {41407557},
issn = {1600-0463},
mesh = {*Biofilms/drug effects/growth &amp; development ; *Gene Editing/methods ; Humans ; *Computational Biology/methods ; CRISPR-Cas Systems ; *Bacteria/genetics/drug effects ; Anti-Bacterial Agents/pharmacology ; Drug Resistance, Bacterial ; Phage Therapy ; Bacteriophages ; },
abstract = {Biofilms are microbial communities enclosed in an extracellular polymeric substance (EPS), significantly contributing to antimicrobial resistance (AMR) in medical, industrial, and environmental settings. Their matrix enhances microbial survival, inhibits antibiotic penetration, and facilitates horizontal gene transfer, worsening the AMR crisis. Conventional antimicrobial treatments often fail against biofilms, necessitating novel therapeutic strategies. Emerging biofilm-targeted interventions, such as nanotechnology-based antimicrobials, bacteriophage therapy, and CRISPR-Cas9 gene editing, offer promising solutions. Nanoparticles improve drug delivery, bacteriophages selectively lyse resistant bacterial populations, and CRISPR-Cas9 disrupts AMR-related genes and biofilm virulence factors. Additionally, AI and ML are advancing biofilm prediction models and antimicrobial optimization, paving the way for precision-targeted interventions. This review explores biofilm biology and next-generation biofilm control strategies, with a focus on AI-driven bioinformatics. Future research should focus on clinical translation, regulatory standardization, and scalable implementation in healthcare and industrial settings to combat biofilm-associated AMR.},
}
@article {pmid41407671,
year = {2025},
author = {Bitew, MA and Paredes-Santos, TC and Maru, P and Krishnamurthy, S and Wang, Y and Sangaré, LO and Duley, S and Yamaryo-Botté, Y and Botté, CY and Saeij, JPJ},
title = {A genome-wide CRISPR screen identifies GRA38 as a key regulator of lipid homeostasis during Toxoplasma gondii adaptation to lipid-rich conditions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11177},
pmid = {41407671},
issn = {2041-1723},
support = {R01 AI173803/AI/NIAID NIH HHS/United States ; R01AI173803//Division of Intramural Research, National Institute of Allergy and Infectious Diseases (Division of Intramural Research of the NIAID)/ ; },
mesh = {*Toxoplasma/genetics/metabolism/pathogenicity/physiology ; Animals ; *Protozoan Proteins/metabolism/genetics ; Homeostasis ; Mice ; *Lipid Metabolism/genetics ; Virulence/genetics ; Phosphatidate Phosphatase/metabolism/genetics ; Toxoplasmosis/parasitology ; Adaptation, Physiological/genetics ; CRISPR-Cas Systems ; Female ; Clustered Regularly Interspaced Short Palindromic Repeats ; Humans ; Genome, Protozoan ; },
abstract = {Intracellular parasites like Toxoplasma gondii scavenge host nutrients, particularly lipids, to support their growth and survival. Although Toxoplasma is known to adjust its metabolism based on nutrient availability, the mechanisms that mediate lipid sensing and metabolic adaptation remain poorly understood. Here, we perform a genome-wide CRISPR screen under lipid-rich (10% Fetal Bovine Serum (FBS)) and lipid-limited (1% FBS) conditions to identify genes critical for lipid-responsive fitness. We identify the Toxoplasma protein GRA38 as a lipid-dependent regulator of parasite fitness. GRA38 exhibits phosphatidic acid (PA) phosphatase (PAP) activity in vitro, which is significantly reduced by mutation of its conserved DxDxT/V catalytic motif. Disruption of GRA38 leads to the accumulation of PA species and widespread alterations in lipid composition, consistent with impaired PAP activity. These lipid imbalances correlate with reduced parasite virulence in mice. Our findings identify GRA38 as a metabolic regulator important for maintaining lipid homeostasis and pathogenesis in Toxoplasma gondii.},
}
@article {pmid41409480,
year = {2025},
author = {Anwar, M and Vinothkanna, A and Jia, AQ},
title = {Fostering plant protection against certain bacterial diseases through quorum-sensing signal molecules: a critical review.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1602573},
pmid = {41409480},
issn = {1664-462X},
abstract = {Quorum sensing (QS) and clustered regularly interspaced short palindromic repeats (CRISPR) systems are envisaged as revolutionary in abating plant bacterial pathogens. Bacterial cell-cell communication and plant pathogen QSSMs (quorum sensing signaling molecules) are dissected for underlying mechanisms in prominent pathogens, viz., Pseudomonas syringae, Erwinia amylovora, and Xanthomonas campestris. Biofilm formation and virulence mechanisms are critically addressed to repurpose potential QS inhibition strategies. CRISPR technologies are combined with CRISPR engineering to produce enhanced disease-resistant varieties, with potential applications. QS-CRISPR interplay for deciphering the key interactive changes in plant health management is prioritized for deliberate future research outcomes. Sustainable agricultural practices are envisaged for successful lab-to-field authentic field trials and large-scale applicability across the globe. Potential technical limitations, the need for stringent agricultural laws, and future innovations are addressed. Moreover, the cost-effectiveness, enhanced crop production, yield, and productivity hindering the above key plant bacterial pathogens are comprehensively addressed against these plant bacterial pathogens. Furthermore, a future outlook characterized by extensive outreach and global implications is substantiated regardless of regional specificity, climate change, and global warming. A decade of research on advancements in adequate plant protection is revisited to incorporate augmented approaches, including artificial intelligence (AI) and machine learning, in sustainable agriculture. The significance of the present review is based on addressing QSSMs and plant protection strategies encompassing modern molecular biological techniques.},
}
@article {pmid41409641,
year = {2024},
author = {Zhang, Z and Ding, S},
title = {Gene editing and reprogramming of human fibroblast cells (hFBs) to human pluripotent stem cells (hiPSCs).},
journal = {Neuromethods},
volume = {210},
number = {},
pages = {39-59},
pmid = {41409641},
issn = {0893-2336},
support = {R01 NS069726/NS/NINDS NIH HHS/United States ; R01 NS123023/NS/NINDS NIH HHS/United States ; },
abstract = {Predictive disease models play significant roles in advancing our knowledge of the pathology of human disease. In this field, animal models have been extensively employed and have provided crucial insights into the pathophysiological mechanisms of human disease. However, they often fail to fully capture many human phenotypes due to significant species differences in genomic responses. Human induced pluripotent stem cells (hiPSCs) are genetically reprogrammed cells that exhibit qualities remarkably similar to those of embryonic stem cells (ESC) and have emerged as a promising source for cell therapy and fundamental research in pathology. The ability to reprogram human fibroblast cells (hFBs) to iPSCs provides an opportunity to model human diseases. However, even hiPSCs from different persons have different genetic background, thus generation of isogenic unaffected control hiPSCs is necessary to study model human disease. Here, we describe methods to generate isogenic hFBs using CRISPR/Cas9 gene editing method, and subsequently reprogram them into iPSCs using commercially available Sendai virus vectors. Specifically, using the CRISPR/Cas9 system and Sendai virus vector, isogenic iPSC lines can be generated. This protocol provides a structured approach for obtaining multiple isogeneic hiPSC lines, which facilitate the modeling of various human diseases.},
}
@article {pmid41410478,
year = {2026},
author = {Wang, X and Yu, G and Luo, Y and Chen, T and Zhang, X and Ye, L and Yang, C and Chen, Q},
title = {The autophagy-related protein PlAtg26b regulates vegetative growth, reproductive processes, autophagy, and pathogenicity in Peronophythora litchii.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2606498},
pmid = {41410478},
issn = {2150-5608},
mesh = {*Autophagy ; Virulence ; Plant Diseases/microbiology/parasitology ; *Autophagy-Related Proteins/genetics/metabolism ; Litchi/microbiology/parasitology ; *Oomycetes/pathogenicity/growth &amp; development/genetics ; CRISPR-Cas Systems ; Reproduction ; Plant Leaves/microbiology ; Mitochondria/metabolism ; Gene Knockout Techniques ; Hyphae/growth &amp; development ; },
abstract = {Peronophythora litchii is an oomycete pathogen responsible for litchi downy blight, a significant threat to global litchi production. Autophagy, a conserved degradation pathway crucial for the growth, development, and pathogenicity of phytopathogenic organisms, remains an area of active investigation. In this study, we characterized the function of the Atg26 homolog PlAtg26b in P. litchii. Using the CRISPR/Cas9 genome editing system, we generated PlATG26b knockout mutants and determined that PlAtg26b localizes to mitochondria under stress conditions. Although deletion of PlATG26b did not impair selective autophagy, it markedly reduced Atg8-PE synthesis, vegetative hyphal growth, asexual and sexual reproduction, and zoospore release. Furthermore, PlATG26b-deficient mutants exhibited significantly reduced virulence on litchi fruits and leaves. Collectively, our findings demonstrate that PlAtg26b plays a pivotal role in the biological development and pathogenicity of P. litchii.},
}
@article {pmid41410797,
year = {2025},
author = {Appolonia, CN and Centore, JT and Shukla, S and Hluck, J and Conlon, RA and Ramakrishnan, P},
title = {A CRISPR/Cas9 assisted strategy for the conditional expression of human NF-kappaB c-Rel cDNA in mouse T cells: design, prospects, and challenges.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {56},
pmid = {41410797},
issn = {1573-9368},
mesh = {Animals ; Humans ; Mice ; *CRISPR-Cas Systems/genetics ; Mice, Transgenic ; *Proto-Oncogene Proteins c-rel/genetics ; Promoter Regions, Genetic ; Mice, Inbred NOD ; Gene Editing ; *T-Lymphocytes/metabolism ; DNA, Complementary/genetics ; *Diabetes Mellitus, Type 1/genetics ; NF-kappa B/genetics ; },
abstract = {Nuclear factor-κB protein c-Rel is a critical regulator of autoimmune diabetes. We found that c-Rel O-GlcNAcylation at serine-350 increases with hyperglycemia, which results in increased transcription of proautoimmune Th1 cytokines, interleukin-2 (IL-2) and interferon-gamma (IFN-γ), and decreased transcription of the T regulatory cell transcription factor forkhead box 3 (FOXP3). To further study the translational relevance of c-Rel S350 O-GlcNAcylation in autoimmune diabetes, we sought to generate transgenic non-obese diabetic (NOD) mice conditionally expressing wildtype or mutant S350A human c-Rel cDNA in T cells downstream of the endogenous mouse REL promoter. We used CRISPR-Cas9 gene editing to insert a unique designer cassette containing floxed mouse c-Rel cDNA-STOP sequence to maintain whole body c-Rel expression, followed by a linker and human c-Rel cDNA-STOP sequence. Using comprehensive PCR analyses and high-throughput sequencing, we confirmed successful insertion of the cassette at the mouse REL locus and the expected deletion of the mouse c-Rel cDNA specifically in T cells following CD4-Cre mating. Additional characterization revealed that the knock-in transgenic mice lacked endogenous mouse c-Rel, further confirming desired interference with its natural start codon. Unexpectedly, these mice lacked mouse and human c-Rel protein expression from inserted cDNAs, which mechanistically correlated with increased CpG methylation of the c-Rel promoter region. Thus, our study presents a unique, universal molecular design and method for the generation of conditional knock-in transgenic mice expressing human genes at the endogenous mouse promoter. It also reveals a potential locus-specific challenge that may arise during the development of such novel transgenic mouse models.},
}
@article {pmid41410807,
year = {2025},
author = {Sarroukh, I and Ibriz, M and Yakkou, L and Lebkiri, N and Fokar, M and Iraqi, D and Gaboun, F and Diria, G and Abdelwahd, R},
title = {The Agrobacterium-mediated genetic transformation: a gateway for efficient CRISPR/Cas9 gene editing in leguminous.},
journal = {Transgenic research},
volume = {34},
number = {1},
pages = {57},
pmid = {41410807},
issn = {1573-9368},
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Transformation, Genetic/genetics ; *Plants, Genetically Modified/genetics/growth &amp; development ; *Agrobacterium tumefaciens/genetics ; },
abstract = {Climate change enhances the damaging consequences of abiotic and biotic stressors, leading to severe soil fertility loss and ecosystem degradation worldwide. Leguminous have contributed significantly to replenishing soil nitrogen via symbiotic nitrogen fixation, contributing approximately 15% of nitrogen input, which is crucial for soil health and enhancing crop production. There is an increasing integration of new biotechnological interventions, such as genome editing, including the CRISPR/Cas9 system, and transgenesis, in addition to classical breeding, to make agriculture more resilient. In this review, we examine several elements that influence the genetic transformation system employing Agrobacterium tumefaciens strains in leguminous to make it an ideal vehicle for CRISPR/Cas9 component delivery. The variables investigated in our study included the incubation period, co-cultivation duration, bacterial density, selectable marker, concentration, and growth regulators used. In addition, the selection and efficiency of the explant choice for transformation should be considered in future studies. However, there have been parallel recommendations for the gradual application of selectable markers such as kanamycin.},
}
@article {pmid41410934,
year = {2025},
author = {Deora, S and Deora, GS and Nigam, S and Harish, },
title = {Hacking heterocysts: advances in the genetic regulation of heterocyst differentiation.},
journal = {Archives of microbiology},
volume = {208},
number = {1},
pages = {80},
pmid = {41410934},
issn = {1432-072X},
mesh = {Nitrogen Fixation/genetics ; *Gene Expression Regulation, Bacterial ; *Cyanobacteria/genetics/metabolism/growth &amp; development ; Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; },
abstract = {Heterocyst differentiation in certain filamentous cyanobacteria is a multifaceted process essential for nitrogen fixation, orchestrated by a sophisticated regulatory network that encompasses several key stages. These include induction, pattern differentiation, commitment, extracellular layer formation, cell-cell communication, and ultimately, nitrogen fixation and metabolism. Key regulators like NtcA and HetR control heterocyst development, while proteins such as PatS, HetN, and PatA modulate pattern formation. Certain non-coding RNAs, such as NsiR1, Yfr1, and NsiR4, also regulate gene expression and contribute to the shutdown of CO2 fixation in differentiating heterocysts. Meanwhile, the heterocysts' unique envelope protects nitrogenase from oxygen, enabling nitrogen fixation. Genetic engineering approaches, including CRISPR-Cas systems, have been employed to increase heterocyst frequency and enhance the production of compounds such as ethanol, butanol and H2. By manipulating genes responsible for heterocyst differentiation, scientists can optimize nitrogen fixation, develop efficient biofertilizers, and unlock opportunities for a more sustainable future in agriculture and biotechnology. This review addresses the current understanding of the regulatory networks and molecular mechanisms that influence the development and function of heterocysts, providing insights into the biology and potential applications of these specialized cells through gene manipulations.},
}
@article {pmid41411128,
year = {2026},
author = {Mochida, T and Fujimoto, N and Asahina, M and Asano, S and Araki, S and Inukai, N and Hotta, A},
title = {Muscle satellite cell editing by LNP-CRISPR-Cas9 to resist muscle injury.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116695},
doi = {10.1016/j.celrep.2025.116695},
pmid = {41411128},
issn = {2211-1247},
mesh = {Animals ; *Satellite Cells, Skeletal Muscle/metabolism ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Mice ; *Muscular Dystrophy, Duchenne/genetics/therapy/pathology ; *Muscle, Skeletal/injuries/metabolism/pathology ; *Nanoparticles/chemistry ; Mice, Inbred C57BL ; RNA, Guide, CRISPR-Cas Systems/genetics ; Disease Models, Animal ; Mice, Inbred mdx ; Male ; Exons/genetics ; Liposomes ; },
abstract = {Muscle satellite cells are essential for skeletal muscle regeneration and represent an attractive therapeutic target for gene delivery in Duchenne muscular dystrophy (DMD). However, efficient in vivo transduction of these cells has remained challenging. Here, we demonstrate that lipid nanoparticle (LNP)-mediated delivery of Streptococcus pyogenes CRISPR-Cas9 mRNA and guide RNA (LNP-CRISPR) induces exon skipping in Pax7-positive satellite cells more efficiently than adeno-associated virus (AAV) vectors following intramuscular or intravenous administration in a DMD mouse model. Furthermore, unlike AAV-CRISPR, LNP-CRISPR-mediated genome editing showed greater resistance to repeated muscle injuries, indicating successful editing of regenerative satellite cells. These results highlight the potential of LNPs as a non-viral platform for durable genome editing in skeletal muscle and lay the foundation for developing safe and sustainable genome-editing therapies for DMD.},
}
@article {pmid41411488,
year = {2026},
author = {Bär, I and Groten, SA and Barraclough, A and Bürgisser, PE and van Kwawegen, C and Lenting, PJ and van Moort, I and Eikenboom, JCJ and Leebeek, FWG and Voorberg, J and van den Biggelaar, M and Bierings, R},
title = {Allele-selective disruption of pathogenic VWF variants in type 2 von Willebrand disease using CRISPR/Cas9.},
journal = {Blood advances},
volume = {10},
number = {5},
pages = {1429-1443},
pmid = {41411488},
issn = {2473-9537},
mesh = {Humans ; *von Willebrand Factor/genetics ; *Alleles ; *CRISPR-Cas Systems ; *von Willebrand Disease, Type 2/genetics/therapy ; Polymorphism, Single Nucleotide ; *Gene Editing/methods ; Genetic Therapy/methods ; Mutation ; },
abstract = {In contrast to major innovations in treating severe hemophilia, the treatment of severe von Willebrand disease (VWD) remains limited to intravenous infusion of von Willebrand factor (VWF) concentrates. To date, no gene therapy-based approaches for the treatment of VWD have been developed, largely owing to the disease's heterogeneous mutational landscape and the challenge of specifically targeting VWF production in endothelial cells. In this study, we developed a novel gene therapy strategy for patients with VWD caused by heterozygous dominant-negative VWF variants. Our strategy permanently inactivates VWF variants by selectively disrupting the pathogenic allele's open reading frame via the introduction of indels by Cas9. To circumvent the challenge of designing variant-specific strategies, we targeted the common single nucleotide polymorphism (SNP) rs1800378 in VWF. We used endothelial colony-forming cells (ECFCs) from patients with VWD2A and VWD2B with heterozygous p.C1190R and p.R1306W variants, respectively, to demonstrate ex vivo proof of principle. Using next-generation sequencing analysis, we show efficient and allele-selective knockout of VWF, while maintaining VWF expression of the nontargeted allele. Variant mapping mass spectrometry that discriminates between wild-type and variant VWF proteoforms confirmed selective reduction of variant allele expression, which was accompanied by reversal of cellular disease phenotypes in ECFCs. This study shows the feasibility of a novel gene editing strategy for VWD that, by virtue of its targeting of a common SNP, can be broadly applicable and can be used to design treatments for VWD without being constrained by the disease-causing variant, pathogenic mechanism, or VWD subtype.},
}
@article {pmid41411621,
year = {2026},
author = {Tankka, AT and Zhang, Y and Einstein, JM and Zhou, CJ and Pham, VN and Naritomi, JT and Nguyen, GG and Mendez-Molina, AN and Hu, Z and Mizrahi, O and Perelis, M and Sarsam, J and Tan, FE and Kaufman, DS and Yang, J and Antal, CE and Yeo, GW},
title = {Integrative CRISPR Screening and RNA Analyses Discover an Essential Role for PUF60 Interactions with 3' Splice Sites in Cancer Progression.},
journal = {Cancer research},
volume = {86},
number = {7},
pages = {1586-1604},
pmid = {41411621},
issn = {1538-7445},
support = {R01 CA268179/CA/NCI NIH HHS/United States ; S10 OD025060/OD/NIH HHS/United States ; U24 HG009889/HG/NHGRI NIH HHS/United States ; S10 OD026929/OD/NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; F32 HL143978/HL/NHLBI NIH HHS/United States ; P30 CA023100/CA/NCI NIH HHS/United States ; U54 CA209891/CA/NCI NIH HHS/United States ; R01 CA174869/CA/NCI NIH HHS/United States ; U24 HG011735/HG/NHGRI NIH HHS/United States ; RF1 MH126719/MH/NIMH NIH HHS/United States ; R01 CA262794/CA/NCI NIH HHS/United States ; R01 HG011864/HG/NHGRI NIH HHS/United States ; T32 CA067754/CA/NCI NIH HHS/United States ; P30CA23100//National Cancer Institute (NCI)/ ; S10OD026929//National Institutes of Health (NIH)/ ; 1S10OD025060//National Institutes of Health (NIH)/ ; U54 CA209891/CA/NCI NIH HHS/United States ; T32CA067754//National Institutes of Health (NIH)/ ; //Gruss-Lipper Family Foundation/ ; F32 HL143978/HL/NHLBI NIH HHS/United States ; R01 CA174869/CA/NCI NIH HHS/United States ; R01 CA262794/CA/NCI NIH HHS/United States ; R01 CA268179/CA/NCI NIH HHS/United States ; U24 HG009889/HG/NHGRI NIH HHS/United States ; RF1 MH126719/MH/NIMH NIH HHS/United States ; R01 HG011864/HG/NHGRI NIH HHS/United States ; R01 HG004659/HG/NHGRI NIH HHS/United States ; 2023-332369//Chan Zuckerberg Initiative (CZI)/ ; V2024-008//V Foundation for Cancer Research (VFCR)/ ; IRG-19-230-48-IRG//American Cancer Society (ACS)/ ; P30CA023100//National Cancer Institute (NCI)/ ; },
mesh = {Humans ; Animals ; Mice ; Female ; Cell Proliferation/genetics ; Disease Progression ; *RNA Splicing Factors/genetics/metabolism ; *CRISPR-Cas Systems ; *Triple Negative Breast Neoplasms/genetics/pathology/metabolism ; Gene Expression Regulation, Neoplastic ; Cell Line, Tumor ; Apoptosis/genetics ; *RNA-Binding Proteins/genetics/metabolism ; RNA Splicing ; Xenograft Model Antitumor Assays ; Repressor Proteins ; },
abstract = {UNLABELLED: RNA-binding proteins (RBP) are important regulators of posttranscriptional gene expression. Understanding which and how RBPs promote cancer progression is crucial for cancers that lack effective targeted therapies, such as triple-negative breast cancer (TNBC). In this study, we employed both in vitro and in vivo pooled CRISPR/Cas9 screening to identify 50 RBP candidates essential for TNBC cell survival. Integrated enhanced cross-linking and immunoprecipitation and RNA sequencing analysis identified that poly(U)-binding splicing factor 60 (PUF60) drives exon inclusion within proliferation-associated transcripts that, when misspliced, induce cell cycle arrest and DNA damage. Furthermore, disrupting PUF60 interactions with 3' splice sites via a substitution in its RNA-binding domain caused widespread exon skipping, leading to downregulation of proliferation-associated mRNAs and inducing apoptosis in TNBC cells. Knockdown of PUF60 or disruption of PUF60-RNA interactions inhibited TNBC cell proliferation and shrunk tumor xenografts in multiple models. Together, these findings reveal the molecular mechanism by which PUF60 supports cancer progression.<br><br>SIGNIFICANCE: Functional screening of RNA-binding proteins is an effective strategy for identifying cancer regulators that revealed PUF60-mediated splicing activity as a driver of oncogenic proliferation and a potential therapeutic target.},
}
@article {pmid41412110,
year = {2025},
author = {Snell, JC and Nelson, BJ and Matreyek, KA},
title = {DIALing in elevated expression setpoints with promoter shortening.},
journal = {Cell systems},
volume = {16},
number = {12},
pages = {101482},
doi = {10.1016/j.cels.2025.101482},
pmid = {41412110},
issn = {2405-4720},
mesh = {*Promoter Regions, Genetic/genetics ; *Gene Expression Regulation/genetics ; Humans ; CRISPR-Cas Systems/genetics ; },
abstract = {DIAL is a novel framework for temporal control of transcript abundances in engineered cells. Targeted excision of DNA spacers in transgenic promoters permits controlled transitions of protein expression between setpoints. DIAL expands the repertoire of bioengineering tools for controlling protein expression, cell fates, and biological systems in general.},
}
@article {pmid41412287,
year = {2026},
author = {Su, Z and Liang, Z and Wu, Q and Xu, S and Li, C and Zheng, H and Wu, C and Ji, W and Niu, Y and Yang, Z},
title = {Metal-organic frameworks for CRISPR/Cas9 gene editing delivery: Innovations in therapeutic and diagnostic applications.},
journal = {Acta biomaterialia},
volume = {210},
number = {},
pages = {516-534},
doi = {10.1016/j.actbio.2025.12.030},
pmid = {41412287},
issn = {1878-7568},
mesh = {*Metal-Organic Frameworks/chemistry/therapeutic use ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Animals ; *Gene Transfer Techniques ; *Genetic Therapy/methods ; },
abstract = {CRISPR/Cas gene editing technology demonstrates significant promise in the treatment of various diseases, and a precise, efficient and safe delivery system is a key to realize gene therapy. Although traditional viral vectors can achieve superior transfection efficiency, viruses suffer from low reproduction efficiency and the risk of random gene integration, further limiting their wide application. Notably, metal-organic frameworks (MOFs), with tunable pore structure, easy surface chemical modification, good biocompatibility and physiological stability, have drawn much attention in the domain of targeted delivery of gene editing systems. Compared to lipid nanoparticles (LNPs) and extracellular vesicles (EVs), MOFs offer superior cargo loading (>80 % for proteins) and protect nucleic acids from degradation, while their stimuli-responsive degradation enables controlled release. This review focus on the cutting-edge advances of intelligent-responsive MOFs in delivering gene editing systems to against diseases, including endogenous responses (e.g., ATP, pH, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Additionally, we further summarize the research of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination. This study will help facilitate the clinical translation of MOFs-based CRISPR/Cas delivery systems in the field of therapy and detection of diseases. STATEMENT OF SIGNIFICANCE: This article reviews the cutting-edge advances of intelligent-responsive MOFs in delivering CRISPR/Cas systems to against diseases, including endogenous responses (e.g., pH, ATP, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Importantly, the potential applications of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination also have been discussed. This study will provide insights for the development of MOFs-based CRISPR/Cas delivery systems in the therapy and detection of clinical diseases.},
}
@article {pmid41412367,
year = {2026},
author = {Jose, J and Hamow, K&#xc1; and Éva, C and Moncsek, B and Kyrpa, T and Gamarra Reinoso, L and Bozsó, Z and Bakonyi, J and Balázs, E and Sági, L},
title = {CRISPR/Cas-mediated polyphenol oxidase gene knockout in potato reveals divergent roles in resistance to bacterial wilt and late blight.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {364},
number = {},
pages = {112944},
doi = {10.1016/j.plantsci.2025.112944},
pmid = {41412367},
issn = {1873-2259},
mesh = {*Solanum tuberosum/genetics/microbiology/enzymology/immunology ; *Catechol Oxidase/genetics/metabolism ; *Ralstonia solanacearum/physiology ; *Plant Diseases/microbiology/immunology/genetics ; *CRISPR-Cas Systems ; *Disease Resistance/genetics ; *Phytophthora infestans/physiology ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; },
abstract = {Polyphenol oxidases (PPOs) play a pivotal role in plant immune responses by catalysing the oxidation of phenolic compounds into cytotoxic quinones and melanin and contributing to the fortification of cell walls. Despite their biological significance, the high expression of PPOs in potatoes is not desirable due to their promotion of tuber browning. This study elucidates the relationship between PPO activity and defense mechanisms against the diverse pathogens Ralstonia solanacearum (Rs) and Phytophthora infestans (Pi) while mitigating enzymatic browning. CRISPR/Cas-mediated editing of the tuber- and root-specific PPO genes in the 'Désirée' and 'Balatoni Rózsa' potato cultivars considerably reduced enzymatic activity and browning. Among four PPO-edited mutant lines, three exhibited increased susceptibility to Rs while responses to Pi remained unchanged, underscoring the importance of PPOs in resistance to Rs. The PPO knockouts resulted in significant shifts in metabolite and hormone profiles characterized by elevated levels of dihydrokaempferol, coniferyl alcohol and taxifolin among other metabolites in the roots of Rs-susceptible mutants. Additionally, reduced PPO activity in these lines correlated with increased concentrations of salicylic acid, jasmonic acid and several antimicrobial compounds and alterations in flavonoid regulation. These findings highlight the complex role of PPOs in plant defense, establishing a positive correlation between PPO activity and resistance to Rs, while offering insights into the trade-offs associated with PPO gene editing in potatoes.},
}
@article {pmid41413028,
year = {2025},
author = {Chen, Q and Jiang, X and Yang, B and Deng, Z and Sun, Y},
title = {Anti-CRISPR protein AcrIIA5 can enhance the activity and security of prime editing.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11435},
pmid = {41413028},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; CRISPR-Associated Protein 9/metabolism/genetics ; Humans ; INDEL Mutation ; HEK293 Cells ; DNA Breaks, Double-Stranded ; },
abstract = {Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known "inhibitor" Acr protein can unexpectedly acting as an "enhancer" of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity.},
}
@article {pmid41413662,
year = {2026},
author = {Green, NFO and Sutton, GJ and Pérez-Burillo, J and Wang, J and Bagot, S and Danon, HG and Walsh, K and Gokool, A and Miles, SA and Yang, G and Herring, CA and Liang, Y and Pfundstein, G and Sytnyk, V and Alinejad-Rokny, H and Lister, R and Rosenbluh, J and Gagnon-Bartsch, JA and Voineagu, I},
title = {CRISPRi screening in cultured human astrocytes uncovers distal enhancers controlling genes dysregulated in Alzheimer's disease.},
journal = {Nature neuroscience},
volume = {29},
number = {3},
pages = {703-716},
pmid = {41413662},
issn = {1546-1726},
support = {2020814//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Astrocytes/metabolism ; *Alzheimer Disease/genetics/metabolism ; *Enhancer Elements, Genetic/genetics ; Cells, Cultured ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis ; Machine Learning ; },
abstract = {Genetic variants associated with complex traits often lie in distal enhancers. While candidate enhancers have been mapped genome wide, their functional state and gene targets in specific cell types remain unclear. Here we present AstroREG, a resource of enhancer-gene interactions in human primary astrocytes, generated by combining CRISPR inhibition (CRISPRi), single-cell RNA-seq and machine learning. By functionally testing nearly 1,000 PsychENCODE enhancers, we identified more than 150 regulatory interactions, revealing enhancers that control key astrocyte functions and genes implicated in Alzheimer's disease. The CRISPRi screen also provided valuable ground-truth data from a primary cell type for training and benchmarking prediction models of enhancer activity. We thus developed EGrf, a random forest (RF) model trained on these data, and applied it genome wide to predict regulatory interactions with high specificity. Together, our data provide a comprehensive functional map of enhancer-mediated regulation in a key glial cell type, shedding light on brain function and disease.},
}
@article {pmid41414648,
year = {2026},
author = {Wang, LR and Zhu, ST and Liao, ZH and Wu, N and Nie, ZK and Ye, C and Shi, TQ},
title = {Establishing a CRISPR/Cas9 Genome Editing System Combined with URA3-Blaster in Botrytis cinerea for Enhanced Abscisic Acid Production.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {2},
pages = {2207-2217},
doi = {10.1021/acs.jafc.5c14153},
pmid = {41414648},
issn = {1520-5118},
mesh = {*Botrytis/genetics/metabolism ; *Abscisic Acid/metabolism ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Metabolic Engineering ; *Fungal Proteins/genetics/metabolism ; },
abstract = {Abscisic acid (ABA) is a key plant growth regulator widely used in agriculture and ecological restoration. Although metabolic engineering of the fungus Botrytis cinerea can enhance ABA production, it has been hindered by inefficient genetic tools. In this study, we first established a recyclable selection marker system in B. cinerea based on orotidine-5'-phosphate decarboxylase. Subsequently, the CRISPR/Cas9 system was optimized, achieving up to 100% editing efficiency, far surpassing traditional homologous recombination. Based on this platform, multiple metabolic engineering strategies were systematically explored to enhance ABA biosynthesis. Increasing acetyl-CoA supply, inhibiting squalene synthesis, and knocking out key secondary metabolism genes Bcpks12 and Bcphs1 all significantly promoted ABA accumulation. Notably, co-overexpression of Bcacly1 and Bcacly2 combined with 1 g/L citrate increased ABA production to 1.36 g/L, representing a 38.66% improvement. Overall, this study provides an efficient genetic toolkit and a solid foundation for the industrial-scale production of ABA via engineered B. cinerea.},
}
@article {pmid41414667,
year = {2025},
author = {Klein, N and Sanchez-Londono, M and Kara, MM and Gomes-Filho, JV and Novak, S and Kholeif, KH and Pekarek, L and Caliskan, N and Randau, L},
title = {Type I-Fv and engineered type IV-A1 CRISPR-Cas effectors facilitate genome reduction in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414667},
issn = {1362-4962},
support = {//German Research Foundation/ ; 360987069//DFG/ ; 505997786//DFG/ ; //Microcosm Earth Center/ ; INST 93/1021-1 FUGG//Helmholtz Association/ ; //Philipps-Universit&#xe4;t Marburg/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing/methods ; *Genome, Bacterial ; DNA Repair ; CRISPR-Associated Proteins/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Class 1 CRISPR-Cas systems utilize multi-subunit effector ribonucleoprotein complexes to identify and target DNA. Upon recognition, type I systems recruit the helicase/nuclease Cas3 for DNA degradation, while type IV-A systems use the helicase CasDinG for transcriptional repression. Here, we developed two recombinant class 1 CRISPR-Cas genome editing tools for inducing large genomic deletions: the compact type I-Fv (also termed I-F2) system from Shewanella putrefaciens and the type IV-A1 system from Pseudomonas oleovorans. In the latter, CasDinG was engineered to include a C-terminal HNH nuclease domain, conferring DNA cleavage activity and enabling analysis of CasDinG processivity. Whole-genome sequencing of Escherichia coli BL21-AI was used to monitor genome reduction and DNA repair mechanisms in response to CRISPR-Cas-induced damage. Small deletions were flanked by microhomologies, consistent with repair via alternative end joining, whereas deletions larger than 10 kb consistently terminated at nearby IS1 elements, implicating these sequences in the repair process. This study introduces compact type I and engineered type IV-A genome editing tools with distinct protospacer-adjacent motif requirements and provides new insights into CasDinG evolution and the DNA repair pathways engaged during CRISPR-Cas-mediated genome editing.},
}
@article {pmid41414668,
year = {2025},
author = {Semsey, S and Søndberg, E and Røen, M and Hallström, B and Petersen, A&#xd8; and Alfastsen, L and Bosch, BR and Wohl, B and Clube, J and van der Helm, E and Groendahl, C and Mougiakos, I},
title = {Characterization and engineering of a type IV-A3 CRISPR-Cas system for genome editing in Escherichia coli.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414668},
issn = {1362-4962},
support = {//SNIPR Biome/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics ; *Gene Editing/methods ; Plasmids/genetics ; Klebsiella pneumoniae/genetics ; Genome, Bacterial ; DNA Helicases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; },
abstract = {CRISPR-Cas systems have revolutionized genome engineering technologies, but type IV CRISPR-Cas systems and their genome engineering potential have been critically underexplored. In this study, we identified a type IV-A3 CRISPR-Cas system from a clinical Klebsiella pneumoniae isolate and characterized its plasmid targeting activity and capacity to suppress chromosomal and plasmid gene expression in Escherichia coli. We revealed the pivotal role of Csf3 (Cas5) and the dispensable roles of Csf1 (Cas8-like) and Csf4 (DinG helicase) subunits in IV-A3 CRISPR-Cas complex formation. The system prevents plasmid propagation via interplay between DinG helicase activity and strategic protospacer positioning relative to plasmid replication and maintenance components. We enabled the IV-A3 CRISPR-Cas system to introduce lethal, sequence-specific double-stranded (ds)DNA breaks in the E. coli chromosome by fusing the nuclease domain of the I-TevI nuclease to the Cas8 N-terminus. Further, we developed a series of base editors, with various editing efficiencies and windows, by fusing the PmCDA1 cytidine deaminase to the Cas8, Cas5, and DinG subunits. Finally, conjugative transfer of the Cas5-PmCDA1 base editor into E. coli deactivated the tryptophan repressor gene, boosting IAA production. Our study provides new insights into type IV-A3 CRISPR-Cas systems and highlights their potential in genome engineering applications.},
}
@article {pmid41414673,
year = {2025},
author = {Chan, J and Wu, Z and Liu, M and Wang, T and Liu, H and Cao, R and Li, X and Li, X and Zhan, S and Cheng, J and Xu, Y and He, M and Feng, Y and Xu, Q and Sun, Y and Chen, L and Hu, P},
title = {Systematic enhancer mapping and functional analysis in zebrafish with optimized CRISPR interference.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41414673},
issn = {1362-4962},
support = {32341061//National Natural Science Foundation of China/ ; 32200414//National Natural Science Foundation of China/ ; 32373113//National Natural Science Foundation of China/ ; 32503168//National Natural Science Foundation of China/ ; 2024M761923//China Postdoctoral Science Foundation/ ; 25ZR1402190//Natural Science Foundation of Shanghai/ ; (32341061//National Natural Science Foundation of China/ ; 32200414//National Natural Science Foundation of China/ ; 32373113//National Natural Science Foundation of China/ ; 32503168//National Natural Science Foundation of China/ ; 2024M761923//China Postdoctoral Science Foundation/ ; 25ZR1402190//Natural Science Foundation of Shanghai/ ; },
mesh = {Animals ; *Zebrafish/genetics ; *Enhancer Elements, Genetic ; *CRISPR-Cas Systems ; Promoter Regions, Genetic ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Noncoding cis-regulatory elements, particularly enhancers, are crucial for controlling gene expression. However, the in vivo use of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) to study enhancer function has been limited in zebrafish, which is widely used in early development and human disease research. Here, we optimized the CRISPRi system in zebrafish to achieve efficient suppression of tyr expression by fine-tuning component concentrations. Applying this optimized system, we functionally annotated distal enhancers of globin genes. Using Hi-C and histone modification assays, we systematically mapped 434 enhancer-promoter (EP) interactions across the genome. Among these EP loops, CRISPRi perturbation identified previously unreported enhancers with regulatory strengths surpassing known elements, demonstrated by disrupted phenotypes in fin and blood cell development. Additionally, several unreported EP loops were validated, underscoring the robustness of our integrated approach. This study not only provides an optimized CRISPRi system for zebrafish but also introduces a powerful platform that integrates computational and experimental strategies for advancing cis-regulatory element annotation in vertebrate gene regulation.},
}
@article {pmid41414712,
year = {2026},
author = {Congdon, ST and Bennett, J and Opinya, R and Agosto, AR and Dossias, O and Kokko, C and Levesque, AA and Koob, AO and Silver, AC and Thomas-Charles, CA},
title = {Investigating and correcting a rare pathogenic mutation in GDF11.},
journal = {HGG advances},
volume = {7},
number = {1},
pages = {100559},
pmid = {41414712},
issn = {2666-2477},
mesh = {Humans ; HEK293 Cells ; *Growth Differentiation Factors/genetics/metabolism ; *Gene Editing/methods ; *Bone Morphogenetic Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Mutation ; Golgi Apparatus/metabolism ; Codon, Nonsense ; },
abstract = {Single-nucleotide variants (SNVs) and small insertions or deletions (indels) underlie most rare monogenic disorders, yet therapeutic strategies to precisely correct these mutations remain limited. Prime editing enables the repair of such pathogenic variants without introducing double-stranded breaks. Here, we applied CRISPR prime editing to model and correct a de novo GDF11 nonsense mutation (Tyr336∗) identified in a participant from the Undiagnosed Diseases Network with growth delay and multisystem abnormalities. Using HEK293T cells, we generated heterozygous (HET) GDF11 Tyr336∗ clones, which exhibited reduced GDF11 protein levels due to post-translational degradation likely mediated by endoplasmic reticulum- and Golgi-associated quality control pathways. These cells displayed marked Golgi abnormalities, including an increased number of compact, irregularly shaped Golgi structures, findings consistent with Golgi fragmentation and stress. Transcriptomic profiling of HET cells revealed a broad dysregulation of gene networks, including downregulation of metabolic and Golgi-linked biosynthetic genes, and upregulation of cell-adhesion and extracellular matrix genes. These transcriptional shifts paralleled the participant's developmental, neural, and cardiovascular phenotypes. To correct the mutation, we tested multiple bespoke prime editing strategies and identified PE7, in combination with a prime editing guide RNA designed by Pridict, as the most effective ribonucleoprotein complex for rescue. Editing efficiency was further enhanced by introducing an additional silent protospacer-adjacent motif-disrupting mutation, likely preventing both Cas9 re-binding and mismatch repair. Together, these findings support a haploinsufficiency mechanism for the GDF11 Tyr336∗ allele and establish a generalizable framework for disease modeling and allele-specific correction of pathogenic variants in human cells.},
}
@article {pmid41415442,
year = {2025},
author = {Sabol, AL and Mengiste, AA and Sreekanth, V and Singh, P and Hendel, SJ and Tran, MTN and Barybin, AM and Chaudhary, S and Harris, RM and Liivak, K and Severance, ZC and Locicero, CM and Kailass, K and Lee, C and Xu, LQ and Butty, VL and Choudhary, A and Shoulders, MD},
title = {Anti-CRISPR-mediated continuous directed evolution of CRISPR-Cas9 in human cells.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41415442},
issn = {2692-8205},
support = {R01 DK132900/DK/NIDDK NIH HHS/United States ; R35 GM136354/GM/NIGMS NIH HHS/United States ; R01 GM137606/GM/NIGMS NIH HHS/United States ; P30 ES002109/ES/NIEHS NIH HHS/United States ; R01 GM132825/GM/NIGMS NIH HHS/United States ; P30 CA014051/CA/NCI NIH HHS/United States ; },
abstract = {Engineering CRISPR-Cas systems for improved or altered function is central to both research and therapeutic applications. Unfortunately most optimization, especially directed evolution in bacterial hosts, fails to capture the functional requirements of the complex mammalian cellular milieu, where activity is usually required. Robust strategies to enable continuous directed evolution of genome-targeting agents directly in human cells remain lacking. Here, we introduce CRISPR-MACE (Mammalian cell-enabled Adenovirus-assisted Continuous Evolution) as a foundational technology to address this need. CRISPR-MACE integrates virus-based continuous evolution with anti-CRISPR-based tunable selection to generate novel Streptococcus pyogenes Cas9 variants with both increased and decreased DNA binding capacity and nearly 1000-fold-enhanced resistance to AcrIIA4, the strongest known inhibitor of SpCas9. Notably, across independent evolution campaigns the same Cas9 gatekeeper mutation reproducibly emerged first, enabling subsequent adaptive steps along two interdependent axes of Cas9 function. In addition to advancing CRISPR technologies, this work establishes key principles and synthetic circuits for continuously evolving CRISPR-Cas systems directly in human cells.},
}
@article {pmid41415454,
year = {2025},
author = {Neupane, S and Pfrender, ME and Wang, L and Xu, S},
title = {Detection of CRISPR-Cas-induced mutations in Daphnia.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41415454},
issn = {2692-8205},
support = {R35 GM133730/GM/NIGMS NIH HHS/United States ; },
abstract = {CRISPR-Cas9 has established itself as a robust tool for conducting loss of function gene research in emerging model species including the freshwater zooplankton Daphnia. However, sensitive detection of mutations, especially in genetic mosaic and pooled samples, remains a challenge. In this study we evaluate two of the most widely used mutation screening techniques, the T7 Endonuclease I (T7EI) assay and Fragment Analysis (FA) for their sensitivity, accuracy, and practical use in detecting CRISPR-induced indels in four targeted genes, DNMT3A, DNMT3B, PERIOD2, and DMRT1 in Daphnia magna. Here, we show that T7EI, although it offers a quick and cost-effective screening method, often produces false positives, especially when examining pooled samples. Conversely, FA facilitates detecting allele size differences at a fine resolution, reproducibility in detecting indels, and distinguishing zygosity and is more reliable as a method to detect mutation. Our comparative analyses convey the importance of carefully selecting the appropriate screening methods depending on research questions.},
}
@article {pmid41416398,
year = {2026},
author = {Hoang, TS and Faraji, F and Mendez-Molina, AN and Adame-Garcia, SR and Sato, K and Ishikawa, T and Vo, PTT and Ramirez, SI and Anguiano Quiroz, PY and Guo, T and Fan, K and Wu, X and Molinolo, AA and Cohen, EEW and Mali, P and Lippman, SM and Gutkind, JS},
title = {Genome-wide CRISPR Screening Reveals a PKA-Driven Resistance Mechanism to Metformin for Oral Cancer Prevention That Can Be Exploited by Combination with NSAIDs.},
journal = {Cancer prevention research (Philadelphia, Pa.)},
volume = {19},
number = {2},
pages = {79-92},
pmid = {41416398},
issn = {1940-6215},
support = {T32 CA121938/CA/NCI NIH HHS/United States ; R01 DE035393/DE/NIDCR NIH HHS/United States ; R25 CA221779/CA/NCI NIH HHS/United States ; U54 CA274502/CA/NCI NIH HHS/United States ; U01 CA290479/CA/NCI NIH HHS/United States ; R01 DE026644/DE/NIDCR NIH HHS/United States ; T32 DC000028/DC/NIDCD NIH HHS/United States ; R01DE026644//National Institute of Dental and Craniofacial Research (NIDR)/ ; U01CA290479//National Cancer Institute (NCI)/ ; SU2C-FARF-FFF//Stand Up To Cancer (SU2C)/ ; 308268//Stand Up To Cancer (SU2C)/ ; T32DT4965//Tobacco-Related Disease Research Program (TRDRP)/ ; T32CA121938//National Cancer Institute (NCI)/ ; T32DC000028//National Institute on Deafness and Other Communication Disorders (NIDCD)/ ; 1061310//American Head and Neck Society (AHNS)/ ; //Takeda Science Foundation (TSF)/ ; //Japan Society for the Promotion of Science (JSPS)/ ; //Rotary Foundation (Rotary)/ ; R25CA221779//National Cancer Institute (NCI)/ ; T34DT8340//Tobacco-Related Disease Research Program (TRDRP)/ ; },
mesh = {*Metformin/pharmacology/therapeutic use ; Humans ; *Anti-Inflammatory Agents, Non-Steroidal/pharmacology/therapeutic use ; *Cyclic AMP-Dependent Protein Kinases/metabolism ; *Mouth Neoplasms/prevention &amp; control/genetics/pathology ; *Drug Resistance, Neoplasm/genetics/drug effects ; CRISPR-Cas Systems ; *Squamous Cell Carcinoma of Head and Neck/genetics/prevention &amp; control/pathology/drug therapy ; Cell Line, Tumor ; Mice ; Drug Synergism ; Animals ; Cell Proliferation/drug effects ; *Antineoplastic Combined Chemotherapy Protocols/pharmacology/therapeutic use ; Signal Transduction/drug effects ; Cyclooxygenase 2 Inhibitors/pharmacology ; },
abstract = {UNLABELLED: Head and neck squamous cell carcinoma (HNSCC) is among the 10 most common cancers worldwide and is associated with high morbidity and poor survival. Diminished HNSCC outcomes are often related to delayed diagnosis and treatment of occult progression of premalignant lesions, underscoring the need for effective and low-risk chemoprevention strategies. In this regard, metformin has shown promising clinical activity for HNSCC prevention. In this study, we performed a genome-wide CRISPR/Cas9 screen of metformin-treated HNSCC cells and identified the activation of PKA signaling as the top resistance pathway. We show that metformin mediates PKA activation in HNSCC cells and that PKA inhibition, when combined with metformin treatment, synergistically inhibits HNSCC growth. We found that metformin-induced PKA activation is mediated by a prostaglandin E2 autocrine loop, which can be blocked using cyclooxygenase-2 (COX2) inhibitors. Importantly, COX2 inhibition using nonsteroidal anti-inflammatory drugs (NSAID) combined with metformin treatment synergistically inhibits HNSCC cell growth and prevents the progression of oral premalignant lesions into invasive HNSCC in a model of tobacco-driven oral carcinogenesis. Together, these findings demonstrate that metformin and NSAID combination therapy may represent a promising therapeutic strategy for HNSCC chemoprevention.<br><br>PREVENTION RELEVANCE: Our findings reveal that using metformin for head and neck cancer chemoprevention leads to compensatory activation of a PKA-driven resistance mechanism that can be blocked by cotreatment with NSAIDs. These findings provide a rationale for combining metformin with NSAIDs as a precision head and neck cancer chemoprevention strategy.},
}
@article {pmid41416522,
year = {2025},
author = {Stella, G and Ye, L and Brady, SF and Marraffini, L},
title = {CARF-HAD phosphatase effectors provide immunity during the type III-A CRISPR-Cas response.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41416522},
issn = {1362-4962},
support = {R01 GM149834/GM/NIGMS NIH HHS/United States ; R01GM149834/GF/NIH HHS/United States ; R35GM122559/GF/NIH HHS/United States ; //HHMI/ ; },
mesh = {*CRISPR-Cas Systems ; *Phosphoric Monoester Hydrolases/metabolism/genetics/chemistry ; Protein Domains ; Adenosine Triphosphate/metabolism ; Escherichia coli/genetics/virology ; Adenine Nucleotides/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; *Bacterial Proteins/metabolism/chemistry/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) systems provide adaptive immunity against phage infection in prokaryotes using an RNA-guided complex that recognizes complementary foreign nucleic acids. Different types of CRISPR-Cas systems have been identified that differ in their mechanism of defense. Upon infection, type III CRISPR-Cas systems employ the Cas10 complex to find phage transcripts and synthesize cyclic oligo-adenylate (cOA) messengers. These ligands bind and activate CARF immune effectors that cause cell toxicity to prevent the completion of the viral lytic cycle. Here, we investigated two proteins containing an N-terminal haloacid dehalogenase (HAD) phosphatase domain followed by four predicted transmembrane helices and a C-terminal CARF domain. We named these proteins Chp for CRISPR-associated HAD phosphatase. We show that, in vivo, Chp localizes to the bacterial membrane and that its activation induces a growth arrest, leads to a depletion of ATP and IMP, and prevents phage propagation during the type III CRISPR-Cas response. In vitro, the CARF domain of Chp binds cyclic tetra-adenylates and the HAD phosphatase domain dephosphorylates dATP, ATP, and IMP. Our findings extend the range of molecular mechanisms employed by CARF effectors to defend prokaryotes against phage infection.},
}
@article {pmid41416796,
year = {2026},
author = {Athipanyasilp, N and Saowpak, S and Chaimayo, C and Angkasekwinai, N and Pattama, A and Athipanyasilp, A and Patchsung, M and Aphicho, K and Uttamapinant, C and Horthongkham, N},
title = {CRISPR-Cas13a SHERLOCK assay for rapid and sensitive detection of chikungunya virus.},
journal = {Microbiology spectrum},
volume = {14},
number = {2},
pages = {e0229825},
pmid = {41416796},
issn = {2165-0497},
support = {R016034012//Faculty of Medicine Siriraj Hospital, Mahidol University/ ; },
mesh = {*Chikungunya virus/genetics/isolation &amp; purification ; *Chikungunya Fever/diagnosis/virology ; Humans ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; RNA, Viral/genetics ; *Molecular Diagnostic Techniques/methods ; Nucleic Acid Amplification Techniques/methods ; Limit of Detection ; Real-Time Polymerase Chain Reaction/methods ; },
abstract = {Chikungunya virus (CHIKV), a major cause of acute febrile illness and joint pain, remains a significant public health threat in tropical regions. Rapid and accurate detection is essential for timely clinical management and outbreak control, particularly in resource-limited settings where real-time PCR (RT-qPCR) is often impractical. We developed and validated a SHERLOCK assay coupled with recombinase polymerase amplification for CHIKV RNA detection. Analytical performance was assessed by determining the limit of detection (LOD), cross-reactivity, clinical sensitivity and specificity, and predictive values. The assay achieved an LOD of 215 copies/reaction with no cross-reactivity against other alphaviruses or flaviviruses. Clinical testing of 146 plasma samples showed a sensitivity and specificity of 94.52% and 100% with lateral-flow readout and 97.26% and 100% with fluorescence readout, respectively. This study establishes a promising CRISPR-Cas13a-based SHERLOCK platform for CHIKV detection, demonstrating high analytical performance, rapid turnaround time, and potential for future adaptation to resource-limited settings.IMPORTANCEEarly and accurate detection of chikungunya virus (CHIKV) is critical for outbreak control, especially in resource-limited settings, where real-time PCR is not feasible. This study demonstrates that the CRISPR-Cas13a-based SHERLOCK platform, combined with RPA, achieves high diagnostic accuracy and a low detection limit, comparable to RT-qPCR. The assay's rapid turnaround time and simple lateral-flow readout make it a promising tool for point-of-care diagnostics during CHIKV outbreaks, potentially improving disease surveillance and clinical decision-making.},
}
@article {pmid41416985,
year = {2026},
author = {Han, DH and Lee, SY and Kim, Y and Oh, J and Park, J and Park, YM and Kim, SG and Kim, TS and Park, JK},
title = {Ultrasensitive Detection of Multiple Foodborne Pathogens Using CRISPR-Cas12a on a Finger-Actuated Microfluidic Device Integrated with a Modular Pressurizing Pump.},
journal = {Analytical chemistry},
volume = {98},
number = {1},
pages = {531-542},
doi = {10.1021/acs.analchem.5c05295},
pmid = {41416985},
issn = {1520-6882},
mesh = {*Lab-On-A-Chip Devices ; *CRISPR-Cas Systems ; *Listeria monocytogenes/isolation &amp; purification/genetics ; Nucleic Acid Amplification Techniques ; Limit of Detection ; *Food Microbiology ; *Escherichia coli O157/isolation &amp; purification/genetics ; Salmonella/isolation &amp; purification/genetics ; *Microfluidic Analytical Techniques/instrumentation ; Milk/microbiology ; Foodborne Diseases/microbiology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Foodborne pathogens pose a serious threat to global health and the economy, causing gastrointestinal illnesses and potentially leading to fatalities. Here, we present a recombinase polymerase amplification (RPA)-CRISPR-Cas12a-based method for the detection of foodborne pathogens using target-specific CRISPR RNAs (crRNAs) on a reusable, reconfigurable finger-actuated microfluidic device. Unlike previous finger-actuated pushbutton-based microfluidic devices, the device incorporates a modular pressurizing pump (MoPP), a standalone, reconfigurable actuation module that not only enhances reusability and reduces cross-contamination risks but also provides a flexible interface that allows user-defined fluidic routing and multiplexed assay workflows. Using a MoPP-integrated finger-actuated microfluidic device, the RPA-CRISPR-Cas12a-based detection of three foodborne pathogens was validated with an optimized crRNA and RPA primer sequence. Genomic DNA (gDNA) extracted from pathogen-spiked milk samples further demonstrated real-world applicability, achieving a limit of detection (LOD) of 1.62, 1.84, and 1.01 CFU/mL for Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes, respectively. The developed microfluidic RPA-CRISPR-Cas12a-based detection platform is expected to be a reconfigurable, user-friendly, and highly sensitive point-of-care testing system for monitoring foodborne pathogens throughout the food supply chain.},
}
@article {pmid41417296,
year = {2025},
author = {Rehman, T and Sharif, A and Khalid, L and Sajid, I},
title = {Whole genome sequencing and genomic characterization of the extensively drug-resistant Acinetobacter baumannii recovered from clinical samples in Lahore, Pakistan.},
journal = {Molecular biology reports},
volume = {53},
number = {1},
pages = {217},
pmid = {41417296},
issn = {1573-4978},
mesh = {*Acinetobacter baumannii/genetics/drug effects/isolation &amp; purification/pathogenicity ; Pakistan ; Humans ; *Drug Resistance, Multiple, Bacterial/genetics ; Whole Genome Sequencing/methods ; *Acinetobacter Infections/microbiology/genetics/drug therapy ; Genome, Bacterial/genetics ; Anti-Bacterial Agents/pharmacology ; Virulence Factors/genetics ; Genomics/methods ; Microbial Sensitivity Tests ; },
abstract = {BACKGROUND: Extensively drug-resistant (XDR) Acinetobacter baumannii has turned into a significant nosocomial pathogen often resistant to all available classes of antibiotics. It has been identified as a key public health issue due to its capacity to get resistance determinants, virulence genes, and mobile genetic elements. This study hypothesized that the XDR A. baumannii isolated in Pakistan would harbor some of the key genomic determinants of the resistance and pathogenicity.<br><br>METHODOLOGY: A total of 11 A. baumannii were obtained from the patient samples and were identified using microbiological, biochemical, and genomic analysis. The sensitivity to antibiotics was determined by Kirby-Bauer disc diffusion assay. While the whole genome sequencing (WGS) was performed for the single XDR Acinetobacter isolate TAB-4 and its in-silico genome analysis on resistant genes, virulence factors, plasmids, mobile genetic elements, prophages, CRISPR-Cas and biosynthetic gene clusters was conducted.<br><br>RESULTS: The isolated strains were 10 multidrug resistant (MDR) and one (TAB-4) was XDR Acinetobacter strain. The AST showed resistance to nearly all classes of antibiotics, and with limited susceptibility to tetracyclines and aminoglycosides. The genome of TAB-4 strain comprised of 3.94&#xa0;Mb, 120 contigs, and a GC content of 39.14%. The major resistant determinants found were blaOXA-23, blaOXA-69, blaNDM-1, blaADC-25, aminoglycoside modifying enzymes, and efflux pumps (adeABC, adeFGH, adeIJK). Virulence-associated genes (ompA, bap, csuA/B-E, plc) were identified along with five prophage regions, multiple CRISPR arrays, and a betalactone biosynthetic gene cluster.<br><br>CONCLUSION: This study to the best of our knowledge reports the first detailed WGS-based characterization of an XDR A. baumannii from Lahore, Pakistan. These genomic findings offer significant insights into the resistance and virulence factors underlying this challenging clinical issue. Hence, there is an urgent need to find new or alternative treatment methods against high-risk pathogens like XDR A. baumannii.},
}
@article {pmid41417728,
year = {2026},
author = {Huber, A and Djajawi, TM and Rivera, IS and Vervoort, SJ and Kearney, CJ},
title = {CRISPR screens define unified hallmarks of cancer cell-autonomous immune evasion.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116738},
doi = {10.1016/j.celrep.2025.116738},
pmid = {41417728},
issn = {2211-1247},
mesh = {Humans ; *Neoplasms/immunology/genetics/pathology ; *Tumor Escape/genetics ; *CRISPR-Cas Systems/genetics ; Tumor Microenvironment/immunology ; Immunotherapy ; Animals ; *Immune Evasion ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; CD8-Positive T-Lymphocytes/immunology ; },
abstract = {Cancer immunotherapy has transformed cancer treatment, yet only a minority of patients achieve durable benefit. Although early efforts to enhance immunotherapy focused on boosting immune effector function, reversing T cell exhaustion, or altering the tumor microenvironment, it is now clear that cancer cell-autonomous mechanisms play a major role in immune escape. Such programs, driven by the cancer cell genome, transcriptome, and epigenome, include desensitization to cytokine signaling, such as interferon (IFN)γ and tumor necrosis factor (TNF); impaired antigen presentation; upregulation of suppressive ligands such as programmed cell death ligand 1 (PD-L1); and epigenetic silencing of immunogenic pathways. The rise of high-throughput functional genomics, especially in vitro and in vivo CRISPR-based screening, has greatly expanded our ability to map these pathways and define how tumors evade CD8[+] T cell-mediated pressure. A deeper understanding of these cancer cell-autonomous immune-evasion mechanisms will be essential for developing new therapeutic strategies that broaden the impact of immunotherapy across diverse cancers.},
}
@article {pmid41417859,
year = {2025},
author = {Bradford, J and Joy, D and Winsen, M and Meurant, N and Wilkins, M and Wilson, LOW and Bauer, DC and Perrin, D},
title = {Democratising high performance computing for bioinformatics through serverless cloud computing: A case study on CRISPR-Cas9 guide RNA design with Crackling Cloud.},
journal = {PLoS computational biology},
volume = {21},
number = {12},
pages = {e1013819},
pmid = {41417859},
issn = {1553-7358},
mesh = {*Cloud Computing ; *Computational Biology/methods ; *RNA, Guide, CRISPR-Cas Systems/genetics ; *CRISPR-Cas Systems/genetics ; Software ; },
abstract = {Organisations are challenged when meeting the computational requirements of large-scale bioinformatics analyses using their own resources. Cloud computing has democratised large-scale resources, and to reduce the barriers of working with large-scale compute, leading cloud vendors offer serverless computing, a low-maintenance and low-cost model that provides ample resources for highly scalable software applications. While serverless computing has broad use, its adoption in bioinformatics remains poor. Here, we demonstrate the most extensive use of high-performance serverless computing for bioinformatics by applying the available technologies to CRISPR-Cas9 guide RNA (gRNA) design. Our adaptation of the established gRNA design tool, named Crackling, implements a novel, cloud-native and serverless-based, high-performance computing environment using technologies made available by Amazon Web Services (AWS). The architecture, compatible with technologies from all leading cloud vendors, and the AWS implementation, contributes to an effort of reducing the barrier to large computational capacity in bioinformatics and for CRISPR-Cas9 gRNA design. Crackling Cloud can be deployed to any AWS account, and is freely available on GitHub under the BSD 3-clause license: https://github.com/bmds-lab/Crackling-AWS.},
}
@article {pmid41417901,
year = {2025},
author = {Chammas, P and Xie, SQ and Sepulveda-Rincon, LP and Leeke, BJ and Dore, MH and Dormann, D and Wagner, RT and Chang, N and Jones, PL and McManus, MT and Karimi, MM and Young, G and Percharde, M},
title = {CRISPRa-mediated disentanglement of the Dux-MERVL axis in the 2C-like state, totipotency, and cell death.},
journal = {Science advances},
volume = {11},
number = {51},
pages = {eadu9092},
pmid = {41417901},
issn = {2375-2548},
mesh = {Animals ; Mice ; Cell Death/genetics ; *Homeodomain Proteins/genetics/metabolism ; *CRISPR-Cas Systems ; Gene Expression Regulation, Developmental ; *Transcription Factors/genetics/metabolism ; *DNA Transposable Elements/genetics ; Zygote/metabolism ; },
abstract = {Transposable elements (TEs) are powerful cis-regulatory drivers of gene expression, particularly during early development when many TEs become de-repressed. MERVL elements are transiently up-regulated in mouse totipotent two-cell (2C) embryos during major zygotic genome activation (ZGA) and 2C-like cells in vitro. One of the most powerful activators of MERVL is the pioneer transcription factor, Dux. However, apparent differences lie in the requirement for Dux versus MERVL activation in embryos. Moreover, sustained Dux activation causes cell toxicity, which may or may not be linked to MERVL. Using a CRISPR activation system, we unpick the relative role of Dux and MERVL in ZGA, totipotent-like characteristics, and cell toxicity. We find that MERVL activation drives a portion of the Dux-dependent transcriptome, sufficient for expanded fate potential, but not other totipotency features. Conversely, Dux-induced pathology is independent of MERVL activation and involves the proapoptotic factor, Noxa. Our study highlights the complexity of the Dux-MERVL transcriptional network and uncovers a previously unknown player in Dux-driven pathology.},
}
@article {pmid41418537,
year = {2026},
author = {Tan, D and Ye, Y and Miao, D and Zhao, C and Wu, S and Shi, J and Yang, J and Fang, K and Lu, F and Lv, Q and Gong, J and Yang, H and Xiao, W and Xiong, Z and Zhang, X and Ruan, H},
title = {UBL3 governs VEGFR inhibitor resistance by activating NOTCH signaling in renal cell carcinoma.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {85},
number = {},
pages = {101332},
doi = {10.1016/j.drup.2025.101332},
pmid = {41418537},
issn = {1532-2084},
mesh = {Humans ; *Carcinoma, Renal Cell/drug therapy/pathology/genetics ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Kidney Neoplasms/drug therapy/pathology/genetics ; Signal Transduction/drug effects ; Animals ; Cell Line, Tumor ; Mice ; Xenograft Model Antitumor Assays ; *Receptors, Vascular Endothelial Growth Factor/antagonists &amp; inhibitors ; *Protein Kinase Inhibitors/pharmacology ; Sunitinib/pharmacology ; Pyridines/pharmacology ; Receptors, Notch/metabolism ; Apoptosis/drug effects ; Mice, Nude ; CRISPR-Cas Systems ; Gene Expression Regulation, Neoplastic ; Anilides ; },
abstract = {BACKGROUND: Targeted therapy is the first-line treatment for patients with metastatic renal cell carcinoma (RCC), with vascular endothelial growth factor receptor inhibitors (VEGFRis) constituting the bulk of regimens used. Although the repertoire of VEGFRis for RCC now spans from sunitinib to cabozantinib, resistance to treatments has emerged as a common and prominent challenge. Thus, identifying novel therapeutic targets has become essential for enhancing the antitumor efficacy of current treatments and inhibiting RCC progression.<br><br>METHOD: To investigate the potential mechanisms underlying VEGFRi resistance in RCC, we performed a genome-wide CRISPR/Cas9 library screen under sunitinib and cabozantinib treatment and identified UBL3 as a key driver of VEGFRi resistance in RCC cells. The critical role of UBL3 in promoting VEGFRi resistance was validated using CCK8 assays, flow cytometry, TUNEL assays, and bioinformatics analyses. To elucidate the molecular mechanisms underlying UBL3, we utilized western blotting, RNA sequencing, chromatin immunoprecipitation, small extracellular vesicles (sEVs) isolation, and Astral-DIA proteomics. The contribution of UBL3 to VEGFRi resistance was further confirmed through comprehensive in vitro and in vivo experiments.<br><br>RESULTS: UBL3 was confirmed to suppress apoptosis and promote VEGFRi resistance through NOTCH signaling activation. Further investigations highlighted the importance of NOTCH signaling in VEGFRi resistance in RCC via the NOTCH-PTEN-AKT and NOTCH-FOS pathways and revealed the mechanisms by which UBL3 activated NOTCH signaling. On the one hand, UBL3 formed complex with NOTCH2 and ADAM17 simultaneously, accelerating ADAM17-mediated cleavage of NOTCH2. On the other hand, UBL3-modified NOTCH2 was sorted into sEVs, which were taken up by recipient cells, activating NOTCH signaling and thereby transmitting VEGFRi resistance. Finally, lipid nanoparticle-mediated delivery of the CRISPR/Cas9 knockout system targeting UBL3 effectively restored the sensitivity of RCC tumors to VEGFRis.<br><br>CONCLUSION: This study emphasized the importance of UBL3 in VEGFRi resistance in RCC and proposed that UBL3 activated NOTCH signaling through two distinct pathways, thereby suppressing cancer apoptosis and promoting resistance to VEGFRis. These findings provided a solid scientific foundation and paved the way for the development of novel therapeutic strategies for patients with advanced RCC.},
}
@article {pmid41418729,
year = {2026},
author = {Wang, Y and Zheng, W and Qiu, B and Chen, Q and Yang, T and Zhou, S and Liu, J and Yang, B},
title = {Generation of a human embryonic stem cell line (SMUDHe010-A-3F) with Cas9 expression cassette integrated at the AAVS1 locus via CRISPR/Cas9-mediated homologous recombination.},
journal = {Stem cell research},
volume = {90},
number = {},
pages = {103882},
doi = {10.1016/j.scr.2025.103882},
pmid = {41418729},
issn = {1876-7753},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Human Embryonic Stem Cells/metabolism/cytology ; *Homologous Recombination/genetics ; Cell Line ; Gene Editing ; Genetic Loci ; Cell Differentiation ; CRISPR-Associated Protein 9/metabolism ; },
abstract = {Cas9, an RNA-guided nuclease, enables precise genome editing by recognizing sgRNA-complementary sequences and cleaving target DNA. In this study, we used CRISPR/Cas9-mediated homologous recombination to integrate a loxP-flanked STOP cassette-controlled Cas9 expression framework (LSL-Cas9) into the AAVS1 safe-harbor locus of human embryonic stem cells. The resulting cell line, SMUDHe010-A-3F, allows Cre-dependent activation of Cas9 but remains inactive in the absence of Cre recombinase. Karyotype and tri-lineage differentiation confirmed genomic stability and pluripotency. This line provides a valuable platform for organoid gene editing and studies of human development and disease.},
}
@article {pmid41418774,
year = {2026},
author = {Ji, R and Chen, Q and Zhang, Y},
title = {Emerging trends in gene and cell therapy: CRISPR in DNA editing and beyond.},
journal = {Cell reports. Medicine},
volume = {7},
number = {1},
pages = {102459},
pmid = {41418774},
issn = {2666-3791},
mesh = {Humans ; *Gene Editing/methods/trends ; *CRISPR-Cas Systems/genetics ; *Genetic Therapy/methods/trends ; *Cell- and Tissue-Based Therapy/methods/trends ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {CRISPR-based gene and cell therapies are rapidly transitioning from experimental platforms to clinical reality, exemplified by the recent approval of CRISPR-derived treatments for β-hemoglobinopathies. This review highlights how advances in genome editing technologies, ranging from CRISPR-Cas nucleases to base and prime editors, are expanding the therapeutic landscape beyond traditional gene knockout approaches. We focus on the clinical translation of these tools, drawing on examples from ongoing and completed human trials to illustrate their potential across diverse disease areas. Furthermore, we discuss critical considerations such as delivery challenges, long-term safety, immune responses, and editing specificity, all of which are critical to the safe and effective integration of CRISPR technologies into modern medicine.},
}
@article {pmid41418786,
year = {2026},
author = {Sherman, A and Benvenisty, N},
title = {Genetic screening of long non-coding RNAs in human embryonic stem cells reveals novel regulators of pluripotency.},
journal = {Stem cell reports},
volume = {21},
number = {1},
pages = {102743},
pmid = {41418786},
issn = {2213-6711},
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; *Human Embryonic Stem Cells/metabolism/cytology ; Cell Differentiation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; Octamer Transcription Factor-3/genetics/metabolism ; *Genetic Testing ; Cell Line ; CRISPR-Cas Systems ; Apoptosis/genetics ; },
abstract = {The human genome encodes thousands of long non-coding RNAs (lncRNAs), transcripts of over 200 nucleotides that lack protein-coding potential. lncRNAs are emerging as key players in diverse cellular processes, particularly in tissue-specific contexts, yet their functionality remained poorly understood. Here, we performed a CRISPR interference (CRISPRi) screen in human embryonic stem cells (hESCs), identifying over 100 essential and about 150 growth-restricting lncRNAs. We show that growth-modifying lncRNAs display distinctive properties, including unique expression signatures, genomic structure, evolutionary conservation, chromosomal distribution, and potential involvement in teratoma formation. Notably, we uncovered two primate-conserved, uncharacterized, essential lncRNAs that regulate neighboring pluripotency transcription factors: lncOCT4, which positively regulates OCT4 and induces p53-mediated apoptosis upon knockdown, and lncVRTN, which acts as a putative negative regulator of VRTN, affecting cell fate determination. These findings shed light on the contribution of lncRNAs to the human-specific pluripotency network and provide insights into lncRNA-mediated regulation of hESC growth and differentiation.},
}
@article {pmid41418951,
year = {2026},
author = {Chen, J and Bian, X and Zheng, X and Peng, B and Li, R and Du, H and Zhou, L and Wen, Y},
title = {The synergistic effect of DNA nanostructures and CRISPR/Cas system for cancer diagnosis and treatment.},
journal = {International journal of biological macromolecules},
volume = {339},
number = {Pt 1},
pages = {149741},
doi = {10.1016/j.ijbiomac.2025.149741},
pmid = {41418951},
issn = {1879-0003},
mesh = {*CRISPR-Cas Systems ; Humans ; *Neoplasms/diagnosis/therapy/genetics ; Gene Editing/methods ; *Nanostructures/chemistry/therapeutic use ; *DNA/chemistry ; Genetic Therapy/methods ; Animals ; },
abstract = {Cancer remains one of the most formidable global public health challenges, exerting a profound and detrimental impact on human health. Despite substantial advancements in cancer research, the escalating incidence and mortality rates underscore the persistent and growing burden on global healthcare systems. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas system, heralded as a revolutionary gene-editing tool, holds immense promise for cancer treatment. However, its efficacy is critically contingent upon developing efficient delivery strategies. DNA nanocarriers, characterized by their programmability, sequence specificity, and design flexibility, emerge as a highly effective vehicle for delivering the CRISPR/Cas system, facilitating the precise transportation of gene-editing tools to the cell nucleus. The integration of DNA nanocarriers with CRISPR/Cas technology provides a new paradigm for precise and controllable gene editing. Through programmable spatial assembly, DNA nanocarriers can protect Cas9 ribonucleoprotein complexs (RNPs), facilitate endosomal escape, and co-localize donor DNA to promote homology-directed repair. These synergistic effects bridge molecular programmability and genetic functionality, paving the way for safer and more efficient genome engineering. This review aims to evaluate the application of DNA nanocarriers in cancer diagnosis comprehensively and to explore their potential utility in cancer therapy when combined with the CRISPR/Cas system, offering novel insights and significant scientific contributions to the field.},
}
@article {pmid41419745,
year = {2025},
author = {Ying, Q and Chen, Y and Shen, L and Xu, Y and Tian, R},
title = {SPLiCR-seq: A CRISPR-Based Screening Platform for RNA splicing Identifies Novel Regulators of IRE1α-XBP1 Signaling Under ER Stress.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {911},
pmid = {41419745},
issn = {2041-1723},
support = {82171416//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*X-Box Binding Protein 1/metabolism/genetics ; *Endoribonucleases/metabolism/genetics ; *Protein Serine-Threonine Kinases/metabolism/genetics ; *RNA Splicing/genetics ; Humans ; Signal Transduction/genetics ; Animals ; *Endoplasmic Reticulum Stress/genetics ; Protein Phosphatase 1/metabolism/genetics/antagonists &amp; inhibitors ; Unfolded Protein Response/genetics ; Mice ; CRISPR-Cas Systems ; HEK293 Cells ; Cell Line, Tumor ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {RNA splicing is fundamental to cellular function, yet systematic investigation of its complex regulation has been limited by existing methods. Here, we present SPLiCR-seq (SPLicing regulator identification through CRISPR screening), a high-throughput CRISPR screening platform that enables direct measurement of RNA splicing outcomes for pooled genetic perturbations, overcoming limitations of traditional fluorescence-based approaches. Applying SPLiCR-seq to investigate XBP1 splicing during the unfolded protein response (UPR), we conduct targeted and genome-wide screens across diverse cellular contexts, revealing both common and cell-type specific regulators. Notably, we identify GADD34 (PPP1R15A) as a novel modulator of IRE1α-XBP1 signaling, demonstrating that it directly interacts with IRE1α and functions independently of its canonical role in eIF2α dephosphorylation. Pharmacological inhibition of GADD34 using Sephin1 effectively suppressed XBP1 splicing and alleviated CAR-T cell exhaustion in an ex vivo model, leading to enhanced tumor-killing capacity across multiple cancer models. This work not only establishes a powerful new tool for systematically studying RNA splicing regulation but also uncovers a promising therapeutic strategy for improving CAR-T cell immunotherapy through modulation of the IRE1α-XBP1 pathway.},
}
@article {pmid41419797,
year = {2025},
author = {Das, IS and Shi, Q and Dreischhoff, S and Polle, A},
title = {Divergent functions of three Kunitz trypsin inhibitor (KTI) proteins in herbivore defense in poplar.},
journal = {BMC plant biology},
volume = {26},
number = {1},
pages = {153},
pmid = {41419797},
issn = {1471-2229},
mesh = {*Populus/genetics/metabolism/physiology/parasitology ; *Herbivory ; *Plant Proteins/genetics/metabolism ; Animals ; Oxylipins/metabolism ; Gene Expression Regulation, Plant ; Cyclopentanes ; *Plant Defense Against Herbivory/genetics ; Peptides ; },
abstract = {BACKGROUND: Climate warming promotes the expansion of insect pests. Among the inducible defense responses activated by attacked plants, Kunitz trypsin protease inhibitors (KTIs) play an outstanding role. KTIs affect food digestion and thereby control the fitness of herbivorous insects. Poplars contain an expanded family of KTIs, whose distinct intrinsic functions are under investigation. Here, we set out to identify KTIs with anti-herbivore activity and assessed the potential growth trade-off incurred by high KTI expression levels.<br><br>RESULTS: Using in-silico database searches, we identified 28 KTIs in the haploid genome of Populus x canescens; 21 of them were responsive to herbivory. The greatest induction by herbivory was observed for KTI_400, KTI_600 and KTI_0882 (P. trichocarpa orthologues Potri.019G124400, Potri.019G124600, Potri.019G088200), whereas a moderate response was found for KTI_53200 (Potri.017G153200 orthologue). Mechanical wounding and methyl-jasmonate treatments resulted in fast and strong induction of KTI_400 and KTI_600 and moderate or lacking responses in KTI_0882 and KTI_53200. Increased KTI expression levels were associated with upregulation of ALLENE OXIDE SYNTHASE, a key enzyme involved in jasmonate biosynthesis. On the contrary, exposure to compounds eliciting ethylene or salicylic acid signaling did not affect KTIs. We generated stable CRISPR-Cas12a-mediated knock-out and p35S-mediated overexpression lines of KTI_400, KTI_600 and KTI_53200 in Populus x canescens. Among the wildtype and transgenic lines, only kti_400&#x2009;+&#x2009;kti_600 double knock-out lines produced greater biomass. Larvae of Helicoverpa armigera, a pest expanding in Europe due to a warmer climate, were allowed to feed on wildtype and transgenic poplar lines. Transgenic poplars overexpressing KTI_400 or KTI_600 resulted in reduced, and their double knockout lines in increased weight gain of the larvae. In contrast, overexpressing or knockout lines of KTI_53200 had no effect on larval weight gain compared with controls.<br><br>CONCLUSION: KTI_400 and KTI_600 are potent, natural in-planta anti-herbivorous agents. Their expression is associated with larval growth reductions. Modulation of KTI_53200 levels had no direct effects on the fitness of leaf-feeding H. armigera or on plant growth. This study sheds light on the potential application of KTI in plant defenses and biocontrol against H. armigera in trees and presents new options to investigate growth-defense theories.},
}
@article {pmid41420106,
year = {2026},
author = {Ge, J and Hirosue, S and Castillon, L and Patel, SA and Wesolowski, L and Dyas, A and Yong, C and de Haan, S and Drost, J and Stewart, GD and Obenauf, AC and Muñoz-Espín, D and Vanharanta, S},
title = {Mechanisms of resistance to VHL loss-induced genetic and pharmacological vulnerabilities.},
journal = {EMBO molecular medicine},
volume = {18},
number = {2},
pages = {599-619},
pmid = {41420106},
issn = {1757-4684},
support = {C9685/A25177//Cancer Research UK (CRUK)/ ; C62187/A29760//Cancer Research UK (CRUK)/ ; BRC-1215-20014//NIHR | NIHR Cambridge Biomedical Research Centre (NIHR Cambridge BRC)/ ; 955951//European Commission (EC)/ ; MC_UU_12022/7//UKRI | Medical Research Council (MRC)/ ; RP_033_20170303//Kidney Research UK/ ; 338420//Research Council of Finland (AKA)/ ; },
mesh = {*Von Hippel-Lindau Tumor Suppressor Protein/genetics/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism/genetics ; CRISPR-Cas Systems ; *Drug Resistance, Neoplasm ; Cell Line, Tumor ; *Antineoplastic Agents/pharmacology ; Epithelial Cells ; },
abstract = {The von Hippel-Lindau tumor suppressor (VHL) is a component of a ubiquitin ligase complex that controls cellular responses to hypoxia. Endogenous VHL is also utilized by proteolysis-targeting chimera (PROTAC) protein degraders, a promising class of anti-cancer agents. VHL is broadly essential for cell proliferation, yet it is a key tumor suppressor in renal cell carcinoma. To understand the functional consequences of VHL loss, and to identify targeted approaches for the elimination of VHL null cells, we have used genome-wide CRISPR-Cas9 screening in human renal epithelial cells. We find that, upon VHL loss, the HIF1A/ARNT complex is the central inhibitor of cellular fitness, suppressing mitochondrial respiration, and that VHL null cells show HIF1A-dependent molecular vulnerabilities that can be targeted pharmacologically. Combined VHL/HIF1A inactivation in breast and esophageal cancer cells can also provide resistance to ARV-771, a VHL-based bromodomain degrader that has anti-cancer activity. HIF1A stabilization can thus provide opportunities for early intervention in neoplastic VHL clones, and the VHL-HIF1A axis may be relevant for the development of resistance to the emerging class of PROTAC-based cancer therapies.},
}
@article {pmid41420208,
year = {2025},
author = {Holmlund, H and Yamauchi, Y and Tekayev, M and Jakobs, S and Robin, A and Fujii, W and Ward, MA},
title = {CRISPR/Cas9-mediated knock-in of the murine Y chromosomal genes Zfy1 and Zfy2.},
journal = {BMC genomics},
volume = {27},
number = {1},
pages = {88},
pmid = {41420208},
issn = {1471-2164},
support = {G12 MD007601/MD/NIMHD NIH HHS/United States ; 17CON-86294//Hawai'i Community Foundation/ ; R01 HD114645/HD/NICHD NIH HHS/United States ; F31 HD111279/HD/NICHD NIH HHS/United States ; HD114645/GF/NIH HHS/United States ; P20 GM103457/GM/NIGMS NIH HHS/United States ; R01 HD072380/HD/NICHD NIH HHS/United States ; NIH F31HD111279/GF/NIH HHS/United States ; },
mesh = {Animals ; *CRISPR-Cas Systems ; Male ; Mice ; *Gene Knock-In Techniques ; Spermatogenesis/genetics ; *Y Chromosome/genetics ; Testis/metabolism ; Gene Editing ; Zinc Fingers/genetics ; },
abstract = {BACKGROUND: The Y-linked mouse zinc finger genes Zfy1 and Zfy2 are critical fertility factors in mice but the mechanisms by which they regulate spermatogenesis remain unclear. We recently produced Zfy1/2 double knock-out mice and observed a complete loss in fertility. However, the biochemical mechanism by which Zfy regulates spermatogenesis is unknown, and ZFY expression has not yet been confirmed at the protein level. As both Zfy homologues share ~ 95% sequence similarity, it is difficult to produce an anti-ZFY antibody specific to either homologue.<br><br>RESULTS: To overcome this technical challenge, we used CRISPR/Cas9 genome editing to develop tagged Zfy1 knock-in (XY[Zfy1-HA]), Zfy2 knock-in (XY[Zfy2-FLAG], XY[Zfy2-3xFLAG], and XY[Zfy2-HA]), and Zfy1/2 double knock-in (XY[Zfy1-HA,Zfy2-MYC]) mice. Successful targeting was confirmed by genotyping and sequencing. The knock-in lines were fertile with normal sperm parameters. Using Western blot on testes, knock-in specific bands were detected matching the predicted ZFY expression patterns. Using immunofluorescence on testis sections from knock-in males, ZFY1 and ZFY2 expression was detected in zygotene spermatocytes, and ZFY2 expression was also detected in spermatids step 7-8 and 9.<br><br>CONCLUSIONS: These novel knock-in mice can be used in future investigations to determine how ZFY controls spermatogenesis.},
}
@article {pmid41420495,
year = {2025},
author = {Pan, G and Wang, L and Zhu, H and Wang, H and Zheng, Z},
title = {Utilization of Miniature CRISPR-AsCas12f1 Nuclease for Efficient Genome Editing in Bacillus subtilis.},
journal = {Biotechnology journal},
volume = {20},
number = {12},
pages = {e70168},
doi = {10.1002/biot.70168},
pmid = {41420495},
issn = {1860-7314},
support = {S2023n06020216//Anhui Province Key Research and Development Plan/ ; 2019YFA0904304//National Key Research and Development Program of China/ ; YZJJ2024QN37//Hefei Institutes of Physical Science, Chinese Academy of Sciences Director's Fund/ ; },
mesh = {*Bacillus subtilis/genetics ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Gene Knockout Techniques ; *Endonucleases/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Genome, Bacterial ; },
abstract = {To address the limitations of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)9 in Bacillus subtilis, such as low transformation efficiency and strong dependence on specific PAM sequences, this study developed a novel genome-editing tool based on AsCas12f1 nuclease derived from Acidibacillus sulfuroxidans. Using the CRISPR-AsCas12f1 system, we successfully achieved gene knockout and targeted insertion in B. subtilis with a knockout efficiency of up to 100%. We further demonstrated that the length of the donor DNA homology arms and the choice of PAM motifs significantly influenced the editing efficiency. To expand the applicability of this system, gene interference and activation experiments were performed using green fluorescent protein (GFP) as a reporter. The system achieved more than 90% gene knockdown efficiency and effectively activated the reported gene transcription, with a maximum activation fold of 3.20. In conclusion, the CRISPR-AsCas12f1 system established in this study provides an efficient and reliable genome editing tool for the functional gene research and industrial applications of B. subtilis.},
}
@article {pmid41421070,
year = {2026},
author = {Salodkar, D and Dongarwar, S and Nair, A and Ashtaputre, P and Reddy, S and Somkuwar, S and Begde, D},
title = {Single-Step CRISPR/Cas13a Assay for detection of small RNAs in Saliva : a proof-of-concept study.},
journal = {Cancer genetics},
volume = {300-301},
number = {},
pages = {67-71},
doi = {10.1016/j.cancergen.2025.12.003},
pmid = {41421070},
issn = {2210-7762},
mesh = {Humans ; *Saliva/chemistry/metabolism ; *CRISPR-Cas Systems/genetics ; *Biomarkers, Tumor/genetics ; Proof of Concept Study ; *MicroRNAs/genetics/analysis ; Sensitivity and Specificity ; *Mouth Neoplasms/genetics/diagnosis ; Leptotrichia/genetics ; Real-Time Polymerase Chain Reaction ; },
abstract = {OBJECTIVE: We describe a proof-of-concept study of a rapid, single-step CRISPR/Cas13a assay using Leptotrichia wadei (LwCas13a) for the detection of small RNA (miRNA) biomarkers in saliva, and compare its performance to real-time PCR (RT-PCR).<br><br>METHODS: The single-step Cas13a assay was evaluated against RT-PCR for its detection efficiency, sensitivity, specificity, and its ability to function in a complex biological matrix. A proof-of-concept test was conducted on patient saliva samples to detect a known oral cancer biomarker, hsa-miR-21-3p RESULTS: The Cas13a assay successfully detected candidate miRNA at picomolar concentrations in both in vitro and saliva samples, demonstrating sensitivity and specificity comparable to RT-PCR. Notably, the assay provided discernible detection of the cancer biomarker directly in patient saliva without the need for RNA extraction or reverse transcription steps.<br><br>CONCLUSION: The proposed single-step CRISPR/Cas13a assay may be developed into a promising platform for developing quick and affordable point-of-care diagnostics for cancer and other diseases, circumventing the need for expensive and time-consuming sample preparation steps.},
}
@article {pmid41421338,
year = {2026},
author = {Du, R and Flynn, MJ and Mahe, K and Honsa, M and Gu, B and Li, D and McGeary, SE and Gradinaru, V and Jungmann, R and Elowitz, MB},
title = {miRNA modules for precise, tunable control of gene expression.},
journal = {Molecular cell},
volume = {86},
number = {1},
pages = {194-212.e7},
doi = {10.1016/j.molcel.2025.11.028},
pmid = {41421338},
issn = {1097-4164},
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; Mice ; Humans ; Dependovirus/genetics ; Transgenes ; *Gene Expression Regulation ; Gene Editing/methods ; Neurons/metabolism ; CRISPR-Cas Systems ; Gene Dosage ; HEK293 Cells ; },
abstract = {Accurate control of transgene expression is important for research and therapy but is challenging to achieve in most settings. MicroRNA (miRNA)-based regulatory circuits can be incorporated within transgenes for improved control. However, the design principles, performance limits, and applications of these circuits in research and biotechnology have not been systematically determined. Here, combining modeling and experiments, we introduce miRNA-based circuit modules, termed "dosage invariant miRNA-mediated expression regulators" (DIMMERs), that establish precise, tunable control of transgene expression across diverse cell types to facilitate imaging, editing, and gene therapy. The circuits use multivalent miRNA regulatory interactions to achieve nearly uniform, tunable protein expression over two orders of magnitude variation in gene dosage. They function across diverse cell types and can be multiplexed for the independent regulation of multiple genes. DIMMERs reduce off-target CRISPR base editing, improve single-molecule imaging, and allow live tracking of adeno-associated virus (AAV)-delivered transgene expression in mouse cortical neurons. DIMMERs thus enable accurate regulation for research and biotechnology applications.},
}
@article {pmid41421670,
year = {2026},
author = {Fan, X and Li, B and Xu, X and Long, B and Jia, Z and Wang, R and Gao, J and Chen, Y and Peng, M and Zhou, M},
title = {Deciphering the regulatory role of the pfs gene on biofilm formation in Lactobacillus plantarum R: Insights from transcriptome and metabolome.},
journal = {Bioresource technology},
volume = {443},
number = {},
pages = {133833},
doi = {10.1016/j.biortech.2025.133833},
pmid = {41421670},
issn = {1873-2976},
mesh = {*Lactiplantibacillus plantarum/genetics/physiology/metabolism ; *Biofilms/growth &amp; development ; *Transcriptome/genetics ; *Metabolome/genetics ; Gene Expression Regulation, Bacterial ; *Bacterial Proteins/genetics/metabolism ; Quorum Sensing/genetics ; *Genes, Bacterial/genetics ; CRISPR-Cas Systems/genetics ; },
abstract = {Lactobacillus plantarum is a widely recognized probiotic that forms biofilms to enhance environmental tolerance and probiotic properties, but the mechanisms regulating its biofilm formation remain unclear. This study successfully used CRISPR-Cas9 to delete the pfs gene in the high biofilm-producing strain L. plantarum R, and first investigated its role by integrated transcriptomic and metabolomic analyses. The pfs gene participates in the activated methyl cycle and AI-2 synthesis, which is involved in quorum sensing and biofilm formation. Deletion of pfs increased biofilm biomass by 91% and markedly enhanced matrix accumulation, including exopolysaccharides, extracellular proteins and extracellular DNA (eDNA). Transcriptomic analysis revealed significant perturbation of cysteine and methionine metabolism and altered expression of key genes involved in exopolysaccharide synthesis. Metabolomic profiling identified 223 differentially expressed metabolites, primarily associated with carbon flux and EPS precursor pathways. In summary, pfs deletion enhances biofilm formation via metabolic reprogramming rather than classical AI-2 dependent QS pathways. This study provides new insights into the pfs gene-mediated regulation of biofilm formation in L. plantarum and establishes a foundation for future strategies to manipulate biofilm formation in industrial applications.},
}
@article {pmid41422102,
year = {2025},
author = {Zhong, C and Yu, L and Zhao, T and Shen, X and Li, Z and Zhu, W and Hu, Z and Tian, R and Han, Z and He, D},
title = {A Plug-in system for reprogramming the editing patterns of base editors.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {910},
pmid = {41422102},
issn = {2041-1723},
support = {32171465//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32500460//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2023M744121//China Postdoctoral Science Foundation/ ; 2023M734090//China Postdoctoral Science Foundation/ ; 2023M734091//China Postdoctoral Science Foundation/ ; },
mesh = {*Gene Editing/methods ; Zebrafish/genetics/embryology ; Animals ; Humans ; CRISPR-Cas Systems/genetics ; Genetic Therapy/methods ; DNA/genetics/metabolism ; HEK293 Cells ; },
abstract = {DNA base editors are transformative genome editing tools that enable nucleotide conversions without inducing double-stranded DNA breaks, making them promising for correcting genetic mutations. Current base editors, however, are limited by fixed editing windows and constrained location of deaminases. To address these constraints, we develop a modular system termed Plug-in Base Editor (Plug-in BE), which dynamically programs deaminase positioning via integrating various epitopes and antibody-fused deaminases. This system expands the editing capabilities of base editors by optimizing deaminase's spatial interaction with DNA, leading to improvements in efficiencies, window restrictions, and safety profiles. We validate Plug-in BE's versatility and high fidelity in cancer gene therapy and zebrafish embryo editing, demonstrating its potential as a powerful and adaptable tool for basic research and therapeutic applications. This innovation can generate a series of base editors without extensive protein evolution, positioning Plug-in BE as a significant advancement in the field of genome editing.},
}
@article {pmid41422144,
year = {2025},
author = {Felício, D and Osório, H and Pereira, C and Brandão, AF and Freixo, JP and Carvalho, I and Sousa, AP and Castro-Caldas, M and Sequeiros, J and Lemos, C and Santos, M},
title = {Missense variant in TTBK2 kinase domain causes loss of function and impaired protein phosphorylation.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2501},
pmid = {41422144},
issn = {2045-2322},
support = {UI/BD/154402/2023//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; UIDB/00215/2020, UIDP/00215/2020, LA/P/0064/2020 , UID/215/2025//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; DL 57/2016 - Norma Transit&#xf3;ria//Funda&#xe7;&#xe3;o para a Ci&#xea;ncia e a Tecnologia/ ; ROTEIRO/0028/2013; LISBOA-01-0145-FEDER-022125//Rede Nacional de Espectrometria de Massa/ ; ZGRACA//Ataxia UK/ ; },
mesh = {Humans ; Phosphorylation ; *Mutation, Missense ; *Protein Serine-Threonine Kinases/genetics/metabolism/chemistry ; Protein Domains ; CRISPR-Cas Systems ; *Loss of Function Mutation ; HEK293 Cells ; },
abstract = {Tau tubulin kinase 2 (TTBK2) is a ubiquitous serine-threonine protein kinase implicated in diverse cellular processes, including microtubule regulation, ciliogenesis, synaptic signaling, and the phosphorylation of key proteins like TDP-43. Despite its relevance, many aspects of TTBK2 function in both physiological and pathological conditions remain poorly understood. Truncating variants in TTBK2 gene cause spinocerebellar ataxia type 11 (SCA11), a rare form of autosomal dominant cerebellar ataxia. However, the functional consequences and pathogenic potential of missense variants have yet to be elucidated. In this study, we developed a CRISPR/Cas9 knock-in cell model harboring a missense variant in TTBK2 kinase domain (NM_173500.4:c.625 C > T; p.Leu209Phe) to evaluate its impact on TTBK2 expression, associated protein levels, and phosphoproteomic profiles. TTBK2 missense variant (TTBK2-L209F) was associated with reduced TTBK2 protein levels, altered levels of cytoskeleton-related proteins, and impaired kinase activity, namely toward TDP-43. Phosphoproteomic analyses identified dysregulation in pathways linked to gene regulation, protein degradation, cytoskeletal organization, and TGF-β signaling. These findings provide valuable insights into the biological roles of TTBK2 in cellular signaling. Moreover, this study underscores the importance of functional studies to better understand the consequences of TTBK2 missense variants, particularly those affecting the kinase domain, and their potential contribution to disease.},
}
@article {pmid41422267,
year = {2025},
author = {Fair, T and Pavlovic, BJ and Swope, D and Castillo, OE and Schaefer, NK and Pollen, AA},
title = {Mapping cis- and trans-regulatory target genes of human-specific deletions.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11380},
pmid = {41422267},
issn = {2041-1723},
support = {F31 HG011569-01A1//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; F31 HG011569/HG/NHGRI NIH HHS/United States ; P51 OD011132/OD/NIH HHS/United States ; DP2MH122400-01//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; DP2 MH122400/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; Pan troglodytes/genetics ; Animals ; CRISPR-Cas Systems ; *Sequence Deletion/genetics ; Gene Expression Regulation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Pluripotent Stem Cells/metabolism ; Genome, Human ; Chromatin/metabolism/genetics ; Cell Proliferation/genetics ; Brain/metabolism ; },
abstract = {Deletion of functional sequence is predicted to represent a fundamental mechanism of molecular evolution. Comparative genetic studies of primates have identified thousands of human-specific deletions (hDels), and the cis-regulatory potential of short (≤31 base pairs) hDels has been assessed using reporter assays. However, how structural variant-sized (≥50 base pairs) hDels influence molecular and cellular processes in their native genomic contexts remains unexplored. Here, we design genome-scale libraries of single-guide RNAs targeting 7.2 megabases of sequence in 6358 hDels and present a systematic CRISPR interference (CRISPRi) screening approach to identify hDels that modify cellular proliferation in chimpanzee pluripotent stem cells. By intersecting hDels with chromatin state features and performing single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes, we discovered 20 hDels controlling gene expression. We highlight two hDels, hDel_2247 and hDel_585, with tissue-specific activity in the brain. Our findings reveal a molecular and cellular role for sequences lost in the human lineage and establish a framework for functionally interrogating human-specific genetic variants.},
}
@article {pmid41423037,
year = {2026},
author = {Ma, L and Wu, B and Li, S and Zhang, X and Zhao, X and Zhang, J and Zhang, M and Zhang, M and Ma, L and Guo, C and Zhang, T},
title = {CRISPR/Cas9-mediated LmSerpin5 knockout causes midgut dysplasia and leads to embryonic lethality in Locusta migratoria.},
journal = {Journal of insect physiology},
volume = {168},
number = {},
pages = {104925},
doi = {10.1016/j.jinsphys.2025.104925},
pmid = {41423037},
issn = {1879-1611},
mesh = {Animals ; *Locusta migratoria/genetics/growth &amp; development/embryology/immunology ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; Nymph/growth &amp; development/genetics ; Immunity, Innate/genetics ; Gene Knockout Techniques ; },
abstract = {Serpins play a crucial role in in various physiological processes of insects. Previous studies have suggested that Serpins regulated processes like egg diapause, melanization, and antimicrobial peptide synthesis in Locusta migratoria, but their overall functional characterization remains insufficient. In this study, the functions of LmSerpin5 in regulating developmental processes and innate immunity were investigated via CRISPR/Cas9-mediated knockout. Homozygous LmSerpin5 mutation caused complete embryonic lethality. By contrast, chimeric mutants showed elevated mortality during embryonic-to-first-instar nymph transition, though chitinous tissue development remained unaffected. Additionally, adult mutants exhibited no external malformations but displayed pathological changes in immune organs, including fat body cells with enlarged lipid droplets and nuclei, and midgut absorptive cells lacking brush borders. Furthermore, pro-nymphal midguts exhibited reduced microvilli density, structural defects, and inflammatory intestinal folds. Molecular analysis confirmed upregulation of Toll pathway downstream genes (LmMyd88, LmPelle and LmTube) in mutant tissues, with midgut-specific activation of LmTube and LmPelle linking structural damage to immune dysregulation. These results demonstrated LmSerpin5 maintains homeostasis through dual mechanisms: ensuring embryonic survival and suppressing excessive Toll activation.},
}
@article {pmid41423170,
year = {2026},
author = {Sheri, V and Verma, PK and Lekkala, S and Janga, MR},
title = {Application of digital PCR and CRISPR/Cas13a-based fluorescent assay for accurate and on-site detection of cotton leafroll dwarf virus.},
journal = {Journal of virological methods},
volume = {341},
number = {},
pages = {115332},
doi = {10.1016/j.jviromet.2025.115332},
pmid = {41423170},
issn = {1879-0984},
mesh = {*Plant Diseases/virology ; *Gossypium/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Plant Leaves/virology ; Viral Load ; *Closteroviridae/isolation &amp; purification/genetics ; RNA, Viral/genetics ; Texas ; *Polymerase Chain Reaction/methods ; },
abstract = {Cotton leafroll dwarf virus (CLRDV) is an emerging viral pathogen posing a significant threat to cotton production in the United States. Early and accurate detection is critical for effective disease surveillance and management. Although traditional reverse transcription PCR (RT-PCR) is commonly employed for CLRDV diagnosis, it suffers from limitations in sensitivity, quantification accuracy, and involves labor-intensive workflows. In this study, we evaluated two advanced molecular diagnostic approaches for detecting CLRDV, digital PCR (dPCR) and CRISPR/Cas13a-based fluorescent assay. Symptomatic cotton leaf samples from Lubbock and Brownfield, Texas, were screened and confirmed positive by RT-PCR. Digital PCR analysis enabled absolute quantification of viral load, revealing significantly higher titers in Brownfield (F2) samples and offered improved sensitivity over RT-PCR, particularly in samples with low viral loads. However, dPCR is resource-intensive and requires specialized instrumentation. To address the need for rapid, field-deployable diagnostics, we developed a CRISPR/Cas13a-based assay targeting the conserved ORF3, ORF2, and ORF3a regions of the CLRDV genome. Adapted from the SHERLOCK platform, this fluorescence-based assay uses collateral cleavage activity of Cas13a to enable highly specific visual detection. While the assay successfully enabled direct detection from crude leaf extracts without RNA purification, the sensitivity analysis was conducted using purified, in vitro transcribed RNA. Fluorescence signals were reliably observed with as few as 50 RNA copies, defining the assay's practical limit of detection. While dPCR is optimal for quantitative laboratory analysis, the CRISPR/Cas13a-based assay offers a rapid, sensitive, and cost-effective tool for field-level detection. Together, these complementary tools enhance CLRDV surveillance and management in cotton.},
}
@article {pmid41423250,
year = {2026},
author = {Sharma, A and Pathangey, L and Chirackal, SS and Shim, KG and Fonseca, R and Swaminathan, S},
title = {Ferritin H Knockout Induces Differential Immunomodulatory Drug Responses in Multiple Myeloma Cell Lines.},
journal = {European journal of haematology},
volume = {116},
number = {4},
pages = {391-401},
doi = {10.1111/ejh.70084},
pmid = {41423250},
issn = {1600-0609},
support = {//Paula and Rodger Riney Foundation/ ; },
mesh = {Humans ; *Multiple Myeloma/genetics/metabolism/drug therapy/pathology ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics ; Gene Knockout Techniques ; *Ferritins/genetics ; Reactive Oxygen Species/metabolism ; Iron/metabolism ; CRISPR-Cas Systems ; *Immunomodulating Agents/pharmacology ; Gene Expression Regulation, Neoplastic/drug effects ; Oxidative Stress ; Oxidoreductases ; },
abstract = {BACKGROUND: Immunomodulatory agents (IMiDs) are a cornerstone in the successful management of multiple myeloma (MM). However, acquired IMiD resistance leading to disease relapses remains a major barrier. Hydrogen peroxide generation and oxidative stress are key mediators that determine IMiD's effectiveness in MM. Iron plays a key role in the generation of oxidative stress; therefore, cellular iron levels are tightly governed. FTH1 is the major iron storage protein that tightly regulates cellular iron availability. Hence, the present study is targeted to investigate the role of FTH1 in MM and IMiD resistance.<br><br>METHODS: IMiD-sensitive and IMiD-resistant MM cells were analyzed for expression of iron-metabolism genes. CRISPR-cas9-mediated knockout of FTH1 was performed and the after-effects were assessed through multiple experiments.<br><br>RESULTS: Initial analysis showed a positive correlation between FTH1 expression and IMiD resistance in MM cells. FTH1-KO reduced IMiD sensitivity in the KMS11 cell line but had no effect on the RPMI8226 cell line. RNA-seq data showed downregulation of ER-stress and calcium signaling genes after FTH1-KO. Further, KMS11-FTH1KO cells exhibited lower intracellular ROS, labile-iron, and mitochondrial superoxide levels along with increased CD63, suggesting activation of L-ferritin secretory pathways.<br><br>CONCLUSION: Data reveals a link between FTH1, labile iron, ROS, and IMiD resistance in MM cells.},
}
@article {pmid41423621,
year = {2025},
author = {Goswami, SG and Gupta, P and Arvinden, VR and Bhargava, N and Iyer, AR and Saravanakumar, V and Yadav, P and Jha, SK and Singh, S and Kumar, A and Singh, P and Gunda, P and Jain, S and Mehta, P and Nakamura, Y and Kurita, R and Bajaj, A and Ramalingam, S},
title = {CRISPR editing of HPFH3 genotype induces γ-globin expression and reverses sickle cell disease and β-thalassemia phenotypes.},
journal = {Stem cell research &amp; therapy},
volume = {17},
number = {1},
pages = {46},
pmid = {41423621},
issn = {1757-6512},
mesh = {*beta-Thalassemia/genetics/therapy/pathology/metabolism ; *Anemia, Sickle Cell/genetics/therapy/pathology/metabolism ; Humans ; *gamma-Globins/genetics/metabolism ; Animals ; *Gene Editing/methods ; *Fetal Hemoglobin/genetics/metabolism ; CRISPR-Cas Systems ; Genotype ; Mice ; Phenotype ; Hematopoietic Stem Cells/metabolism ; },
abstract = {BACKGROUND: Hereditary persistence of Fetal Hemoglobin (HPFH) is a benign condition known to mitigate symptoms in individuals with co-inherited β-hemoglobinopathies, such as β-thalassemia (BT) and sickle cell disease (SCD), through the reactivation of fetal hemoglobin (HbF). HPFH typically arises from deletions of varying sizes affecting the β-globin gene cluster or point mutations in the promoters of the γ-globin genes. While the therapeutic benefits of point mutations have been extensively studied, the potential of deletional forms of HPFH remains underexplored in preclinical settings.<br><br>METHOD: In this study, we generated benign deletional HPFH3 genotype in SCD and BT patient-derived HSPCs using CRISPR/Cas9 and showed that therapeutically relevant levels of HbF reactivation result in the alleviation of the pathological phenotypes.<br><br>RESULTS: In edited cells derived from SCD patients, we observed reduced sickling and oxidative stress, while in edited from BT cells, restoration of the &#x3b1;-globin/&#x3b2;-globin ratio improved erythroid lineage maturation and reduced ROS levels. Importantly, HPFH3-edited HSPCs retained their genome integrity and showed no detrimental effect on their regeneration or differentiation into erythroid, myeloid, T, and B cell lineages in immunodeficient NBSGW mice post-xenotransplantation. Additionally, we showed a reduced interaction between the LCR and HBB, suggesting that the HPFH3 deletion specifically promoted LCR interactions with HBG1/2, likely due to the absence of the HBB locus.<br><br>CONCLUSIONS: Collectively, our preclinical findings suggest that the generation of the HPFH3 genotype has the potential to significantly enhance HbF levels, offering a promising universal therapeutic strategy for treating both SCD and &#x3b2;-thalassemia.},
}
@article {pmid41423823,
year = {2025},
author = {Stamilla, A and Recchia, D and Stelitano, G and Maci, L and Marturano, MC and De Rossi, E and Chiarelli, LR and Pasca, MR and Degiacomi, G},
title = {Uncovering Insights Into the Biology of Mycobacterium tuberculosis Using Genetic Tools.},
journal = {MicrobiologyOpen},
volume = {14},
number = {6},
pages = {e70206},
pmid = {41423823},
issn = {2045-8827},
support = {20205B2HZE_003//University of Pavia; Italian Ministry of University and Research/ ; 2022JTPP53//University of Pavia; Italian Ministry of University and Research/ ; PE00000007//NextGenerationEUMUR PNRR/ ; INF-ACT//NextGenerationEUMUR PNRR/ ; },
mesh = {*Mycobacterium tuberculosis/genetics/drug effects/pathogenicity ; Humans ; *Genetic Engineering/methods ; Tuberculosis/microbiology ; Drug Discovery ; Genome, Bacterial ; },
abstract = {Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, is one of the most challenging pathogens due to its complex physiology, diverse clinical manifestations, and growing multidrug resistance. The global rise of drug-resistant Mtb strains has prompted the search for innovative genetic and molecular strategies to accelerate drug discovery and vaccine development. Progress in Mtb research has long been hindered by its slow replication rate and impermeable cell envelope, which limit the efficacy of genetic manipulation. This review outlines methodological advances that have transformed the study of Mtb pathogenesis and drug resistance mechanisms. Traditional homologous recombination-based approaches, including allelic exchange and specialized transduction, laid the groundwork for targeted mutagenesis but were limited by low efficiency. The advent of phage-derived recombineering systems, such as the Che9c RecET, has substantially improved the precision and throughput of genetic modification. Hybrid systems such as ORBIT, which combines oligonucleotide-mediated recombineering with Bxb1 integrase, have further enabled rapid and versatile genome engineering across mycobacterial species. Parallel developments in conditional gene expression systems (e.g., the use of TetR/Pip-based promoters) have facilitated the functional analysis of essential genes and the validation of novel drug targets. The advent of CRISPR-Cas technologies has represented a paradigm shift, by enabling programmable, high-fidelity gene regulation and functional genomics even in slow-growing mycobacteria. Together, these genetic innovations are transforming Mtb research by accelerating drug discovery and vaccine design, and shedding light on host-pathogen interactions.},
}
@article {pmid41424172,
year = {2025},
author = {Ikram, M and Farhan, M and Derakhshani, B and Kumar, S and Khan, N and Gupta, R and Usman, B and Liu, P},
title = {Machine Learning and CRISPR-Based Validation Elucidate OsWOX13 Involvement in Rice Heat Stress Tolerance and Flowering.},
journal = {Physiologia plantarum},
volume = {177},
number = {6},
pages = {e70714},
doi = {10.1111/ppl.70714},
pmid = {41424172},
issn = {1399-3054},
support = {YSPTZX202206//Special Project for the Academician Team Innovation Center of Hainan Province/ ; ZDYF2022XDNY185//Key Research Program of Hainan Province/ ; },
mesh = {*Oryza/genetics/physiology ; *Machine Learning ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Heat-Shock Response/genetics ; *Flowers/physiology/genetics ; *Thermotolerance/genetics ; CRISPR-Cas Systems/genetics ; Proteomics ; },
abstract = {Rice (Oryza sativa) is a staple food for billions of people globally, but it faces significant yield losses due to heat stress. However, rice responses to heat stress remain understudied as compared to other stress factors. In this study, we combined meta-transcriptomics, machine learning, functional validation, and proteomic analysis to identify the key genes involved in heat stress tolerance in rice. We identified 409 meta-differentially expressed genes (meta-DEGs) between heat-tolerant and susceptible genotypes, which were associated with detoxification, oxidative stress, protein folding, phenylpropanoid biosynthesis, glutathione metabolism, and plant hormone signal transduction. We trained five machine learning models, of which Random Forest (RF) and eXtreme Gradient Boosting (XGBoost) outperformed the others. Using SHAP analysis, the top 14 genes for each model were identified, including the OsWOX13 gene, which was detected simultaneously across both models, indicating a positive regulator and a strong candidate for heat stress tolerance. Functional validation of OsWOX13 via CRISPR/Cas9-mediated knockout (KO) confirmed its positive role in heat stress, with a delay in flowering and survival rate of ~20% compared to ~60% for WT under heat stress. Physiological and antioxidant enzymatic activities showed a significant (p ≤ 0.05) reduction in ABA accumulation levels, increased MDA accumulation, and decreased SOD and POD activities in KO lines compared to WT. The proteomic analysis identified upregulated heat shock proteins (HSF8, BIP1, BIP5, and HSP81-1) and downregulated flowering-associated proteins (ROC6 and 4CL4) in mutant lines in response to heat stress. These results indicate that OsWOX13 enhances heat tolerance by regulating ABA signaling and antioxidant defense mechanisms. Taken together, this study highlights the efficiency of machine learning models in the identification of stress responsive genes and provides OsWOX13 as a strong positive candidate for heat stress tolerance and breeding climate-resilient rice varieties.},
}
@article {pmid41424849,
year = {2026},
author = {Liu, M and Fu, X and Zhang, H and Pan, J and Jia, Q and Zhang, C and An, F},
title = {Endothelial KSR2 regulated by genetic variation protects against atherosclerosis through AMPKα1 stabilization.},
journal = {Theranostics},
volume = {16},
number = {5},
pages = {2598-2626},
pmid = {41424849},
issn = {1838-7640},
mesh = {Animals ; *Atherosclerosis/genetics/metabolism/pathology ; Mice ; *AMP-Activated Protein Kinases/metabolism/genetics ; Humans ; Polymorphism, Single Nucleotide/genetics ; Mice, Knockout ; Male ; Endothelial Cells/metabolism ; Diet, High-Fat/adverse effects ; Mice, Inbred C57BL ; Apolipoproteins E/genetics ; Mice, Knockout, ApoE ; Disease Models, Animal ; *Adaptor Proteins, Signal Transducing/genetics/metabolism ; Apoptosis/genetics ; CRISPR-Cas Systems ; },
abstract = {Rationale: The single nucleotide polymorphism (SNP) rs11830157 within the scaffold protein kinase suppressor of Ras 2 (KSR2) locus is strongly associated with the incidence of coronary artery disease (CAD), yet its functional role remains undefined. This study aimed to investigate the potential impact of rs11830157 polymorphism on atherosclerosis and to elucidate the underlying molecular mechanisms. Methods: Dual-luciferase reporter assays, chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assays (EMSA), and CRISPR/Cas9 gene-editing techniques were used to investigate the regulatory role of the SNP rs11830157. To assess the role of KSR2 in atherosclerosis, we utilized global KSR2 knockout mice fed a high-fat diet ad libitum, pair-fed global KSR2 and Apoe (Apolipoprotein E) double knockout mice, and mice with endothelial-specific KSR2 overexpression mediated by AAV9-ICAM2. Results: Genetic analyses identified SNP rs12822146, in linkage disequilibrium with rs11830157 and located within an endothelial enhancer, as a regulator of KSR2 expression via differential binding of the transcriptional repressor XBP1s. KSR2 expression was significantly reduced in endothelial cells within atherosclerotic plaques in both humans and mice. Using multiple KSR2 gene-edited mouse models, we demonstrated that endothelial KSR2 protects against atherosclerosis by suppressing inflammation and apoptosis. Mechanistic studies revealed that KSR2 competes with CRBN for binding to the K52 site of AMPKα1, inhibiting CRL4A[CRBN] E3 ubiquitin ligase complex-mediated K48-linked polyubiquitination and proteasomal degradation of AMPKα1. The subsequently activated AMPK signaling pathway maintains glycolytic balance in endothelial cells, ultimately exerting anti-inflammatory and anti-apoptotic effects. Conclusions: Our findings provide the first comprehensive molecular explanation of the rs12822146-KSR2-atherosclerosis axis, with important implications for both primary prevention and secondary treatment of CAD.},
}
@article {pmid41424917,
year = {2025},
author = {Sharma, S and Saroha, NK and Sehrawat, A and Tang, G and Singh, D and Teotia, S},
title = {Emerging tools in plant genome editing.},
journal = {Frontiers in genome editing},
volume = {7},
number = {},
pages = {1588089},
pmid = {41424917},
issn = {2673-3439},
abstract = {Plant genome editing has undergone a transformative shift with the advent of advanced molecular tools, offering unprecedented levels of precision, flexibility and efficiency in modifying genetic material. While classical site-directed nucleases such as ZFNs, TALENs and CRISPR-Cas9 have revolutionized genome engineering by enabling targeted mutagenesis and gene knockouts, the landscape is now rapidly evolving with the emergence of novel systems that go beyond the conventional double strand break (DSB)-mediated approaches. Advanced and recent tools include LEAPER, SATI, RESTORE, RESCUE, ARCUT, SPARDA, helicase-based approaches like HACE and Type IV-A CRISPR system, and transposon-based techniques like TATSI and piggyBac. These tools unlock previously inaccessible avenues of genome and transcriptome modulation. Some of these technologies allow DSB-free editing of DNA, precise base substitutions and RNA editing without altering the genomic DNA, a significant advancement for regulatory approval and for species with complex genomes or limited regeneration capacity. While LEAPER, RESCUE and RESTORE are the new advents in the RNA editing tool, SATI allows DSB-free approach for DNA editing, ARCUT offers less off-target and cleaner DNA repairs and Type IV-A CRISPR system induces gene silencing rather than editing. The transposon-based approaches include TATSI, piggyBac and TnpB, and helicases are used in HACE and Type IV-A CRISPR system. The prokaryotic Argonaute protein is used in SPARDA tool as an endonuclease to edit DNA. The transient and reversible nature of RNA editing tools such as RESTORE and LEAPER introduces a new layer of epigenetics-like control in plant systems, which could be harnessed for tissue-specific and environmentally-responsive trait expression. Simultaneously, innovations like ARCUT and SPARDA utilize chemically-guided editing, minimizing reliance on biological nucleases and reducing off-target risks. Their modularity and programmability are enabling gene function studies, synthetic pathway designs and targeted trait stacking. These advances represent a novel synthesis of genome engineering and systems biology, positioning plant genome editing not just as a tool of modification but as a platform for designing adaptive and intelligent crops, tailored to future environmental and nutritional challenges. Although, many of these recent tools remain to be applied on plant systems, they are proven to be effective elsewhere and hold a great potential to be effective in creating climate-resilient crops.},
}
@article {pmid41425600,
year = {2025},
author = {Fazeli, A and Ullrich, E and Cathomen, T and Bexte, T},
title = {Engineering with care: safety assessment platforms for CRISPR-modified natural killer cells.},
journal = {Frontiers in immunology},
volume = {16},
number = {},
pages = {1711414},
pmid = {41425600},
issn = {1664-3224},
mesh = {*Killer Cells, Natural/immunology/metabolism/transplantation ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; Animals ; *Immunotherapy, Adoptive/methods/adverse effects ; },
abstract = {CRISPR-based gene editing has become a transformative tool to enhance immune cell therapies. In particular, engineering natural killer (NK) cells with CRISPR/Cas systems has gained traction due to their ability to mediate strong anti-tumor responses in an MHC-unrestricted, non-alloreactive manner. Early trials show the feasibility and safety of allogeneic NK cells, paving the way as scalable "off-the-shelf" products. CRISPR/Cas9 edits genomes by inducing DNA double-strand breaks (DSBs), mainly repaired through non-homologous end joining (NHEJ) or homology-directed repair (HDR). While effective, CRISPR carries risks of off-target (OT) activity that may disrupt essential genes, cause chromosomal rearrangements, or trigger oncogenic changes - posing threats to product integrity and patient safety. These concerns intensify with multiplex editing, where multiple loci are modified to improve function, persistence, and immune evasion. Since unmodified NK cells are typically short-lived, many clinical-stage products are engineered to express IL-15 or related constructs, extending their half-life and amplifying risks associated with unintended changes. This underscores the urgent need for robust safety assessments. In this review, we summarize the current landscape of safety assessment platforms for evaluating gene edited NK cells. We highlight predictive in silico tools, biochemical in vitro assays, and emerging cell-based detection systems to identify and quantify CRISPR-induced OT events. Particular attention is given to their suitability, limitations, and practical use in primary NK cells and multiplex editing strategies. Our aim is to support the design of safe, effective editing workflows for NK cell therapies - ensuring rigor as the field advances rapidly toward clinical application.},
}
@article {pmid41427723,
year = {2026},
author = {Ortiz-Severin, J and Geoffroy, P and Aravena, P and Hodar, C and Palma, DE and González, M and Cambiazo, V},
title = {Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {1},
pages = {e0156025},
pmid = {41427723},
issn = {1098-5336},
support = {1211893//ANID-Fondecyt/ ; 2024T2DID//Doctoral Fellowship/ ; ICN2021_044//Millennium Science Initiative Program/ ; },
mesh = {*Piscirickettsia/genetics ; Fish Diseases/microbiology ; Piscirickettsiaceae Infections/microbiology/veterinary ; Animals ; *CRISPR-Cas Systems ; Bacterial Proteins/genetics ; Gene Silencing ; },
abstract = {UNLABELLED: Piscirickettsia salmonis is the causative agent of salmonid rickettsial septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous reporter (sfGFP) and an endogenous regulator (Fur) that controls intracellular iron homeostasis in bacteria. The inducible expression of dCas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains when the expression of the fur gRNA was induced with IPTG. By RNA-seq, we detected a significant increase in the expression of genes encoding the Fe[2+] and Fe[3+] acquisition systems and iron mobilization in the fur1 knockdown after IPTG induction. All the genes with over 2-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The implementation of the Mobile-CRISPRi system in P. salmonis has been demonstrated to be effective, thus providing a tool with potential application for the analysis of gene function in this pathogen. It is anticipated that these analyses will be valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis.<br><br>IMPORTANCE: Salmonid rickettsial septicemia (SRS) is an infectious disease caused by the marine bacterium Piscirickettsia salmonis. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen P. salmonis, inserting exogenous genes into the bacterium's chromosome to ensure their constitutive and inducible expression and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Fur's relevance to the iron metabolism in the pathogen.},
}
@article {pmid41428463,
year = {2026},
author = {Ye, T and Xue, M and Chen, H and Yue, S and Yuan, M and Yu, J and Cao, H and Hao, L and Wu, X and Yin, F and Xu, F},
title = {Allosteric Aptamer CRISPR/Cas Activation Enables Non-competitive ATP Detection and Meat Freshness Assessment.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1592-1601},
doi = {10.1021/acs.jafc.5c12150},
pmid = {41428463},
issn = {1520-5118},
mesh = {*Adenosine Triphosphate/analysis ; *Aptamers, Nucleotide/chemistry/genetics ; *CRISPR-Cas Systems ; *Biosensing Techniques/methods/instrumentation ; Animals ; *Meat/analysis ; *Meat Products/analysis ; },
abstract = {CRISPR/Cas-based aptasensors (Cas-aptasensors) hold great promise for detecting non-nucleic acid targets, yet their intrinsic competitive recognition mechanism imposes a trade-off between transduction efficiency and background leakage. In this study, we developed a Cas-aptasensor that employs a non-competitive recognition mechanism. In our design, the aptamer-target interaction accelerates the toehold-mediated strand displacement reaction and exposes a second toehold domain. The CRISPR/Cas system is ultimately activated via a cascade strand-displacement reaction, which is hindered in the absence of the target and, thus, delays activation. We demonstrated the applicability of this non-competitive Cas-aptasensor for the detection of ATP, achieving a detection limit as low as 1.0 nM within 45 min. Furthermore, we successfully applied this method to ATP detection in complex matrices and to assess the freshness of diverse meat products across different storage temperatures. Overall, this work advances the design of Cas-aptasensors and expands their potential applications in food safety monitoring.},
}
@article {pmid41428486,
year = {2026},
author = {Ribeiro Gomes, AR and Hamel, N and Mastwal, S and Wright, N and Ide, DC and Richie, CT and Usdin, TB and Wang, KH and Leopold, DA},
title = {Targeted gene transfer into developmentally defined cell populations of the primate brain.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116756},
pmid = {41428486},
issn = {2211-1247},
support = {ZIA MH002898/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Animals ; *Gene Transfer Techniques ; *Brain/metabolism/embryology ; Callithrix ; Female ; Rats ; Dependovirus/genetics ; Transgenes/genetics ; Gene Editing/methods ; Genetic Vectors ; Male ; CRISPR-Cas Systems ; },
abstract = {The primate brain possesses unique physiological and developmental features, yet its systematic investigation has been hampered by a paucity of transgenic germline models and tools. Here, we present a minimally invasive method to introduce transgenes widely across the primate cerebral cortex using ultrasound-guided fetal intracerebroventricular viral injections (FIVIs). FIVI enables efficient and long-lasting transgene expression following intrauterine delivery of recombinant adeno-associated viruses (rAAVs). In the marmoset, we demonstrate that adjusting gestational timing, rAAV serotype, and transcriptional regulatory elements enables selective targeting of defined cell populations, including layer-restricted labeling and Cre-dependent intersectional access. Pilot experiments in rats further demonstrate the potential of FIVIs for prenatal CRISPR-based gene editing and labeling of peripheral somatosensory and retinal pathways. By mimicking key desirable features of germline transgenic models, this efficient and targeted method for gene transfer into the fetal primate brain expands the experimental opportunities for basic and translational neuroscience research across the lifespan.},
}
@article {pmid41428487,
year = {2026},
author = {Deng, C and Hu, J and Chen, Q and Zhou, S and Ni, J},
title = {Expanded global groundwater microbial diversity reveals bioprospecting potential.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116760},
doi = {10.1016/j.celrep.2025.116760},
pmid = {41428487},
issn = {2211-1247},
mesh = {*Groundwater/microbiology ; Phylogeny ; *Bacteria/genetics/classification ; *Microbiota/genetics ; *Bioprospecting ; Archaea/genetics/classification ; Genome, Bacterial/genetics ; Genome, Archaeal/genetics ; Biodiversity ; Metagenomics ; Metagenome ; },
abstract = {Although the terrestrial subsurface harbors a substantial fraction of Earth's microbial biomass, the genomic diversity of groundwater microbiomes and their potential for bioprospecting remain poorly characterized. Here, we recovered 44,320 bacterial and archaeal genomes from in-house and publicly available metagenomic datasets, establishing a large-scale groundwater microbiota catalog (GWMC) spanning 167 phyla, including four candidate phyla and over 12,000 previously uncharacterized species. This unprecedented phylogenetic diversity was accompanied by a bimodal genome size distribution (0.3-12.8 Mbp), revealing divergent strategies of genomic allocation. By mining extensive genomic resources, we found that small genomes prioritized molecular defense and redox regulation, whereas large genomes frequently harbored greater biosynthetic potential. Notably, we establish the largest selenoprotein catalog to date and highlight groundwater as an overlooked hotspot of microbial selenium metabolism. Overall, this work advances our understanding of microbial diversity in aquifers and uncovers underexplored genomic resources with potential for biotechnology and biomedicine.},
}
@article {pmid41428728,
year = {2025},
author = {Gervais, NC and Rogers, RKJ and Robin, MR and Shapiro, RS},
title = {HyperdCas12a-based multiplexed genetic regulation in Candida albicans.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428728},
issn = {1362-4962},
support = {RGPIN-2018-4914//Natural Sciences and Engineering Research Council of Canada/ ; //NSERC/ ; //Canada Research Chair/ ; RGPIN-2018-4914//NSERC/ ; },
mesh = {*Candida albicans/genetics/metabolism/drug effects ; *CRISPR-Cas Systems ; *Gene Expression Regulation, Fungal ; Ergosterol/biosynthesis ; Fungal Proteins/genetics/metabolism ; Drug Resistance, Fungal/genetics ; },
abstract = {Complex microbial phenotypes involve the combined activity of diverse gene regulatory networks. However, the majority of reverse genetics approaches in microbial pathogenesis research have focused on single-gene perturbation studies, in part due to the lack of available genetic tools in many pathogens. Developing enhanced versions of CRISPR-Cas platforms holds significant promise for improving the scalability of microbial functional genomics research. Here, we demonstrate highly efficient, inducible, and multiplexed activation and repression in the major human fungal pathogen Candida albicans by translating the hyperdCas12a variant to the fungal kingdom. This represents the first application of a CRISPR-Cas12 system in a human fungal pathogen. We profile the effectiveness of our new CRISPR activation and CRISPR interference tools and achieve tunable levels of target modulation. Further, we demonstrate that perturbing combinations of genes in the drug efflux and ergosterol biosynthesis pathways reveals important redundancies and synergistic properties in drug resistance circuitry. Our hyperdCas12a platform is thus an efficient system for the rapid generation of combinatorial mutants that will enable the mechanistic understanding of genetic interactions involved in diverse phenotypes in C. albicans. The enhanced activity with hyperdCas12a in fungi suggests it could be translated to other microbes as a powerful tool for studying genetic interactions.},
}
@article {pmid41428733,
year = {2025},
author = {Yang, Z and Yu, M and Li, P and Li, Z and Teng, Y and Zhou, Y and Zhao, M and Liu, C and Zhao, Z and Wang, Z and Li, J and Jing, Y and Li, Y and Zhao, H and Song, W and Bian, C and Zhao, H and Chen, J and Xin, B and Lai, J},
title = {Casδ, an evolutionary transitional CRISPR system enables efficient genome editing across animals and plants.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428733},
issn = {1362-4962},
support = {//Agriculture Science and Technology/ ; 2023YFD1202900//National Key Research and Development Program of China/ ; PC2023A01004//Pinduoduo-China Agricultural University/ ; //Agriculture Science and Technology/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; Animals ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Genome, Plant ; RNA, Guide, CRISPR-Cas Systems/genetics ; Zea mays/genetics ; Evolution, Molecular ; Oryza/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) adaptive immune systems provide sequence-specific mechanisms for targeting foreign DNA or RNA and have been widely used in genome editing and DNA detection. Type V CRISPR-Cas systems are characterized by a single RNA-guided RuvC domain-containing effector, Cas12. Here, through comprehensive mining of large-scale genomic and metagenomic data from microbial sources, we identified a new Class 2 CRISPR-Cas effector superfamily, designated Casδ, comprising three members with protein sizes ranging from 867 to 936 amino acids. Biochemical analyses revealed that Casδ-1 functions as a single RNA-guided endonuclease with specific recognition of 5'-RYR-3' protospacer-adjacent motifs, where R represents A or G, and Y represents T or C. Casδ-1 exhibits robust double-stranded DNA cleavage activity and target-dependent trans-cleavage activity. Casδ-1 mediates efficient genome editing across species, achieving up to 60% indel rates in human cells while generating homozygous knockout lines in two agriculturally important monocot species (Oryza sativa and Zea mays) through stable transformation. Structural and evolutionary analyses reveal Casδ as an evolutionary transitional nuclease bridging Cas12n and canonical type V systems, featuring a C-terminal loop that is essential for activity. Collectively, Casδ is an evolutionarily distinct, compact (<1000 aa), tracrRNA-free CRISPR system enabling versatile cross-kingdom genome editing.},
}
@article {pmid41428734,
year = {2025},
author = {Zhu, C and Xiao, D and Wang, Y and Han, H and Qin, C and Liu, S and Chen, X and Xiao, H and Chen, X and Shi, J and Tang, J and Shen, J and Song, H},
title = {Molecular basis of NFIB-mediated regulation of oncogenic transcription.},
journal = {Nucleic acids research},
volume = {53},
number = {22},
pages = {},
pmid = {41428734},
issn = {1362-4962},
support = {Z2023033//Hunan Health Commission Key Clinical Specialty Major Research Project/ ; NFPS-JJ-501348//Chinese National Key Clinical Specialty Program/ ; 82272508//National Natural Science Foundation of China/ ; 005/2023/SKL//State Key Laboratory of Mechanism and Quality of Chinese Medicine/ ; 0007/2022/AKP, 0068/2023/ITP2, 0143/2025/ITP2//Macau Science and Technology Development Fund/ ; MYRG-GRG2024-00283-ICMS-UMDF, SRG2023-00054-ICMS//University of Macau/ ; 005/2023/SKL//State Key Laboratory of Mechanism and Quality of Chinese Medicine/ ; },
mesh = {Humans ; *NFI Transcription Factors/genetics/chemistry/metabolism ; *Gene Expression Regulation, Neoplastic ; DNA/metabolism/chemistry/genetics ; Cell Line, Tumor ; *Transcription, Genetic ; CRISPR-Cas Systems ; Protein Binding ; *Neoplasms/genetics/pathology/metabolism ; Cell Proliferation/genetics ; Oncogenes ; Models, Molecular ; Cell Movement/genetics ; Transcriptional Activation ; },
abstract = {The Nuclear Factor I (NFI) family of transcription factors orchestrates key regulatory programs in development, differentiation, and metabolism, with dysregulation implicated in diverse pathological conditions, including cancer. Among the paralogs, NFIB has emerged as an oncogenic driver in multiple tumor types, yet the mechanisms through which it engages DNA and directs oncogenic transcriptional programs remain undefined. Here, using cancer cells with high NFIB expression, we demonstrate that NFIB promotes malignant phenotypes, as CRISPR-Cas9 knockout impairs proliferation, migration, and invasion. Transcriptomic profiling reveals that NFIB regulates a cancer-enriched gene network that includes FGFR3 and PDGFRB. Biophysical analyses show that NFIB, including its DNA-binding domain, functions as a monomer and binds DNA with strict 1:1 stoichiometry. High-resolution crystal structures of NFIB DNA-binding domain bound to ChIP-seq-derived DNA motifs reveal a monomeric binding mode mediated by conserved base-specific interactions with the TGGCA sequence, providing an atomic view of NFIB-DNA recognition. Mutational disruption of key DNA-contacting residues abolishes DNA binding and transcriptional activation, linking atomic-level recognition to oncogenic transcriptional regulation. Together, these findings elucidate the structural mechanism underlying NFIB function in cancer and establish a framework for therapeutic strategies targeting NFIB-driven malignancies.},
}
@article {pmid41430049,
year = {2025},
author = {Dong, Q and Chen, P and Guo, Z and Wei, H and Zeng, Y and Zhang, J and Men, Y and Liu, W and Sun, Y and Yang, J},
title = {Computational design of allulose-responsive biosensor toolbox for auto-inducible protein expression and CRISPRi mediated dynamic metabolic regulation.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11562},
pmid = {41430049},
issn = {2041-1723},
support = {32271545//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Biosensing Techniques/methods ; *Transcription Factors/metabolism/genetics/chemistry ; *CRISPR-Cas Systems ; Escherichia coli/metabolism/genetics ; Synthetic Biology/methods ; Glucose/metabolism ; },
abstract = {Biosensors based on transcription factors (TFs) have shown extensive applications in synthetic biology. Due to the complex multi-domain structure of effector-TF-DNA, computational design of TFs remains a challenge. Here, we present the successful structure-guided computational design of the access tunnel, ligand binding, allosteric transition process for an allulose-responsive PsiR. It enables a 20-fold increase in sensitivity, reducing the EC50 of PsiR-allulose biosensors (PABs) from 16 mM to 0.8 mM, and delivers a PAB box possessing the detection range from 10 μM to 100 mM. We further validate its broader applicability in enhancing sensitivity of LacI-IPTG biosensor. Based on the developed PABs, we present the inducer-free allulose-mediated auto-inducible protein expression system, and demonstrate an allulose-triggered CRISPR interference circuit for dynamic metabolic regulation. It facilitates a 68% increase in allulose titer and achieves a high yield of 0.43 g/g glucose. This work provides the versatile TF toolbox for developing allulose-triggered regulation circuits in biotechnology application.},
}
@article {pmid41430241,
year = {2025},
author = {Nieto-Sanchez, A and Martinez-Lage, M and Puig-Serra, P and Carpintero, S and Alonso-Yanez, A and Ojeda-Walczuk, P and Ibañez-Navarro, M and Pita, G and Moya, FJ and Moreno, C and Martin, MC and Alonso, R and Nuñez-Torres, R and Sanchez-Arevalo Lobo, VJ and Alonso-Guirado, L and Malats, N and Gonzalez-Neira, A and Fernandez, L and Roda-Navarro, P and Torres-Ruiz, R and Rodriguez-Perales, S},
title = {Selective genome editing of amplified oncogenes triggers immunogenic cell death and tumor remodeling.},
journal = {Molecular cancer},
volume = {25},
number = {1},
pages = {21},
pmid = {41430241},
issn = {1476-4598},
mesh = {Humans ; Animals ; *Gene Editing/methods ; CRISPR-Cas Systems ; Mice ; *Oncogenes/genetics ; Cell Line, Tumor ; *Immunogenic Cell Death/genetics ; *Gene Amplification ; *Neoplasms/genetics/pathology/immunology ; Tumor Microenvironment/genetics ; Xenograft Model Antitumor Assays ; },
abstract = {Oncogene amplifications fuel some of the most lethal, therapy‑refractory cancers, yet remain clinically untargeted. We report a single‑guide CRISPR/Cas9 strategy that converts the sheer copy‑number excess of oncogene amplicons into an Achilles' heel. A solitary intronic double‑strand break is innocuous in diploid genomes but collapses oncogene amplification‑positive cells across neuroblastoma, small‑cell lung and colorectal carcinoma models, driving > 90% loss of viability, G2/M blockade and catastrophic DNA‑damage signalling. Amplified‑locus cleavage rewires transcription toward cell death activation, necroptosis and cGAS-STING-mediated immunogenic cell death, enabling dendritic‑cell cross‑priming and T‑cell activation and proliferation. In xenografts, delivery of the intronic sgRNA shrinks tumours by 90%, prolongs survival and remodels the innate tumour microenvironment. Deep sequencing confirms negligible off‑target editing, and combination with doxorubicin achieves supra‑additive killing. These findings establish amplification density, not sequence content, as a tractable, tumour‑exclusive target and unveil a dual‑action platform that is simultaneously cytotoxic and immunostimulatory. Editing of tumor amplifications therefore offers a blueprint for translating copy‑number aberrations into precision genome‑editing therapies for treatment‑resistant cancers.},
}
@article {pmid41430372,
year = {2025},
author = {Kim, GE and Lee, SY and Kang, YJ and Bin Jin, H and Park, HH},
title = {AcrIIA19 binds to the WED domain and inhibits various Cas9 orthologs at multiple stages.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {136},
pmid = {41430372},
issn = {2399-3642},
support = {RS-2025-02316334//National Research Foundation of Korea (NRF)/ ; RS-2025-16065724//National Research Foundation of Korea (NRF)/ ; },
mesh = {*CRISPR-Associated Protein 9/metabolism/chemistry/antagonists &amp; inhibitors/genetics ; *CRISPR-Cas Systems ; Staphylococcus aureus/genetics/enzymology ; Protein Domains ; Streptococcus pyogenes/genetics/enzymology ; Protein Binding ; *Bacterial Proteins/metabolism/chemistry/genetics ; Gene Editing ; RNA, Guide, CRISPR-Cas Systems/metabolism ; Crystallography, X-Ray ; },
abstract = {Anti-CRISPR (Acr) proteins are natural inhibitors of clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) systems, providing valuable tools for regulating genome editing. Here, we present the crystal structure of AcrIIA19, a plasmid-encoded Type II-A CRISPR-Cas system inhibitor that targets Cas9. AcrIIA19 adopts a previously uncharacterized fold and forms a stable homodimer. Biochemical assays revealed that AcrIIA19 binds selectively to the wedge (WED) domain of Cas9, a conserved structural interface critical for single guide RNA-DNA duplex stabilization and catalysis. This interaction disrupts Cas9 activity at multiple stages, independent of the order of complex assembly. Notably, AcrIIA19 exhibits broad-spectrum inhibition across divergent Cas9 orthologs, including Streptococcus pyogenes and Staphylococcus aureus Cas9, by exploiting a conserved WED domain vulnerability. Our findings establish AcrIIA19 as a versatile Cas9 inhibitor and highlight the WED domain as a strategic target for developing species-agnostic CRISPR regulatory tools in biotechnology and therapeutic applications.},
}
@article {pmid41431195,
year = {2026},
author = {Spezzani, E and Capelli, L and Di Lena, D and Chamorro-Garcia, A and Ippodrino, R and Porchetta, A and Bertucci, A},
title = {MARPLE: A Proximity-Triggered CRISPR-Cas13 Platform for Ultrasensitive Antibody Detection.},
journal = {Advanced science (Weinheim, Baden-Wurttemberg, Germany)},
volume = {13},
number = {13},
pages = {e17799},
pmid = {41431195},
issn = {2198-3844},
support = {//National Recovery and Resilience Plan/ ; MUR 2023-2027//'Departments of Excellence' program of the Italian Ministry for University and Research/ ; 31108//Fondazione AIRC per la ricerca sul cancro ETS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Antibodies/analysis ; Immunoassay/methods ; },
abstract = {Monitoring clinically relevant antibodies-as biomarkers of disease or therapeutic response-is essential for informed clinical decision-making. Traditional immunoassays like ELISA offer reliable quantification but often involve multistep workflows and limited point-of-care utility. New approaches coupling antibody recognition with signal amplification are therefore highly desirable. The CRISPR-Cas13 system, known for its potent collateral cleavage activity, has emerged as a powerful diagnostic tool for nucleic acid detection. However, its application to protein biomarkers such as antibodies remains underdeveloped. Here, we introduce MARPLE (Modular Antibody Recognition via Proximity-triggered Linker Exchange), a modular CRISPR-Cas13-based platform for ultrasensitive antibody detection. MARPLE harnesses antibody-induced proximity to trigger a strand displacement reaction that releases a sequestered RNA target, activating Cas13-mediated collateral cleavage of fluorescent RNA reporters. This cascade enables detection of antibodies at femtomolar concentrations. We demonstrate MARPLE's versatility across diverse targets-including anti-digoxigenin, anti-cholesterol, anti-HA, trastuzumab, and anti-MUC1-highlighting applications in infectious disease monitoring, cancer diagnostics, and therapeutic drug tracking. The assay is isothermal, one-pot, and retains robust performance in complex matrices such as human serum. These features establish MARPLE as a promising tool for immunodiagnostics, extending CRISPR-based sensing beyond nucleic acids to protein biomarker detection.},
}
@article {pmid41431758,
year = {2026},
author = {Hwang, S and Ko, H and Lee, HY and Choi, J},
title = {Nanocarriers for the delivery of the CRISPR/Cas9 system.},
journal = {Nanomedicine (London, England)},
volume = {21},
number = {3},
pages = {429-448},
pmid = {41431758},
issn = {1748-6963},
support = {//Korean Government/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Humans ; Animals ; *Nanoparticles/chemistry ; *Gene Transfer Techniques ; *Drug Carriers/chemistry ; },
abstract = {The clustered regularly interspaced short palindromic repeat/associated protein 9 (CRISPR/Cas9) system has been used for the precise manipulation of target DNA, making efficient genome editing in cells a reality. The CRISPR/Cas9 system has shown great potential in biomedical applications, such as disease treatment, transcription regulation, and genome-wide screening, and is opening a new era in biotechnology. However, the efficient and selective delivery of the CRISPR/Cas9 system remains a critical obstacle. Literature search conducted using Web of Science, Scopus, PubMed and Google Scholar for articles published from 2015 to 2024. In this review, we discuss several delivery methods for the CRISPR/Cas9 system, focusing on techniques using nanocarriers. Specifically, we comprehensively discussed the challenges, future directions, and potential of various delivery methods for the CRISPR/Cas9 system.},
}
@article {pmid41431922,
year = {2026},
author = {Chen, K and Zhu, J and Fan, C and Zhou, A and Li, B and Ge, H and Ning, X},
title = {A Nanoimprinted Photothermal Chip for On-Demand Spatiotemporal Activation of CRISPR/Cas9 Gene Editing.},
journal = {Nano letters},
volume = {26},
number = {1},
pages = {532-542},
doi = {10.1021/acs.nanolett.5c05571},
pmid = {41431922},
issn = {1530-6992},
support = {//National Key Research and Development Program of China/ ; //National Natural Science Foundation of China/ ; //Natural Science Foundation of Jiangsu Province/ ; //Innovation Fund of China Acoustics Valley (Suzhou), the Key Research and Development Program of Science and Technology Innovation (Social Development) in Danyang City/ ; //Clinical Medicine Special Research Fund Project of Nantong University/ ; //the Guiding Science and Technology Plan Project of Social Development in Zhenjiang City/ ; //Jiangsu Funding Program for Excellent Postdoctoral Talent/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; Animals ; Mice ; Gold/chemistry ; Sheep ; Infrared Rays ; Humans ; },
abstract = {Precise control of gene editing in target cells is essential for CRISPR/Cas9 applications. Here, we present a nanoimprinted photothermal chip (NPC) engineered for on-demand delivery and activation of CRISPR/Cas9 complexes with high spatial and temporal precision. Fabricated by nanoimprint lithography and subsequent surface modification, NPC features a customized PEGylated plasmonic gold nanopillar array, which provides both optimal cellular adhesion and efficient photothermal conversion. Upon NIR irradiation, NPC generates spatially confined thermal microdomains that transiently permeabilizes cell membranes, thereby facilitating cytosolic delivery of CRISPR/Cas9 complexes and synchronously modulating genome-editing kinetics. In vitro studies demonstrate robust gene knockout in both mouse and sheep cell lines while preserving high cell viability and editing fidelity. Remarkably, NPC-mediated PD-1 gene disruption in cytotoxic T cells markedly enhance their antitumor activity. Overall, this work establishes NPC as a transformative platform for precise and controllable CRISPR/Cas9 gene editing with broad therapeutic potential.},
}
@article {pmid41432281,
year = {2026},
author = {Liu, XL and Liu, L and Cheng, L},
title = {Sequence-independent optical regulation of CRISPR/Cas editing using star-shaped crRNA dendrimers.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {6},
pages = {1877-1881},
doi = {10.1039/d5cc06011g},
pmid = {41432281},
issn = {1364-548X},
mesh = {*Dendrimers/chemistry ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Associated Proteins/metabolism/genetics ; *RNA/chemistry ; Humans ; Bacterial Proteins/metabolism/genetics ; Endodeoxyribonucleases ; },
abstract = {Precise spatiotemporal control of CRISPR/Cas editing is vital for studying dynamic processes and ensuring therapeutic safety. We present a single-site photolabile crRNA dendrimer platform enabling robust, sequence-independent optical regulation of Cas9 and Cas12a. This simple, universal strategy achieves rapid OFF-to-ON control with minimal leakage, advancing programmable, light-responsive genome editing for biomedical applications.},
}
@article {pmid41432353,
year = {2026},
author = {Tang, X and Ju, D and Hu, H},
title = {A Dual CRISPR-Cas/Cre-loxP Genome Engineering Strategy for Stable Uricase Expression in Food-Grade Probiotics.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {331-341},
doi = {10.1021/acssynbio.5c00774},
pmid = {41432353},
issn = {2161-5063},
mesh = {*Probiotics/metabolism ; *CRISPR-Cas Systems/genetics ; *Lactococcus lactis/genetics/enzymology ; Animals ; *Urate Oxidase/genetics/metabolism ; Gene Editing/methods ; Mice ; Integrases/genetics/metabolism ; Genome, Bacterial/genetics ; Genetic Engineering/methods ; },
abstract = {The development of robust, food-grade microbial chassis with tailored metabolic functions is critical for advancing synthetic biology applications in health and nutrition. Here, we report a dual genome engineering strategy that integrates CRISPR-Cas9-mediated knock-in with Cre/loxP-driven genome reduction to streamline the genome of Lactococcus lactis NZ9000 and enable stable expression of a high-activity uricase variant. The resulting strain, NZ9000::UA[T]-ΔD6, demonstrated enhanced enzymatic performance in vitro, achieving 2.34 U/mL activity and complete degradation of ∼500 μM urate within 20 h. Beyond improved catalytic output, this dual-system approach established a genetically stable and biosafe probiotic chassis with moderate colonization capacity in the murine gut. The integration of CRISPR-Cas and Cre/loxP techniques in this work is intended to enhance the expression of heterologous genes in the chassis strain, while providing a versatile platform for the rational design of food-grade probiotics and offering a general strategy for constructing living biotherapeutic agents with targeted metabolic activities.},
}
@article {pmid41432359,
year = {2026},
author = {Fruitet, E and de Fouchier, A and Heckel, DG and Groot, AT},
title = {Multiple CRISPR/Cas9 modifications of an esterase reveal its role in influencing acetate esters in the pheromone blend of a moth.},
journal = {Insect molecular biology},
volume = {35},
number = {2},
pages = {166-176},
doi = {10.1111/imb.70016},
pmid = {41432359},
issn = {1365-2583},
support = {//Instituut voor Biodiversiteit en Ecosysteem Dynamica, Universiteit van Amsterdam/ ; //Universiteit van Amsterdam/ ; //International Max Plank Research School, Max-Planck-Instituts f&#xfc;r chemische &#xd6;kologie/ ; //Max-Planck-Gesellschaft/ ; },
mesh = {*Moths/genetics/metabolism/enzymology ; Animals ; CRISPR-Cas Systems ; Female ; Male ; *Esterases/genetics/metabolism ; *Acetates/metabolism ; Esters/metabolism ; *Insect Proteins/genetics/metabolism ; *Sex Attractants/metabolism ; Quantitative Trait Loci ; },
abstract = {Sexual signalling by pheromones is essential for mate finding and mate choice in moths and plays an important role in reproductive isolation. Acetates (i.e., acetate esters) produced by females of Heliothis (Chloridea) subflexa Fabricius, 1777 (Lepidoptera: Noctuidae) attract conspecific males but repel Heliothis virescens Fabricius, 1777 (Lepidoptera: Noctuidae) males. A QTL (quantitative trait locus) harbouring carboxylesterases and lipases was previously shown to affect acetates, and CRISPR/Cas9-induced knockouts increased acetate amounts by blocking hydrolysis of the esters as expected. A second, unlinked QTL, containing a cluster of three different carboxylesterases (CXEs), unexpectedly yielded decreased acetate amounts. In one of these genes, esterase CXE24, we found a naturally occurring transposable element insertion in exon 8. A CRISPR/Cas9-induced frameshift at the same position yielded the same results. The paradox was resolved by a CRISPR/Cas9-induced frameshift in exon 2 of CXE24 which increased acetate amounts. The frameshift in exon 2 produced a truncated protein lacking the substrate binding site and the catalytic triad, while the frameshift in exon 8 removed only the third residue of the catalytic triad. In silico modelling showed that the exon-8-truncated protein could not hydrolyse the esters by itself, which likely explains the decreased acetate amounts. To place our findings in an evolutionary context, we explored variation in the esterase cluster in 16 species of Lepidoptera with completely sequenced genomes. Geographic and temporal variation in acetates has been observed in H. subflexa, and variation in the frequency of the transposable element could be a possible explanation.},
}
@article {pmid41432570,
year = {2026},
author = {Hu, J and Li, X and Gao, Y and Guo, Y and Liu, Y and Wang, C and Xu, G and Du, C and Liu, S and Zhao, Z and Wang, Y and Wu, Y and Dong, X and Li, C and Wan, J},
title = {Cas9-Embedding Hyperactive TadA8e Confers Efficient and Highly Specific A-To-G Base Editing in Rice.},
journal = {Plant biotechnology journal},
volume = {24},
number = {4},
pages = {2576-2591},
doi = {10.1111/pbi.70511},
pmid = {41432570},
issn = {1467-7652},
support = {2023ZD04074//the Biological Breeding-Major Projects/ ; 2023YFD1202900//the National Key Research and Development Program/ ; ZSBBL-KY2023-04//the Zhongshan Biological Breeding Laboratory/ ; NAUSY-ZZ03//the Guidance Foundation of the Sanya Institute of Nanjing Agricultural University/ ; BK20230038//the Jiangsu Province Natural Science Foundation/ ; //the Nanjing U35 program/ ; 2023AB006-02//the Bingtuan Key Science and Technology Program of Xinjiang Province/ ; KYT2024005//the Fundamental Research Funds for the Central Universities/ ; 31872806//the National Natural Science Foundation of China/ ; //Ministry of Education of the People's Republic of China/ ; },
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; *Adenosine Deaminase/genetics/metabolism ; Plants, Genetically Modified ; Plant Proteins/genetics/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {Adenine base editors (ABEs) produce precise A-to-G conversion in the genomic target sites without causing double-strand breaks. However, the hyperactive adenosine deaminase TadA8e raises safety concerns on genome-wide off-target edits. We engineered 11 chimeric proteins for ABEs (CP-ABEs) by embedding hyperactive TadA8e within Cas9 nickase to minimise the sgRNA-independent off-target effects. Four CP-ABEs exhibited robust on-target activity with minimal sgRNA-independent off-target edits. Then we developed four chimeric high-fidelity ABEs (CH-ABEs) to minimise both sgRNA-dependent and sgRNA-independent off-target effects by employing high-fidelity Cas9 variants. The CH-ABEs achieved reductions of up to 7.0-fold and 79.4-fold in the respective off-target edits, while generating 22.0%-72.4% homozygous and biallelic rice mutants. Whole-genome and whole-transcriptome sequencing (WGS/WTS) confirmed the specificity of CH-ABEs. Incorporating Sniper2L into CH-ABEs further enhanced both specificity and on-target activity. Two PAM-less SpRY variants (SpRY-K2, SpRY-KK) expanded the targeting scope of CP-ABEs and boosted activity by 80.0%. Furthermore, we demonstrated that CP-ABE8e-RY[KK] could discriminate paralogous targets in rice and successfully applied it to create herbicide-resistant rice by precisely installing the OsALS-K591E mutation.},
}
@article {pmid41433155,
year = {2026},
author = {Hemani, D and Grissom, JH and Chi, RJ},
title = {Protocol for marker-free genome editing in Saccharomyces cerevisiae using universal donor templates and multiplexed CRISPR-Cas9.},
journal = {STAR protocols},
volume = {7},
number = {1},
pages = {104280},
pmid = {41433155},
issn = {2666-1667},
mesh = {*Saccharomyces cerevisiae/genetics ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Genome, Fungal/genetics ; },
abstract = {Here, we present a protocol for marker-free genome editing in Saccharomyces cerevisiae by combining PCR-based selectable marker cassettes with CRISPR-Cas9. We describe steps for generating gene deletions using MX6 markers and excising the markers by introducing a reusable guide RNA (gRNA)-Cas9 plasmid and universal repair templates, allowing multiplex removal in a single step. Final verification by PCR yields marker-free strains that can be iteratively edited using the same selectable markers. For complete details on the use and execution of this protocol, please refer to Grissom et al.[1].},
}
@article {pmid41433963,
year = {2026},
author = {Schneider, O and Zehl, M and Miele, M and Pace, V and Brungs, C and Cheng, JF and Hummelbrunner, S and Dirsch, VM and Zotchev, SB},
title = {Heterologous Expression and CRISPR/Cas9-Assisted Manipulation of the Hybrid Gene Cluster Specifying the Biosynthesis of Meroterpenoids and Phenazines.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {137-148},
pmid = {41433963},
issn = {2161-5063},
mesh = {*Terpenes/chemistry/metabolism ; Phenazines/chemistry/metabolism ; CRISPR-Cas Systems/genetics ; Streptomyces/genetics/metabolism ; Anti-Bacterial Agents/biosynthesis ; },
abstract = {A hybrid gene cluster, mfq, predicted to govern the biosynthesis of both meroterpenoids and phenaziterpenes, was cloned from the genome of Streptomyces sp. S4.7 and introduced into the heterologous host Streptomyces coelicolor M1154. The biosynthesis of the meroterpenoids marfuraquinocins C and D, previously isolated from Streptomyces niveus SCSIO 3406, as well as a new congener, marfuraquinocin E, which exhibited antibacterial activity, was activated upon overexpression of the regulatory protein MfqF. However, production of neither phenaziterpenes nor phenazines was detected. The structure of marfuraquinocin E was elucidated, revealing the attachment of a terpene moiety at C-2, in contrast to C-6 as seen in the known congeners A-D. Using the CRISPR/Cas9 system, several genes in the mfq cluster were inactivated, confirming the role of MfqW as a prenyltransferase specific to the meroterpenoid pathway. Both gene overexpression and further knockouts provided the first insights into the complex regulation of this hybrid gene cluster. To restore the presumably deficient phenazine biosynthetic pathway, a gene encoding a PhzF homologue from another gene cluster in S4.7 was heterologously expressed alongside the mfq cluster, leading to the production of 1,6-phenazine dicarboxylic acid upon MfqF overexpression. This work lays the foundation for elucidating the complete biosynthetic pathway of marfuraquinocins and its potential coregulation with that of phenazines.},
}
@article {pmid41435708,
year = {2026},
author = {Fang, J and Chen, Q and Ran, M and Chen, R and Chen, W and Cui, J and Wang, J and Zhong, K and Shi, L and Lu, C and Jiang, H},
title = {RPA-CRISPR/Cas12a-coupled microfluidic biosensor enabling on-site, sensitive quantification of Vibrio parahaemolyticus.},
journal = {Biosensors &amp; bioelectronics},
volume = {296},
number = {},
pages = {118327},
doi = {10.1016/j.bios.2025.118327},
pmid = {41435708},
issn = {1873-4235},
mesh = {*Vibrio parahaemolyticus/isolation &amp; purification/genetics/pathogenicity ; *Biosensing Techniques/instrumentation ; *Nucleic Acid Amplification Techniques/instrumentation ; Limit of Detection ; *CRISPR-Cas Systems/genetics ; Animals ; Seafood/microbiology ; Lab-On-A-Chip Devices ; *Vibrio Infections/microbiology/diagnosis ; Equipment Design ; },
abstract = {Vibrio parahaemolyticus is a major cause of seafood-associated gastroenteritis and aquatic animal diseases, posing persistent threats to public health and aquaculture. Rapid and accurate on-site quantitative detection is essential for risk assessment and early intervention. Although qPCR and digital PCR provide reliable quantification, their reliance on complex instrumentation limits field deployment. RPA-CRISPR-based isothermal assays offer a low-equipment alternative; however, existing approaches lack robust methodological strategies to achieve standard-curve-based quantitative reliability under field-deployable conditions. Here, we propose a methodological framework that enables in-run calibration and variance control for quantitative isothermal amplification by integrating reaction-volume locking and simultaneous standard-curve generation, implemented here within a closed centrifugal microfluidic system. A multi-unit microfluidic platform preloaded with gradient concentration standard plasmids allows concurrent construction of standard curves and sample analysis in a single run, reducing the impact of environmental and batch-to-batch variability. Signal generation was achieved using a one-pot RPA-CRISPR/Cas12a assay, in which balanced amplification and cleavage kinetics were obtained by screening crRNAs targeting suboptimal PAM sites and optimizing reaction conditions. The platform achieved a detection limit of 6.08 copies/μL and a linear quantitative range of 10[0]-10[4] copies/μL (R[2] > 0.96), with performance comparable to qPCR (AUC = 0.984), and acceptable intra- and inter-assay variability under the tested conditions, with relative standard deviations of 2.63-6.07 %, at a cost of approximately $3.30 per test. Validation using spiked and real seafood samples demonstrated reliable on-site quantification. This work establishes a transferable quantitative methodology for RPA-CRISPR-based isothermal assays, advancing field-deployable pathogen detection in aquaculture and food safety.},
}
@article {pmid41435996,
year = {2026},
author = {Gopalakrishnan, R and Kannan, K and Gunasekaran, R and Ramachandran, P and Ganapathy, D and Pitchiah, S},
title = {A comparative review of vector insertion techniques in Saccharomyces cerevisiae.},
journal = {Journal of microbiological methods},
volume = {241},
number = {},
pages = {107378},
doi = {10.1016/j.mimet.2025.107378},
pmid = {41435996},
issn = {1872-8359},
mesh = {CRISPR-Cas Systems ; Gene Editing/methods ; *Genetic Engineering/methods ; *Genetic Vectors/genetics ; Genome, Fungal ; Homologous Recombination ; *Mutagenesis, Insertional/methods ; *Saccharomyces cerevisiae/genetics ; Synthetic Biology/methods ; },
abstract = {Saccharomyces cerevisiae, a model organism in genetics and molecular biology has been extensively engineered using various vector insertion techniques. This review compares and contrasts three prominent techniques: In vivo homologous recombination (HR), Cre-lox recombination and CRISPR/Cas9. In vivo HR leverages the organism's innate DNA repair machinery for easy vector integration and targeted genome modifications. Cre-lox recombination offers high specificity and efficiency at loxP sites, making it ideal for targeted gene excision or integration. CRISPR/Cas9 has revolutionized genome engineering with its precision and ability to target multiple loci simultaneously. Each technique has its strengths and limitations, including site dependency, off-target effects, and strain-specific variability. This review provides a comprehensive overview of these vector insertion techniques, highlighting their applications, advantages, and limitations in S. cerevisiae genome engineering and synthetic biology.},
}
@article {pmid41436498,
year = {2025},
author = {Jiang, J and Jiang, Z and Luo, Q and Chen, X and Zhuang, J and Chen, J and Mu, Q and Qiu, J and Li, Y and Chen, S and Zhang, P and Yu, K and Chen, S and Liu, GS and Zhuang, J},
title = {Loss of ELF2 drives topotecan resistance in retinoblastoma revealed by genome-wide CRISPR-Cas9 screening.},
journal = {Cell death &amp; disease},
volume = {17},
number = {1},
pages = {128},
pmid = {41436498},
issn = {2041-4889},
support = {82472143//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82372131//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024A1515012562//Natural Science Foundation of Guangdong Province (Guangdong Natural Science Foundation)/ ; GNT2029648//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {*Topotecan/pharmacology/therapeutic use ; Humans ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Retinoblastoma/genetics/drug therapy/pathology/metabolism ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; Cell Line, Tumor ; Topoisomerase I Inhibitors/pharmacology ; Xenograft Model Antitumor Assays ; Apoptosis/drug effects ; *Retinal Neoplasms/genetics/drug therapy/pathology ; Female ; },
abstract = {The topoisomerase I inhibitor topotecan is an effective chemotherapeutic agent for retinoblastoma; however, treatment resistance remains a major clinical challenge, and its mechanisms remain elusive. Using genome-wide CRISPR-Cas9 knockout screening, we identified ELF2 as a key gene involved in topotecan resistance. Here, we show that surviving retinoblastoma cells exposed to topotecan showed progressively decreased ELF2 expression, accompanied by reduced apoptosis. In a mouse xenograft model, ELF2 disruption diminished the antitumor efficacy of topotecan, with ELF2-knockout cells exhibiting reduced topotecan-induced apoptosis. RNA sequencing further revealed that the MT-CYB pathway, associated with ATP synthesis, contributes to ELF2-mediated resistance. Importantly, clinical analysis demonstrated a correlation between ELF2 expression and tumor volume in retinoblastoma patients treated with topotecan. Together, these findings interrogate the mechanisms underlying topotecan resistance in retinoblastoma and suggest ELF2 as a potential therapeutic target to overcome drug resistance.},
}
@article {pmid41436729,
year = {2025},
author = {Wu, Y and Cai, Z and Cross, D and Noble, JR and Prest, K and Littleboy, J and Cohen, SB and Edlundh, B and Koh, JMS and Xu, R and Noor, Z and Bastami, M and Valentini, S and Richardson, L and Barthorpe, S and Arymanesh, N and Robinson, PJ and Hains, PG and Garnett, MJ and Zhong, Q and Reddel, RR and MacKenzie, KL},
title = {Large-scale drug sensitivity, gene dependency, and proteogenomic analyses of telomere maintenance mechanisms in cancer cells.},
journal = {Nature communications},
volume = {16},
number = {1},
pages = {11337},
pmid = {41436729},
issn = {2041-1723},
support = {IIRS-18-164//National Breast Cancer Foundation (NBCF)/ ; GNT1170739//Department of Health | National Health and Medical Research Council (NHMRC)/ ; RG24-05//Cancer Council NSW (Cancer Council New South Wales)/ ; 2017/TPG001, REG171150//Cancer Institute NSW (Cancer Institute New South Wales)/ ; /WT_/Wellcome Trust/United Kingdom ; 206194/WT_/Wellcome Trust/United Kingdom ; CMP-01//NSW Ministry of Health (NSW Health)/ ; GNT1170739, H2020-SC1-DTH-2018-1//Department of Health | National Health and Medical Research Council (NHMRC)/ ; },
mesh = {Humans ; *Neoplasms/genetics/drug therapy/metabolism ; *Telomere Homeostasis/genetics/drug effects ; Cell Line, Tumor ; *Telomere/metabolism/genetics ; Telomerase/metabolism/genetics ; *Proteogenomics/methods ; CRISPR-Cas Systems ; *Antineoplastic Agents/pharmacology ; Proteomics ; Drug Resistance, Neoplasm/genetics ; },
abstract = {Replicative immortality is a hallmark of cancer, driven by the activation of telomere maintenance mechanisms, that is yet to be therapeutically exploited. To expedite discoveries that will enable the development of therapeutics that target telomere maintenance mechanisms, this study provides a resource of telomere biology metrics for a pan-cancer panel of 976 cell lines. We generate proteomic data from data-independent-acquisition mass spectrometry for most of these cell lines and integrate pre-existing multi-omic, drug sensitivity, and molecular dependency data from CRISPR/Cas9 knock-out screens. The data illustrate a broad range and heterogeneity in telomere biology, including states that diverge from the binary model of telomere maintenance activation involving either telomerase or the Alternative Lengthening of Telomeres mechanism. Using the telomere biology metrics and multi-omic data, we derive proteomic and transcriptomic predictors of Alternative Lengthening of Telomeres and telomerase activity levels. Our investigations also reveal molecular vulnerabilities associated with the Alternative Lengthening of Telomeres mechanism and drug sensitivity correlating with telomerase activity levels. These findings illustrate opportunities for leveraging this resource to realize the potential for telomere biology-directed cancer therapeutics and companion diagnostics.},
}
@article {pmid41439322,
year = {2026},
author = {Wang, Z and Yang, F and Zeng, S and Sun, R and Hu, Q and Du, Y},
title = {An integrated valved microfluidic platform for rapid and simultaneous nucleic acid detection.},
journal = {Lab on a chip},
volume = {26},
number = {2},
pages = {507-514},
doi = {10.1039/d5lc01096a},
pmid = {41439322},
issn = {1473-0189},
mesh = {Humans ; *Lab-On-A-Chip Devices ; *Nucleic Acid Amplification Techniques/instrumentation ; Human papillomavirus 16/genetics/isolation &amp; purification ; Human papillomavirus 18/genetics/isolation &amp; purification ; *DNA, Viral/analysis/genetics ; *Nucleic Acids/analysis ; *Microfluidic Analytical Techniques/instrumentation ; CRISPR-Cas Systems ; },
abstract = {Applying CRISPR-based diagnostics to point-of-care pathogen detection remains challenging because of the multi-step and time-consuming sample preparation process. This study presents a low-cost, integrated valved microfluidic device that combines recombinase polymerase amplification (RPA), CRISPR signal amplification, and lateral flow readout for simultaneous nucleic acid detection. The core advantage of the platform lies in its ability to sequentially control the entire multi-step assay through simple valve operation, significantly minimizing user intervention. All key reagents, including the RPA mix, Cas12a/crRNA complex, and proteinase K lysis buffer, are pre-lyophilized, ensuring stability and ready-to-use functionality. The platform demonstrates a sensitivity of 20 copies/reaction for HPV16/18 plasmids and accurately genotypes HPV in lysates of cervical cancer cells within one hour, showing complete concordance with quantitative PCR results. This integrated device, achieving a user-friendly protocol and visual readout, provides a powerful tool for nucleic acid-based point-of-care testing and self-testing in resource-limited settings.},
}
@article {pmid41439415,
year = {2026},
author = {Zhao, R and Wan, P and Huang, H and Li, Q and Zeng, Z and Xiong, W},
title = {Harnessing CRISPR-Cas9 and CRISPRi systems to reverse antibiotic resistance in a clinical multidrug-resistant Escherichia coli isolate.},
journal = {The Journal of antimicrobial chemotherapy},
volume = {81},
number = {1},
pages = {},
doi = {10.1093/jac/dkaf442},
pmid = {41439415},
issn = {1460-2091},
support = {2022YFD1800400//National Key R&amp;D Program/ ; },
mesh = {*Escherichia coli/drug effects/genetics/isolation &amp; purification ; *CRISPR-Cas Systems ; *Drug Resistance, Multiple, Bacterial/genetics ; Microbial Sensitivity Tests ; *Anti-Bacterial Agents/pharmacology ; Escherichia coli Proteins/genetics ; Escherichia coli Infections/microbiology ; Humans ; ATP-Binding Cassette, Sub-Family C Proteins/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Deletion ; },
abstract = {OBJECTIVES: To evaluate the CRISPR-Cas9 and CRISPR interference (CRISPRi) systems as an antibiotic re-sensitization strategy for reversing multidrug resistance in a clinical Escherichia coli isolate.<br><br>MATERIALS AND METHODS: The CRISPR-Cas9 system was applied for precise deletion of the acrB gene from clinical E. coli isolate GP53 and homologous recombination (HR) was provided for accurate repairs of double-strand breaks. An arabinose-inducible CRISPRi system was developed and optimized using fluorescent reporter strain GH01. Multiple guide RNAs (gRNAs) targeting acrB were designed, and the most effective gRNA was selected based on its transcriptional suppression of gene acrB. The minimum inhibitory concentrations (MICs) of selected antibiotics in GP53&#x394;acrB, CRISPRi strains, WT and WT combined with efflux pump inhibitor PA&#x3b2;N were evaluated.<br><br>RESULTS: The CRISPR-Cas9 system precisely deleted the acrB gene in clinical E. coli isolate GP53 with 11.46% knockout efficiency. The constructed arabinose-inducible CRISPRi system effectively repressed fluorescent protein expression in strain GH01. Although dCas9 expression increased with L-arabinose concentration, the transcriptional repression efficiency of the target gene under 1&#x2005;mM induction reached a significant inhibitory level. The CRISPRi system targeting gene acrB exhibited 44.9%, 5.4% and 23.5% inhibition rates on the transcriptional levels with 1&#x2005;mM L-arabinose for three distinct gRNAs. Both the knockout and CRISPRi strains successfully restored susceptibility of the multidrug-resistant E. coli GP53 to quinolones and tetracyclines, outperforming the effect of PA&#x3b2;N combination therapy.<br><br>CONCLUSIONS: In this study, CRISPR-based systems effectively reversed multidrug resistance in a clinical E. coli isolate, advancing the applications of CRISPR systems in controlling bacterial multidrug resistance.},
}
@article {pmid41440288,
year = {2025},
author = {Hwang, SB and Song, YJ and Park, PG},
title = {A Novel Diagnostic Tool for West Nile Virus Lineage 1a and 2 Using a CRISPR-Cas12a System.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440288},
issn = {2079-6374},
support = {GCU-202502820001//Gachon University/ ; },
mesh = {*West Nile virus/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques ; *West Nile Fever/diagnosis/virology ; Animals ; Humans ; },
abstract = {The West Nile Virus (WNV), transmitted by Culex mosquitoes as a major vector, has been reported worldwide. Also, West Nile neuroinvasive disease (WNND) caused by WNV lineage 1a and 2 neuroinvasive infections has been constantly reported with high fatality rates. Nevertheless, there are no treatments and vaccinations, so diagnosis in the early stages is important. Recently, a molecular diagnostic technique using DNA endonuclease-targeted CRISPR trans reporter (DETECTR) with the CRISPR-Cas12a system integrated with isothermal nucleic acid amplification has newly emerged. In this study, we designed a 2-Step WNV DETECTR with reverse transcription-recombinase polymerase amplification (RT-RPA) for rapid and sensitive WNV diagnosis. It successfully detected down to 1.0 × 10[2] RNA copies for both WNV lineage 1a and 2 with demonstrating similar sensitivity to qRT-PCR without cross-reactivity to other viruses. Additionally, we designed a 1-Step WNV DETECTR, incorporating all processing steps into a single tube, capable of detecting down to 1.0 × 10[3] RNA copies for both lineages. Furthermore, we developed a more streamlined method, the 1-Step with Filter WNV DETECTR, which achieved detection limits comparable to the 2-Step method, while reducing the processing time by 5 min. This study also explored the potential of the Punch-it™ NA-Sample Kit as an efficient alternative lysis method by comparing the detection differences across various lysis methods. Through this method, we achieved rapid and simple amplification and detection processes suitable for field diagnostics with high specificity and sufficient sensitivity. Therefore, DETECTR methods presented themselves as promising alternatives to conventional diagnostic tools, potentially overcoming financial and technical constraints in diverse medical settings.},
}
@article {pmid41440293,
year = {2025},
author = {Safenkova, IV and Kamionskaya, MV and Sotnikov, DV and Biketov, SF and Zherdev, AV and Dzantiev, BB},
title = {Advancing Lateral Flow Detection in CRISPR/Cas12a Systems Through Rational Understanding and Design Strategies of Reporter Interactions.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440293},
issn = {2079-6374},
support = {25-16-00246//Russian Science Foundation/ ; 1.1.13//State assignment of the State Research Center of Applied Microbiology and Biotechnology/ ; },
mesh = {*CRISPR-Cas Systems ; *Biosensing Techniques/methods ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Kinetics ; },
abstract = {CRISPR/Cas12a systems coupled with lateral flow tests (LFTs) are a promising route to rapid, instrument-free nucleic acid diagnostics due to conversion target recognition into a simple visual readout via cleavage of dual-labeled single-stranded DNA reporters. However, the conventional CRISPR/Cas12a-LFT system is constructed in a format where the intact reporter should block nanoparticle conjugate migration and can produce false-positive signals and shows strong dependence on component stoichiometry and kinetics. Here, we present the first combined experimental and theoretical analysis quantifying these limitations and defining practical solutions. The experimental evaluation included 480 variants of LFT configuration with reporters differing in the concentration of interacting components and the kinetic conditions of the interactions. The most influential factor leading to 100% false-positive results was insufficient interaction time between the components; pre-incubation of the conjugate with the reporter for 5 min eliminated these artifacts. Theoretical analysis of the LFT kinetics based on a mathematical model confirmed kinetic constraints at interaction times below a few minutes, which affect the detectable signal. Reporter concentration and conjugate architecture represented the second major factors: lowering reporter concentration to 20 nM and using smaller gold nanoparticles with multivalent fluorescent reporters markedly improved sensitivity. The difference in sensitivity between various LFT configurations exceeded 50-fold. The combination of identified strategies eliminated false-positive reactions and enabled the detection of up to 20 pM of DNA target (the hisZ gene of Erwinia amylovora, a bacterial phytopathogen). The strategies reported here are general and readily transferable to other DNA targets and CRISPR/Cas12a amplification-free diagnostics.},
}
@article {pmid41440302,
year = {2025},
author = {Chen, J and Liu, S and Chen, S and Mai, J and Abudukadi, M and Chen, Y and Lu, J and Li, G and Ge, C},
title = {Rapid Visual Detection of Mycoplasma Hominis Using an RPA-CRISPR/Cas12a Assay.},
journal = {Biosensors},
volume = {15},
number = {12},
pages = {},
pmid = {41440302},
issn = {2079-6374},
mesh = {*Mycoplasma hominis/isolation &amp; purification/genetics ; *Biosensing Techniques ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques ; Humans ; RNA, Ribosomal, 16S/genetics ; },
abstract = {Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor's response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.},
}
@article {pmid41441637,
year = {2025},
author = {Meng, Y and Chen, J and Liu, L},
title = {Functional Coupling and Evolutionary Relationships Between Toxin-Antitoxin Systems and CRISPR-Cas Systems.},
journal = {Toxins},
volume = {17},
number = {12},
pages = {},
pmid = {41441637},
issn = {2072-6651},
support = {20720250095//Fundamental Research Funds for the Central Universities/ ; 20720240046//Fundamental Research Funds for the Central Universities/ ; 32371346//National Natural Science Foundation of China/ ; 32301007//National Natural Science Foundation of China/ ; 2024J011007//Natural Science Foundation of Fujian Province/ ; 2023J01023//Natural Science Foundation of Fujian Province/ ; 2023J05008//Natural Science Foundation of Fujian Province/ ; },
mesh = {*CRISPR-Cas Systems ; *Toxin-Antitoxin Systems/genetics ; *Evolution, Molecular ; *Bacteria/genetics/metabolism ; Bacterial Proteins/genetics/metabolism ; *Bacterial Toxins/genetics ; },
abstract = {Bacteria encode a broad range of survival and defence systems, including CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas systems, restriction-modification systems, and toxin-antitoxin (TA) systems, which are involved in bacterial regulation and immunity. The traditional view holds that CRISPR-Cas systems and TA systems are two independent defense lines in prokaryotes. However, groundbreaking studies in recent years have revealed multi-level functional coupling between them. This review systematically elaborates on this mechanism, focusing on three types of TA systems that mediate the core correlation of CRISPR-Cas systems: CreTA maintains the evolutionary stability of CRISPR-Cas systems through an addiction mechanism; CreR enables self-regulation of CRISPR-Cas expression; and CrePA provides herd immunity by triggering abortive infection after the CRISPR-Cas system has been destroyed by Anti-CRISPRS protein. Additionally, we discuss the evolutionary homology between the type III toxin AbiF and the type VI CRISPR effector Cas13, offering a new perspective for understanding the origin of CRISPR-Cas systems. These findings not only reveal the functional coupling of prokaryotic defense systems but also provide a powerful theoretical framework and practical solutions for addressing stability challenges in CRISPR technology applications.},
}
@article {pmid41441922,
year = {2025},
author = {Govender, P and Ghai, M and Karpoormath, R},
title = {Advances in biosensors for bacterial detection and identification.},
journal = {World journal of microbiology &amp; biotechnology},
volume = {42},
number = {1},
pages = {6},
pmid = {41441922},
issn = {1573-0972},
support = {PMDS2205108850//National Research Foundation/ ; },
mesh = {*Biosensing Techniques/methods ; Humans ; *Bacteria/isolation &amp; purification/genetics/classification ; Artificial Intelligence ; Wearable Electronic Devices ; Bacterial Infections/diagnosis/microbiology ; },
abstract = {Bacterial detection and identification is paramount as it plays a key role in safeguarding human health, food safety and security. Over the past decade, biosensors have emerged as a powerful tool for bacterial detection due to their ability to provide rapid, sensitive, specific and cost-effective monitoring of bacteria. Biosensors rely on the interaction between the target analyte and biological recognition elements, which triggers a measurable signal that can be quantified, thus enabling the detection of bacteria. In recent years, nanoparticles have become a focal point in biosensor research due to their unique physical and chemical properties, enhancing their sensitivity, specificity and functionality. Artificial intelligence, microfluidics and wearable biosensor technologies are shaping the next-generation real-time bacterial monitoring tools. AI-based biosensors interpret complex biological signals and provide automated detection of bacterial pathogens. Similarly, wearable biosensors are emerging as a promising option for non-invasive detection and monitoring of wound infections. Additionally, the integration of CRISPR/Cas systems into biosensing platforms has revolutionized molecular diagnostics by enabling highly specific detection of pathogenic bacteria. In forensic sciences, biosensors are being explored for the identification of body fluids based on their unique bacterial signatures, which can assist in crime scene reconstruction and post-mortem interval estimation. Most studies that have reported on biosensors for detection of bacteria, have targeted a single analyte or bacterial species. Given the growing interest and demand for multiplexed biosensors, future research should focus on developing biosensors capable of detecting multiple bacteria simultaneously, without compromising the accuracy. Biosensors with dual functionality will be instrumental in providing an integrated solution to detect, manage and control bacterial pathogens, thereby mitigating any potential threat to human health.},
}
@article {pmid41442816,
year = {2026},
author = {Zhu, Y},
title = {The potential and innovative applications of CRISPR gene editing technology in enzyme gene development.},
journal = {Enzyme and microbial technology},
volume = {195},
number = {},
pages = {110799},
doi = {10.1016/j.enzmictec.2025.110799},
pmid = {41442816},
issn = {1879-0909},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems ; Humans ; *Enzymes/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {The CRISPR gene editing technology is simple in design and highly efficient, making it the most widely used gene editing tool today. At present, CRISPR gene editing technology has shown a certain application value in enzyme development, but its application potential has not been fully developed. CRISPR gene editing technology can not only be used to knockin enzyme genes and knockout genes that are not conducive to enzyme expression, but can also be applied to single-base editing of enzyme genes, tandem sgRNA for multi-enzyme gene editing, sgRNA library for enzyme screening, endogenous enzyme gene modification, transcriptional activation or inhibition of enzyme gene expression, and fluorescence imaging of enzyme genes. Especially, this review innovatively proposes for the first time that CRISPR gene editing technology can be used for site specific fusion of enzyme genes, cell surface display of endogenous enzymes, and knockin of super long DNA for simultaneous expression of multiple enzymes, providing new ideas for maximizing the value of CRISPR gene editing technology in enzyme development in the future.},
}
@article {pmid41443126,
year = {2026},
author = {Rahimi, A and Rahimmanesh, I and Abedpoor, N and Boshtam, M and Bidram, E and Javanmard, SH and Khanahmad, H and Rafiee, L and Bigham, A and Rafienia, M and Karbasi, S and Shariati, L},
title = {The MCM/Lys-Cys nanodevices for the efficient gene delivery: An approach towardsMCP1gene manipulation using CRISPR technology.},
journal = {Colloids and surfaces. B, Biointerfaces},
volume = {260},
number = {},
pages = {115377},
doi = {10.1016/j.colsurfb.2025.115377},
pmid = {41443126},
issn = {1873-4367},
mesh = {Humans ; *Silicon Dioxide/chemistry ; *Gene Transfer Techniques ; *Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Chemokine CCL2/genetics ; Cell Proliferation ; Cell Line, Tumor ; Female ; *Cysteine/chemistry ; Breast Neoplasms/genetics/pathology/therapy ; *Lysine/chemistry ; Cell Movement ; Particle Size ; Surface Properties ; Gene Editing ; Plasmids/genetics ; },
abstract = {Breast cancer continues to be the most common malignancy among women worldwide, requiring novel therapeutic approaches. This research investigates an innovative gene delivery strategy employing mesoporous silica nanoparticles (MCM-41) modified with lysine and cysteine (Lys-Cys) for the effective delivery of CRISPR-Cas9 plasmids aimed at the monocyte chemoattractant protein-1 (MCP-1/CCL2) gene. Bioinformatics analysis of the TCGA-BRCA dataset revealed substantial deregulation of CCL2 in breast cancer, underscoring its involvement in tumor growth and inflammation. The MCM/Lys-Cys nanocarrier demonstrated remarkable biocompatibility and effectively encapsulated a plasmid containing GFP, promoting superior cellular uptake in MDA-MB-231 breast cancer cells compared to conventional techniques. Functional experiments demonstrated that CRISPR/Cas9-mediated suppression of CCL2 markedly decreased cell proliferation, migration, and invasion, highlighting the promise of this targeted gene therapy strategy in breast cancer management. The findings indicate that the MCM/Lys-Cys nanosystem presents a viable non-viral approach for precise gene editing, potentially boosting therapeutic efforts against breast cancer by modulating inflammatory pathways.},
}
@article {pmid41443566,
year = {2026},
author = {Ghosh, P and Wadsworth, BC and Terry, L and Evans, TA},
title = {Evolutionary conservation of midline axon guidance activity between Drosophila and Tribolium Frazzled.},
journal = {Developmental biology},
volume = {531},
number = {},
pages = {1-9},
doi = {10.1016/j.ydbio.2025.12.015},
pmid = {41443566},
issn = {1095-564X},
mesh = {Animals ; *Axon Guidance/physiology/genetics ; *Tribolium/embryology/genetics/metabolism ; *Drosophila Proteins/genetics/metabolism ; *Drosophila melanogaster/embryology/genetics/metabolism ; Axons/metabolism ; Signal Transduction ; Netrin Receptors/genetics/metabolism ; Gene Expression Regulation, Developmental ; Biological Evolution ; CRISPR-Cas Systems ; },
abstract = {The regulation of midline crossing of axons is of fundamental importance for the proper development of nervous system connectivity in bilaterian animals. A number of conserved axon guidance signaling pathways coordinate to attract or repel axons at the nervous system midline to ensure the proper regulation of midline crossing. The attractive Netrin-Frazzled/DCC (Net-Fra) signaling pathway is widely conserved among bilaterians, but it is not clear whether the mechanisms by which Net and Fra promote midline crossing are also conserved. In Drosophila, Fra can promote midline crossing via Netrin-dependent and Netrin-independent mechanisms, by acting as a canonical midline attractive receptor and also through a non-canonical pathway to inhibit midline repulsion via transcriptional regulation. To examine the conservation of Fra-dependent axon guidance mechanisms among insects, in this paper we compare the midline attractive roles of the Frazzled receptor in the fruit fly (Drosophila melanogaster) and flour beetle (Tribolium castaneum) using CRISPR/Cas9-mediated gene editing. We replace the Drosophila fra gene with sequences encoding Drosophila Fra (DmFra) or Tribolium Fra (TcFra) and examine midline crossing of axons in the ventral nerve cord of embryos carrying these modified alleles. We show that Tribolium Fra can fully substitute for Drosophila Fra to promote midline crossing of axons in the embryonic nervous system, suggesting that the mechanisms by which Frazzled regulates midline axon guidance are evolutionarily conserved within insects.},
}
@article {pmid41444327,
year = {2025},
author = {Oh, D and Seok, C and Park, HW and Park, S and Lee, J and Choi, H and Jawad, A and Ham, J and Jang, H and Lee, SC and Oh, BC and Moon, C and Park, KH and Hyun, SH and Kim, D},
title = {Generation and ophthalmological characterization of oculocutaneous albinism type 1 pig models by selection-free genome editing.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {44564},
pmid = {41444327},
issn = {2045-2322},
support = {RS-2025-00518006//National Research Foundation of Korea/ ; 2020R1A2C2101714//National Research Foundation of Korea/ ; RS-2024-00398561 , RS-2024-00399475//Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries/ ; 20023068//Ministry of Agriculture, Food and Rural Affairs/ ; HR22C1363//Korea Health Industry Development Institute/Republic of Korea ; },
mesh = {Animals ; *Albinism, Oculocutaneous/genetics/pathology ; *Gene Editing/methods ; Disease Models, Animal ; Swine ; Monophenol Monooxygenase/genetics ; CRISPR-Cas Systems ; Humans ; Electroretinography ; Phenotype ; Melanins/metabolism ; },
abstract = {Oculocutaneous albinism type 1 (OCA1) is an autosomal recessive disorder caused by mutations in the tyrosinase (TYR) gene, resulting in melanin deficiency and severe visual impairments. Although mouse models provide insights into OCA1 pathogenesis, they exhibit significant anatomical and physiological differences from humans, particularly in ocular structure and function, thereby limiting their ability to recapitulate human OCA1 phenotypes. Therefore, in this study, we generated a porcine OCA1 model by selection-free genome editing via somatic cell nuclear transfer to characterize ophthalmological features and evaluate their translational relevance to human OCA1. Our approach utilized TYR-targeting CRISPR/Cas9 ribonucleoproteins without the need for single-cell-derived clonal expansion, thus streamlining the generation process. After somatic cell nuclear transfer with TYR knockout donor cells, the embryos demonstrated normal in vitro embryonic development comparable to the control, resulting in four healthy OCA1 piglets that exhibited characteristic OCA1 phenotypes with complete melanin loss in ocular and cutaneous tissues. Comprehensive ophthalmological analyses revealed significant structural abnormalities, including marked reduction in retinal layer thickness and elevated intraocular pressure. Remarkably, electroretinography revealed selective impairment of the rod bipolar pathway with reduced b-wave amplitudes and increased oscillatory potentials, indicating disturbances in synaptic processing. Overall, our study demonstrates the efficiency and reliability of selection-free genome editing for generating porcine OCA1 models. Moreover, the ophthalmological findings provide valuable insights for exploring retinal dysfunction and pigmentation mechanisms and advancing the preclinical evaluation of potential therapeutic interventions for human OCA1.},
}
@article {pmid41444344,
year = {2025},
author = {Li, X and Guo, J and Yang, H and Wu, Y and Xie, Z and Li, D},
title = {A CRISPR-assisted passive microfluidic chip for rapid, visual detection of multiple respiratory viruses.},
journal = {Scientific reports},
volume = {16},
number = {1},
pages = {2033},
pmid = {41444344},
issn = {2045-2322},
support = {GJHZ20220913143207014//Science and Technology Innovation Commission of Shenzhen/ ; 12274197//China National Natural Science Fund/ ; 2022B1515020093//Guangdong Scientific and Technological Project/ ; },
mesh = {Humans ; SARS-CoV-2/genetics/isolation &amp; purification ; *Lab-On-A-Chip Devices ; *CRISPR-Cas Systems ; COVID-19/diagnosis/virology ; *Respiratory Tract Infections/virology/diagnosis ; Coinfection/diagnosis/virology ; Nucleic Acid Amplification Techniques ; Influenza B virus/genetics/isolation &amp; purification ; Influenza A virus/genetics/isolation &amp; purification ; },
abstract = {In recent years, viral co-infections, particularly with respiratory viruses, have resulted in more complex symptoms, a greater disease burden, and increased challenges in clinical decision-making. These complexities underscore the urgent need for improved diagnostic tools in the managing acute respiratory infections. To address the limitations of conventional qPCR and current POCT methodologies, we developed a passively driven microfluidic chip capable of rapidly screening multiple respiratory viruses. This platform is particularly suited for the point-of-care diagnosis of viral co-infections. Our device integrates nucleic acid amplification and CRISPR-based detection within a single, passively operated system. By utilizing a rapid, 10-minute sample preparation protocol and a 35-minute on-chip assay, this platform enables the multiplex detection of influenza A/B, human parainfluenza virus, and SARS-CoV-2. The total assay time from sample to answer is approximately 45 min, with equipment requirements minimized to a heating block. The assay demonstrated a detection sensitivity of about 10 copies/µL for viral RNA in dilution series experiments. The sensitivity of the assay was 98.44% (95% CI: 91.6%-99.96%), and the specificity was 100% (95% CI: 79.4%-100%). The system combines CRISPR-Cas12a-mediated sensing with reverse transcription recombinase polymerase amplification (RPA) for highly specific nucleic acid detection. The chip design utilizes capillary action and gravity-driven flow for autonomous fluid control, while lyophilized reagent preloading ensures storage stability and minimizes user intervention.},
}
@article {pmid41445188,
year = {2026},
author = {Shmuel-Eidelman, M and Cohen-Fultheim, R and Eisenberg, E and Levanon, EY},
title = {Off-target RNA editing hotspots caused by base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {2361-2371},
pmid = {41445188},
issn = {1525-0024},
mesh = {*RNA Editing ; Humans ; *Gene Editing/methods ; CRISPR-Cas Systems ; Algorithms ; RNA, Guide, CRISPR-Cas Systems/genetics ; Exons ; },
abstract = {Base editors, composed of engineered deaminases fused with Cas proteins and a guide RNA, enable precise, programmable alteration of single nucleotides within the genome and transcriptome. This innovative technology holds promising therapeutic potential for correcting disease-causing point mutations. However, its clinical translation hinges on both high efficacy and accuracy. Non-specific unintended edits by base editors remain a critical challenge. Efforts to mitigate off-target activity have focused mostly on detecting recurrent RNA deaminations at specific sites. Complementarily, our methodology quantifies the total burden of RNA alterations, which is particularly effective for capturing stochastic off-target edits that evade conventional detection. Here, we applied the RNA editing index algorithm to quantify off-target levels across individual genes and identified 2,844 adenine base editors and 1,253 cytosine base editor hotspot genes susceptible to aberrant editing. Exon-level analysis revealed localized regions within genes that are particularly prone to off-target editing, including regions where edits introduce premature stop codons, a critical risk for therapeutic applications. By uncovering these previously unrecognized off-target landscapes, our study deepens our understanding of base editor specificity and provides a framework for optimizing their precision, accelerating the development of safer next-generation editing tools.},
}
@article {pmid41445368,
year = {2026},
author = {Wei, J and Jiang, C and Chen, Y and Yang, X and Li, Q},
title = {Functional characterization of Hsk1 and Chit1 genes in the virulence of Metarhizium guizhouense Xct1 via CRISPR-Cas9-mediated gene editing.},
journal = {Pest management science},
volume = {82},
number = {4},
pages = {3625-3639},
doi = {10.1002/ps.70484},
pmid = {41445368},
issn = {1526-4998},
support = {//SCCXTD-2024-04/ ; },
mesh = {*CRISPR-Cas Systems ; Virulence/genetics ; Gene Editing ; Animals ; *Metarhizium/genetics/pathogenicity ; *Fungal Proteins/genetics/metabolism ; Spodoptera/growth &amp; development/microbiology ; *Protein Serine-Threonine Kinases/genetics/metabolism ; Larva/growth &amp; development/microbiology ; Pest Control, Biological ; },
abstract = {BACKGROUND: The entomopathogenic fungus Metarhizium guizhouense Xct1 exhibits high virulence against early-instar Spodoptera frugiperda larvae (>90% mortality in preliminary studies), yet the molecular mechanisms, particularly the roles of key genes such as the chitin-degrading enzyme (Chit1) and serine/threonine kinase (Hsk1) are poorly understood. Functional studies using CRISPR-Cas9 are lacking, limiting its biocontrol application.<br><br>RESULTS: Chit1 and Hsk1 genes were amplified from M.&#x2009;guizhouense Xct1. Chit1 showed high homology to M.&#x2009;anisopliae, whereas Hsk1 exhibited greater genetic diversity. Expression analysis revealed peak Chit1 expression on Day (D)4 and peak Hsk1 expression on D2. A CRISPR-Cas9 system was established, and knockout of Chit1 resulted in thickened cell walls [119&#x2009;nm versus 87&#x2009;nm in wild-type (WT)] and reduced virulence [median lethal time (LT50)&#x2009;=&#x2009;7.4&#x2009;days versus 4.8&#x2009;days in WT]. Overexpression of Chit1 improved virulence (LT50&#x2009;=&#x2009;3.3&#x2009;days). Hsk1 knockout was lethal, confirming its essential role, while overexpression did not alter virulence (LT50&#x2009;=&#x2009;4.8&#x2009;days).<br><br>CONCLUSIONS: Chit1 is a critical virulence factor, influencing cell-wall integrity and insecticidal activity, while Hsk1 is essential for fungal viability. This study presents the first CRISPR-Cas9-mediated functional analysis of these genes, revealing that Chit1 overexpression enhances biocontrol efficacy against S.&#x2009;frugiperda. &#xa9; 2025 Society of Chemical Industry.},
}
@article {pmid41447470,
year = {2026},
author = {Liu, T and Ye, B and Zhang, Y and Yan, X},
title = {Finely Tuned CRISPRi Module for Upgrading the Performance of Constitutive Promoters in the Bacillus subtilis Protein Expression System.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1046-1052},
doi = {10.1021/acs.jafc.5c03424},
pmid = {41447470},
issn = {1520-5118},
mesh = {*Bacillus subtilis/genetics/metabolism/growth &amp; development ; *Promoter Regions, Genetic ; *Bacterial Proteins/genetics/metabolism ; Xylose/metabolism ; Gene Expression Regulation, Bacterial ; CRISPR-Cas Systems ; Fermentation ; },
abstract = {Bacillus subtilis is a critical host for protein production, with many industrial strains relying on strong constitutive promoters. However, this kind of promoter typically imposes a heavy burden on the host from the early stage of fermentation, leading to reduced growth rate and biomass. To overcome the drawbacks of these promoters, we developed a xylose-inducible CRISPRi module to dynamically control the activity of these promoters. The strength of this module was finely tuned via promoter engineering and the xylose concentration. The addition of xylose inhibited the target promoter and favored cell growth at an early stage, while the consumption of xylose recovered the strength of the promoter and facilitated protein expression, resulting in better balance between cell growth and protein production. The yield of a target protein was increased by 38% using this module. Our work provides a simple and effective method to upgrade industrial strains driven by strong constitutive promoters.},
}
@article {pmid41447528,
year = {2026},
author = {Liu, P and Yuan, Q and Yang, X and Wang, Q and Chang, T and Bi, Y and Wu, P and Zhang, T and Yang, J and Guo, S and Xue, C and Zheng, Z and Xin, B and Ma, H and Wang, Y},
title = {A synthetic biology toolkit for the plasmid-dependent and thermophilic methylotroph Bacillus methanolicus.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116788},
doi = {10.1016/j.celrep.2025.116788},
pmid = {41447528},
issn = {2211-1247},
mesh = {*Plasmids/genetics/metabolism ; *Bacillus/genetics/metabolism ; *Synthetic Biology/methods ; CRISPR-Cas Systems/genetics ; Gene Editing ; Methanol/metabolism ; Arginine/metabolism ; Metabolic Engineering ; },
abstract = {Bacillus methanolicus, a unique plasmid-dependent and thermophilic methylotroph, is an ideal chassis for one-carbon (C1) biomanufacturing. Despite its evolutionary uniqueness and industrial promise, the synthetic biology toolkit remains limited in comparison to that of conventional model microorganisms. Here, we present a comprehensive toolkit comprising a high-efficiency electroporation protocol, a CRISPR-Cas9 method enabling robust and multiplex genome editing, diverse neutral loci for gene integration, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, restriction-modification machinery, and the functional significance of chromosomal methylotrophic genes. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR-Cas9 editing, we engineered L-arginine feedback regulation to achieve L-arginine overproduction from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and C1 biomanufacturing.},
}
@article {pmid41448060,
year = {2026},
author = {Long, Y and Sun, S and Mei, H and Zhou, D and Zhou, H and Fang, Z and Li, X and Li, N and Zhuang, T and Guo, C},
title = {RT-LAMP-CRISPR/Cas12b-based hand-pressure-actuated microfluidic chip for rapid and portable detection of severe fever with thrombocytopenia syndrome virus.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129277},
doi = {10.1016/j.talanta.2025.129277},
pmid = {41448060},
issn = {1873-3573},
mesh = {*Phlebovirus/genetics/isolation &amp; purification ; *Severe Fever with Thrombocytopenia Syndrome/diagnosis/virology ; *Nucleic Acid Amplification Techniques/instrumentation/methods ; Humans ; *Lab-On-A-Chip Devices ; *Molecular Diagnostic Techniques/instrumentation/methods ; *CRISPR-Cas Systems ; Pressure ; RNA, Viral/genetics/blood ; },
abstract = {Severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, highly pathogenic tick-borne virus causing severe viral hemorrhagic fever, posing a significant public health threat. Rapid and accurate detection of SFTSV in resource-limited settings is critical for early diagnosis and effective control of severe fever with thrombocytopenia syndrome (SFTS). Here, we developed the RT-LAMP-CRISPR/Cas12b-based Hand-Pressure-Actuated Microfluidic Chip for Rapid and Portable Detection of Severe Fever with Thrombocytopenia Syndrome Virus (HARD). The hand-pressure-actuated microfluidic chip integrates reverse transcription loop-mediated isothermal amplification (RT-LAMP), clustered regularly interspaced short palindromic repeats (CRISPR) and its associated proteins (CRISPR associated proteins, Cas) 12b in order to achieve rapid, low-cost, and contamination-free point-of-care testing. The HARD system achieves a detection limit of 5 copies per reaction, utilizing direct RNA lysis from blood samples and a hand warmer as a heat source, enabling electricity-free operation. Clinical validation with blood samples from vector-borne infectious diseases demonstrated high concordance with laboratory RT-qPCR, with 88.9 % sensitivity, 100 % specificity, and 95 % accuracy. Thus, the HARD platform offers a rapid, portable, and efficient solution for the early diagnosis of SFTSV in resource-limited settings, with potential for broader application in POCT for infectious diseases.},
}
@article {pmid41449724,
year = {2025},
author = {Plesser, E and Goldenberg, L and Kelly, G and Bdolach, E and Arad, T and Bejerano, E and Masok, O and Carmeli-Weissberg, M and Shaya, F and Sherman, A and Eyal, Y and Carmi, N},
title = {Targeting the "bitterness gene" by genome editing abolishes synthesis of bitter flavanones in citrus; prospects for new varieties and extended climates for cultivation.},
journal = {The Plant journal : for cell and molecular biology},
volume = {124},
number = {6},
pages = {e70654},
pmid = {41449724},
issn = {1365-313X},
mesh = {*Flavanones/metabolism/biosynthesis ; *Citrus/genetics/metabolism ; *Gene Editing ; Plant Leaves/metabolism/genetics ; Fruit/genetics/metabolism ; Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Taste/genetics ; Hesperidin/metabolism/analogs &amp; derivatives ; },
abstract = {Bitterness in citrus fruit is conferred by flavanone-neohesperidosides, whose accumulation is catalyzed by a single enzyme flavanone-7-O-glucosides-1,2-rhamnosyltransferase (1,2RhaT), expressed in both leaves and fruit. To eliminate citrus bitterness, we used CRISPR/Cas9 genome editing to inactivate the 1,2RhaT gene in grapefruit (Citrus paradisi) and "Carrizo" citrange (Citrus sinensis × Citrus trifoliata). Edited lines displayed frameshift mutations that introduced premature stop codons, effectively abolishing the synthesis of the bitter neohesperidosides naringin, neohesperidin, and poncirin. Metabolomic analyses in leaves from 1,2RhaT-mutant lines confirmed the absence of bitter flavanone-neohesperidosides and a compensatory increase in the tasteless flavanone-rutinosides hesperidin, didymin, and narirutin. Since 1,2RhaT is encoded by a single gene, our findings in leaves are expected to be identical for fruit and thus demonstrate a strategy for developing non-bitter citrus cultivars while retaining health-benefitting flavonoid levels. Furthermore, cold-hardy citrus species that are currently unacceptably bitter due to high flavanone-neohesperidoside levels may become useful sources for introduction of cold-hardiness following inactivation of the 1,2RhaT gene. This approach thus paves the way for expanding grapefruit markets and breeding cold-hardy, palatable citrus varieties that are better suited to a wider range of climates.},
}
@article {pmid41450580,
year = {2025},
author = {Graves, LE and Christina, S and Mullany, KL and Alexander, IE and Falhammar, H},
title = {Exploration of the potential of genomic editing in the treatment of congenital adrenal hyperplasia.},
journal = {Frontiers in endocrinology},
volume = {16},
number = {},
pages = {1719376},
pmid = {41450580},
issn = {1664-2392},
mesh = {*Adrenal Hyperplasia, Congenital/genetics/therapy ; *Gene Editing/methods/trends ; Humans ; Steroid 21-Hydroxylase/genetics ; *Genetic Therapy/methods/trends ; Animals ; CRISPR-Cas Systems ; },
abstract = {Despite life-saving glucocorticoids, therapeutic options for congenital adrenal hyperplasia (CAH) remain sub-optimal. Adrenal crisis continues to be the highest cause of mortality in individuals with CAH and even with recommended treatment regimens complications from the disease and treatments themselves persist. These patients have limited treatment options and advanced therapeutics could be a solution. Development of genetic therapies have exponentially increased in recent years. The advent of CRISPR/Cas technology has brought previously inconceivable treatment options to reality. Genomic editing could repair the defective 21-hydroxylase gene and provide a cure for 21-hydroxylase deficiency, the most common CAH variant, eliminating the current need for constant patient intervention. There are a number of technologies within reach for CAH, however, delivery of the genomic editing reagents to the elusive adrenocortical progenitor cells remains challenging. Here we discuss the complexity of CAH genetics, which has implications for choice of genomic editing strategy, and potential future strategies for the development of a cure of CAH.},
}
@article {pmid41451278,
year = {2025},
author = {Narra, M and Ray, A and Polley, B and Yang, H and Bhowmik, PK},
title = {AI-driven advances in plant biotechnology: sharpening the edge of plant tissue culture and genome editing.},
journal = {Frontiers in plant science},
volume = {16},
number = {},
pages = {1718810},
pmid = {41451278},
issn = {1664-462X},
abstract = {The advent of artificial intelligence (AI) holds great promise for revolutionizing the fields of plant tissue culture and genome editing. Plant tissue culture is recognized as a powerful tool for rapid multiplication and crop improvement. However, the complex interactions between genetic and environmental factors generate large volumes of data, posing challenges for traditional statistical analysis methods. To address this, researchers are now employing machine learning (ML)-based and artificial neural networks (ANN) approaches to predict and optimize in vitro culture protocols thereby improving precision, sustainability, and efficiency. Integrating AI technologies such as machine learning (ML), artificial neural networks (ANN), and deep learning (DL) can significantly advance the development of data-driven models for CRISPR/Cas9 genome editing. Today, AI-driven methods are routinely applied to enhance precision in predicting on- and off-target sequence locations and editing outcomes. Additionally, predicting protein structures can provide a directed evolution framework that facilitates the creation of improved gene editing tools. However, the application of AI-based CRISPR modeling in plants is not yet fully explored. In this context, we aim to examine representative ML/DL/ANN models of CRISPR/Cas based editing employed in various organisms. This review significantly compiles a diverse set of studies and provides a clear overview of how AI is transforming the fields of plant tissue culture and genome editing. It emphasizes AI's potential to increase the efficiency and precision of biotechnological practices, making them more accessible and cost-effective. While outlining current findings, the paper sets the stage for future research, encouraging further exploration into the integration of AI with plant biotechnology.},
}
@article {pmid41451985,
year = {2026},
author = {Wang, Z and Hu, T and Liu, W and Zhou, H and Lv, X and Li, H and Li, X and Huang, X and He, L},
title = {Development and clinical validation of an ERA-CRISPR/Cas12a assay for the rapid detection of 14 high-risk HPV types.},
journal = {Microbiology spectrum},
volume = {14},
number = {2},
pages = {e0303625},
pmid = {41451985},
issn = {2165-0497},
support = {82100344//National Natural Science Foundation of China/ ; 2024A22//Tongji Hospital/ ; },
mesh = {Humans ; *Papillomavirus Infections/diagnosis/virology ; *CRISPR-Cas Systems ; Sensitivity and Specificity ; Female ; *Molecular Diagnostic Techniques/methods ; *Nucleic Acid Amplification Techniques/methods ; *Papillomaviridae/genetics/isolation &amp; purification/classification ; Uterine Cervical Neoplasms/virology/diagnosis ; Reproducibility of Results ; DNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {UNLABELLED: Persistent infection with high-risk human papillomavirus (HR-HPV) is the leading cause of cervical cancer, highlighting the critical need for early detection to improve prevention. Although real-time quantitative polymerase chain reaction (RT-qPCR) remains the gold standard for HR-HPV detection, its dependence on sophisticated equipment, complex procedures, and trained personnel limits accessibility. Here, we developed a simplified assay for 14 HR-HPV types by integrating direct lysis, enzyme-mediated isothermal rapid amplification (ERA), and CRISPR-Cas12a-mediated cleavage into a streamlined workflow that requires only a basic isothermal heating device. The optimized system achieved a sensitivity of 50 copies per reaction with no cross-reactivity, while a refined lysis buffer containing 20% Chelex-100 minimized inhibition from vaginal swab samples, thereby enhancing detection performance. Validation with 152 clinical samples demonstrated 97.62% sensitivity and 100% specificity, confirming the reliability of the method. This user-friendly and cost-effective assay requires minimal equipment, enabling rapid and field-deployable HR-HPV detection, and offers a practical alternative to conventional laboratory-based approaches, particularly in resource-limited settings.<br><br>IMPORTANCE: High-risk human papillomavirus (HR-HPV) is the principal etiological agent of cervical cancer, and early detection remains central to effective disease prevention. Current PCR-based assays, however, rely on specialized laboratories and trained personnel, limiting their deployment in many settings. Here, we report a streamlined CRISPR-Cas12a assay that integrates direct sample lysis, ERA, and CRISPR-based detection into a single workflow operable with only a simple heating device to determine the presence of 14 HR-HPV types. The assay achieves high analytical sensitivity, strong specificity, and robust clinical performance while maintaining low cost and ease of use. This platform enables rapid HR-HPV detection and scalable screening, particularly in resource-constrained environments, with the potential to facilitate earlier intervention and reduce cervical cancer incidence.},
}
@article {pmid41452514,
year = {2025},
author = {Lal, SK and Khatoon, G and Kumar, A and Kumar, K and Kumar, R and Pan, X and Kumar, S and Bhadana, VP and Pandey, A and Kumar, M and Soren, KR and Panditi, V},
title = {Genome Editing Enhanced Abiotic Stress ToleranceIn Cereal Crops.},
journal = {Functional &amp; integrative genomics},
volume = {26},
number = {1},
pages = {13},
pmid = {41452514},
issn = {1438-7948},
mesh = {*Gene Editing/methods ; *Edible Grain/genetics/growth &amp; development ; *Stress, Physiological/genetics ; CRISPR-Cas Systems ; *Crops, Agricultural/genetics ; *Genome, Plant ; Plants, Genetically Modified/genetics ; },
abstract = {Cereals are crucial sources of food for human and animal populations worldwide. Their grain and fodder primarily serve as sources of energy and nutrition. Cereal production is hampered because of the prevalent abiotic stress worldwide. Abiotic stresses such as drought, salinity, extreme temperatures, and heavy metal toxicity significantly reduce global cereal crop production. Previously, traditional breeding and transgenic technology have been promising and potent approaches used to mitigate unfavourable abiotic stresses, enhancing crop production to some extent. The recent advent of more potent genome-editing technologies, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), has revolutionized the pace of crop improvement programs. Genome-editing technology using engineered nucleases offers significant opportunities for crop improvement. Genome editing tools include Meganucleases, Zinc Finger Nucleases (ZFN), Transcription activator-like effector nucleases (TALENs), and CRISPR/CRISPR-associated protein (Cas). Among all genome-editing tools, CRISPR/Cas9 has been widely used to improve crop cultivars due to its specificity, simplicity, robustness, and flexibility. Recent progress in genome-editing technology have improved various plant traits in cereals. Among these traits, cereal genotypes have shown substantial advances in the last decade, particularly in enhanced tolerance to abiotic stress, enabled by genome-editing tools. This review summarizes the recently developed cereal cultivars for abiotic stress tolerance that employ different genome-editing technologies, including the most recent additions, prime editing and base editing. These improved cereal cultivars perform better and maintain higher yields under adverse abiotic stresses.},
}
@article {pmid41453247,
year = {2026},
author = {Park, SE and Jeong, JH and Kim, YG and Park, HH},
title = {Structural analysis of predicted anti-CRISPR, ACZ01644.},
journal = {Biochemical and biophysical research communications},
volume = {797},
number = {},
pages = {153199},
doi = {10.1016/j.bbrc.2025.153199},
pmid = {41453247},
issn = {1090-2104},
mesh = {*CRISPR-Cas Systems ; Crystallography, X-Ray ; Models, Molecular ; *Viral Proteins/chemistry/metabolism/genetics ; Bacteriophages ; Protein Conformation ; DNA Cleavage ; Amino Acid Sequence ; Protein Multimerization ; },
abstract = {The CRISPR-Cas system provides adaptive immunity in bacteria and archaea against invading genetic elements, while anti-CRISPR (Acr) proteins have evolved in phages to counteract this defense. Here, we report the first structural and biochemical characterization of ACZ01644, a protein previously predicted to be an Acr. The crystal structure of ACZ01644 reveals a unique cone-shaped architecture composed of five α-helices and five β-strands forming a compact core, which represents a fold distinct from any known Acr family. Biochemical analyses demonstrated that ACZ01644 assembles as a trimer in solution, suggesting a potential functional relevance of this oligomeric state. However, in vitro assays revealed that ACZ01644 does not inhibit Cas9-mediated DNA cleavage, indicating that its inhibitory activity, if present, may involve other CRISPR subtypes or yet unidentified cofactors. Our findings reveal an unprecedented structural scaffold among putative Acr proteins and provide a foundation for future studies to elucidate its biological role in CRISPR-Cas regulation.},
}
@article {pmid41453683,
year = {2026},
author = {Akhter, R and Kitab, B and Kayesh, MEH and Shimizu, R and Onuma, H and Yamamoto, N and Ogawa, S and Sugiyama, M and Tanaka, Y and Sato, Y and Kohara, M and Tsukiyama-Kohara, K},
title = {Optimization of lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes for in vivo delivery of a CRISPR/Cas9 system targeting hepatitis B virus.},
journal = {Virus research},
volume = {363},
number = {},
pages = {199682},
pmid = {41453683},
issn = {1872-7492},
mesh = {*Hepatitis B virus/genetics/drug effects/physiology ; *CRISPR-Cas Systems ; Animals ; *Nanoparticles/chemistry/administration &amp; dosage ; Mice ; Humans ; *Ribonucleoproteins/administration &amp; dosage/genetics/chemistry ; Virus Replication/drug effects ; *Lipids/chemistry ; Genetic Therapy/methods ; *Hepatitis B, Chronic/therapy/virology ; RNA, Guide, CRISPR-Cas Systems/genetics/administration &amp; dosage ; CRISPR-Associated Protein 9 ; Liposomes ; },
abstract = {Patients with chronic hepatitis B virus (HBV) infection may benefit from clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene therapy. We previously identified a guide RNA (WJ11) that suppressed HBV replication in vitro and in vivo; however, we were unable to achieve delivery at clinically feasible doses in vivo using an adeno-associated virus (AAV) vector. Lipid nanoparticle (LNP)-based WJ11/Cas9 ribonucleoprotein-oligonucleotide complex delivery suppressed HBV replication by 2-3-fold more than did AAV-based delivery. In the present study, we investigated the HBV replication-suppressive effects of LNP/WJ11/Cas9 complexes after intravenous administration to persistently HBV genotype C-infected humanized chimeric mice. CL4H6 (ionizable lipid) LNPs were selected as the first candidate for WJ11/Cas9 delivery based on their reported high encapsulation efficiency; however, no significant anti-HBV effect was noted in serum or hepatic tissue. The ionizable lipid candidate CL4F11_ε-3 improved absolute serum HBV values to a certain degree but had no significant effect on hepatic HBV DNA or covalently closed circular (ccc)DNA levels. CL4F11_ζ-2 LNP/WJ11/Cas9, a new complex prepared through structural optimization of the ionizable lipid and heat treatment of WJ11, showed suppressive effect for serum viral load along with a reduction of hepatic HBV DNA, HBV cccDNA, HBsAg, and HBcrAg levels when compared with controls. Therefore, LNP-based delivery of this CRISPR/Cas9 formula holds promise for the treatment of chronic HBV infection.},
}
@article {pmid41454508,
year = {2025},
author = {Li, J and Wang, X and Wang, X and Qu, H and Gao, L and Zhao, Z and Luo, P and Zheng, Y},
title = {A Rapid and Sensitive CRISPR-Cas12a for the Detection of Legionella pneumophila.},
journal = {Polish journal of microbiology},
volume = {74},
number = {4},
pages = {484-493},
pmid = {41454508},
issn = {2544-4646},
mesh = {*Legionella pneumophila/isolation &amp; purification/genetics ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Bacterial Proteins/genetics ; Sensitivity and Specificity ; Legionnaires' Disease/microbiology/diagnosis ; Water Microbiology ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Legionella pneumophila is a common environmental bacterium that can cause severe respiratory disease. In this study, a reliable, rapid, and convenient detection method for L. pneumophila was established using a combination of recombinase polymerase amplification (RPA) and CRISPR/Cas12a technology. First, we designed three pairs of RPA primers and two types of crRNA based on the L. pneumophila-specific mip gene. Subsequently, we optimized the primers and amplification time for the RPA reaction, the crRNA for the CRISPR/Cas12a reaction, as well as the concentration of the fluorescent probe. We successfully constructed an RPA-CRISPR/Cas12a fluorescence detection system and a portable RPA-CRISPR/Cas12a LFB. The detection systems achieved a sensitivity of 5 copies/μl and high specificity. One hundred sixty environmental water samples tested by RPA-CRISPR/Cas12a LFB showed no significant difference compared to the qPCR method, providing a reliable tool for future on-site detection.},
}
@article {pmid41454745,
year = {2026},
author = {Mehnath, S},
title = {Engineering stimuli-responsive nanocarriers for CRISPR/Cas9 genome editing: next-generation cancer therapeutics.},
journal = {The Journal of pharmacy and pharmacology},
volume = {78},
number = {3},
pages = {},
doi = {10.1093/jpp/rgaf127},
pmid = {41454745},
issn = {2042-7158},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Neoplasms/genetics/therapy ; Animals ; *Nanoparticles ; Drug Delivery Systems/methods ; Drug Carriers ; Genetic Therapy/methods ; },
abstract = {OBJECTIVES: To highlight recent developments in CRISPR/Cas9 genome-editing strategies for cancer therapy and to evaluate how nanocarrier-based delivery systems enable controlled, spatiotemporal manipulation of genetic information to overcome off-target effects, cytotoxicity, and limitations in clinical translation.<br><br>KEY FINDINGS: CRISPR/Cas9 has emerged as a simple and programmable tool for correcting cancer-associated mutations and regulating adaptive immune responses; however, challenges such as off-target effects, unintended mutations in healthy cells, and cytotoxicity hinder its clinical application. Nanocarriers address these limitations through refined spatiotemporal delivery of Cas9 nuclease and sgRNA using internal and external stimuli-responsive functional groups. These systems improve cancer-cell specificity by engineering guide RNAs, prevent premature clearance, enhance systemic circulation and intracellular delivery, enable nuclear targeting, and regulate Cas9 activity. Stimuli such as light, heat, ultrasound, magnetic fields, pH, redox conditions, glutathione, and oxygen play key roles in controlled activation and release.<br><br>SUMMARY: This review critically evaluates the structural design of nanocarriers, advanced spatiotemporal regulation strategies, and safety and efficacy concerns in CRISPR/Cas9-based cancer therapeutics. It discusses the role of cell-specific promoters, small-molecule stimulation, and stimuli-responsive delivery systems in improving genome-editing precision and therapeutic outcomes. The review also outlines future opportunities for exploiting CRISPR/Cas9 in advanced biomedical applications to enhance the effectiveness of next-generation cancer therapy.},
}
@article {pmid41454886,
year = {2026},
author = {Yang, H and Gao, X and Jin, ZC and Zhang, R and Ning, B and Yan, X},
title = {Simple and Versatile Toolkit for Genetic Manipulation of Bacillus licheniformis.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {262-270},
doi = {10.1021/acssynbio.5c00699},
pmid = {41454886},
issn = {2161-5063},
mesh = {*Bacillus licheniformis/genetics ; Plasmids/genetics ; *Genetic Engineering/methods ; DNA Transposable Elements/genetics ; Mutagenesis ; CRISPR-Cas Systems/genetics ; },
abstract = {Bacillus licheniformis is a spore-forming bacterium with probiotic, environmental, and industrial applications. Many wild strains with diverse functions have been described in recent years. Nevertheless, the lack of efficient and universal genetic manipulation tools hinders the study and engineering of these strains. Here, a versatile and simple genetic manipulation toolkit is established for B. licheniformis. The cornerstone of this toolkit is a conjugative DNA transfer system. This system could effectively transfer temperature-sensitive plasmid pTSMK into all ten tested B. licheniformis strains, with efficiencies ranging from 10[-5] to 10[-3]. Based on this DNA transfer system, the tools for maker-free knockout and knock-in, CRISPRi, as well as transposon mutagenesis, were built. A transposition frequency of 7.68 × 10[-3] was observed. The toolkit developed in this study fulfills most tasks in the engineering of this species and will promote the basic and applied research of B. licheniformis.},
}
@article {pmid41454921,
year = {2025},
author = {Sanjay, G and Seetharam, RN and Singdevsachan, SK and Sathya, M},
title = {Microbial Systems Enhancing CAR-Based Therapies: A Synthetic Biology Paradigm for Next-Generation Cancer Immunotherapy.},
journal = {Current microbiology},
volume = {83},
number = {2},
pages = {106},
pmid = {41454921},
issn = {1432-0991},
support = {Hoynoza Technologies Pvt. Ltd//Hoynoza Technologies Pvt. Ltd/ ; },
mesh = {Animals ; Humans ; *Immunotherapy/methods ; *Immunotherapy, Adoptive/methods ; *Neoplasms/therapy/immunology ; *Receptors, Chimeric Antigen/genetics/immunology ; *Synthetic Biology/methods ; },
abstract = {Chimeric antigen receptor (CAR)-based immunotherapies face significant translational challenges in solid tumor applications, particularly regarding manufacturing scalability, tumor targeting specificity, and antigen heterogeneity. This systematic review evaluates microbial systems as innovative platforms to address these limitations through synthetic biology-driven approaches, with a focus on bridging preclinical advances to clinical implementation. Analysis of 389 peer-reviewed studies (2015-2025) reveals that engineered probiotic strains (e.g., Escherichia coli Nissle 1917) achieve selective tumor colonization while functioning as programmable factories for:1. Synthetic antigen production and single-chain variable fragment (scFv) expression,2. Costimulatory domain delivery enabling antigen-agnostic CAR-T activation,3. Tumor microenvironment modulation via immunostimulatory chemokines. Microbial platforms demonstrate superior manufacturing economics (70-90% cost reduction vs. conventional methods) and enhance CAR-T functionality through epigenetic reprogramming by microbial metabolites (e.g., short-chain fatty acids). CRISPR/Cas-engineered genetic circuits further enable precise spatiotemporal control of therapeutic payloads.Microbial systems represent transformative platforms for scalable, programmable CAR immunotherapy with significant potential for solid tumor targeting. Key barriers to clinical translation include biocontainment challenges, incomplete mechanistic understanding of tumor homing specificity, and safety validation requirements. Strategic integration of synthetic biology with microbial chassis offers a viable pathway toward accessible next-generation cancer therapies.},
}
@article {pmid41455263,
year = {2026},
author = {Liu, F and Cheng, AX and Zhang, C and Li, J and Huang, YF and Zhang, YP and Li, CP and Zhao, H},
title = {Amplification-free, CRISPR-Cas12a-mediated fluorescence biosensor using mesoporous silica nanomaterials for ultrasensitive detection of nucleic acid biomarkers.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129289},
doi = {10.1016/j.talanta.2025.129289},
pmid = {41455263},
issn = {1873-3573},
mesh = {*Biosensing Techniques/methods ; *Silicon Dioxide/chemistry ; *CRISPR-Cas Systems ; Humans ; *Nanostructures/chemistry ; Porosity ; Limit of Detection ; Fluorescent Dyes/chemistry ; Rhodamines/chemistry ; Fluorescence ; ErbB Receptors/genetics ; Gold/chemistry ; Metal Nanoparticles/chemistry ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Sensitive detection of nucleic acid biomarkers is crucial in many fields, including biomedical diagnosis, veterinary medicine, and food safety. Thus, developing an accurate and cost-effective detection method for nucleic acid biomarkers is essential. Here, we developed a sensitive CRISPR-Cas12a-based fluorescence biosensor using mesoporous silica nanomaterials (MSNs). A large quantity of rhodamine B (RB) was enriched on the MSNs to synthesize RB@MSN nanocomposites, which served as fluorescent probe materials, and Au NPs acted as fluorescence quenching materials. Combined with the high specific recognition capability of the CRISPR-Cas12a system, we detected three important nucleic acids without requiring amplification: the EGFR exon 19 deletion mutation (EGFR 19Del, found in circulating tumor DNA), African swine fever virus (ASFV), and human papilloma virus (HPV). Under optimal conditions and using quantitative analysis, there were strong linear correlations between the concentrations of the targets and their respective fluorescence intensities. The lowest detection limits were 55 aM for EGFR 19Del, 51 aM for ASFV, and 24 aM for HPV. By enriching and encapsulating MSNs with RB, our method avoided the problems of fluorescence modifications in typical CRISPR-Cas12a systems, such as professional outsourcing requirements and easily quenched fluorescence. Moreover, the results exhibited good repeatability and stability. This method provides a novel approach to nucleic acid fluorescence detection using the CRISPR-Cas12a system.},
}
@article {pmid41455873,
year = {2025},
author = {Bibi, R and George, M and Sarkar, K},
title = {RNA-guided STAT3 modification fine tunes the epigenetic and epitranscriptomic regulation of CD4 + T helper cell differentiation during non-small cell lung cancer (NSCLC).},
journal = {Medical oncology (Northwood, London, England)},
volume = {43},
number = {2},
pages = {102},
pmid = {41455873},
issn = {1559-131X},
support = {11019/07/2018-Sch//Ministry of Tribal Affairs, Govt. of India/ ; EMDR/SG/15/2023-5901//Indian Council of Medical Research/ ; },
mesh = {Humans ; *STAT3 Transcription Factor/genetics/metabolism ; *Carcinoma, Non-Small-Cell Lung/genetics/immunology/pathology ; *Lung Neoplasms/genetics/immunology/pathology ; *Epigenesis, Genetic ; Cell Differentiation/genetics ; Tumor Microenvironment/immunology ; *CD4-Positive T-Lymphocytes/immunology ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems ; Transcriptome ; },
abstract = {The accurate control of immune responses in the tumor microenvironment is crucial for augmenting anti-cancer immunity. This work examined the function of STAT3 in modulating epigenetic and epitranscriptomic pathways during the differentiation of CD4 + T helper cells in non-small cell lung cancer (NSCLC). Employing CRISPR/Cas9 genome editing, STAT3 was specifically eliminated in CD4[+]T cells derived from NSCLC patients. Functional investigations demonstrated that the reduction of STAT3 markedly enhanced the production of T helper 1 (TH1) cytokines, notably IFN-γ, while concurrently diminishing immunosuppressive signaling. Epigenetic analysis revealed significant modifications in DNA and RNA methylation patterns, along with heightened R-loop formation-alterations linked to augmented transcriptional activity of anti-tumor immune genes. Moreover, STAT3-deficient CD4[+]T cells demonstrated an enhanced ability to activate cytotoxic T lymphocytes, facilitating the targeted eradication of tumor cells. All of these effects together made the NSCLC microenvironment's immune system better at fighting cancer. Our results identify STAT3 as a crucial regulator of the genetic and epigenetic frameworks that influence T cell functionality in lung cancer. By combining RNA-guided genome editing with immune functional tests, we show that blocking STAT3 in a specific way could bring back strong anti-tumor immunity. This research underscores the therapeutic potential of STAT3-targeted therapies, presenting an innovative approach to alter T cell destiny and improve immune-mediated tumor eradication in non-small cell lung cancer (NSCLC). These methods could lead to the next generation of immunotherapies that improve clinical outcomes by fine-tuning both epigenetic and epitranscriptomic circuits.},
}
@article {pmid41456387,
year = {2026},
author = {Zhang, J and Liu, WJ and Ma, F and Zhang, CY},
title = {Self-priming amplification-integrated orthogonal CRISPR-Cas system for multiplexed profiling of piRNAs in clinical samples.},
journal = {Biosensors &amp; bioelectronics},
volume = {297},
number = {},
pages = {118334},
doi = {10.1016/j.bios.2025.118334},
pmid = {41456387},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Breast Neoplasms/genetics/diagnosis ; Nucleic Acid Amplification Techniques/methods ; Female ; *Colorectal Neoplasms/genetics/diagnosis ; Piwi-Interacting RNA ; },
abstract = {CRISPR/Cas-based biosensors hold great diagnostic potential, but they are often limited in clinical practice by insufficient sensitivity and the lack of multiplexed analysis capability. To address these issues, we develop a self-priming amplification-integrated orthogonal CRISPR-Cas (SPA-OCRISPR) system for multiplexed detection of piRNAs. This assay employs an innovative four-way junction probe that seamlessly integrates target recognition with a primer-free self-priming amplification, significantly simplifying the workflow and enhancing the ligation efficiency. High-fidelity SplitR ligase-mediated transduction guarantees the excellent specificity, efficiently eliminating the need for reverse transcription. The orthogonal trans-cleavage activities of Cas12a and Cas13a are harnessed to generate distinct fluorescent signals with minimal cross-interference for multiplexed analysis. This strategy can achieve attomolar-level sensitivity and good specificity. Moreover, it can successfully quantify breast cancer-associated piRNAs (piR-651 and piR-36026) in clinical tissues, and accurately discriminate cancerous samples from healthy ones. Importantly, this strategy exhibits good generality and it can be extended to detect colorectal cancer-related piRNAs (piR-823 and piR-54265) through simply modifying the recognition sequences of split probes, underscoring its broad potential in multiplexed profiling and clinical cancer diagnostics.},
}
@article {pmid41456781,
year = {2026},
author = {Puri, B and Gaikwad, AB},
title = {Targeting LncRNAs with CRISPR/Cas9 for Kidney Therapeutics: A Review.},
journal = {International journal of biological macromolecules},
volume = {339},
number = {Pt 1},
pages = {149932},
doi = {10.1016/j.ijbiomac.2025.149932},
pmid = {41456781},
issn = {1879-0003},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Long Noncoding/genetics ; Gene Editing/methods ; Animals ; *Kidney Diseases/therapy/genetics ; Genetic Therapy/methods ; },
abstract = {Long noncoding RNAs (lncRNAs) have emerged as key players in the pathogenesis of kidney diseases, including acute kidney injury (AKI), AKI-to-chronic kidney disease (CKD) transition, CKD, diabetic kidney disease (DKD), renal cell carcinoma (RCC), polycystic kidney diseases (PKD), and lupus nephritis (LN). Although the roles of lncRNAs in disease progression have been investigated in preclinical models, their underlying mechanisms remain poorly understood. The therapeutic potential of lncRNA-based therapies remains largely unexplored in clinical settings. Recently, an advancement in clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing technology offers a novel strategy for treating sickle cell anemia and β-thalassemia. Additionally, CRISPR/Cas9 is currently being evaluated in clinical trials for various diseases, including kidney diseases like RCC. However, the application of CRISPR/Cas9 to target lncRNAs is still in the early stages. Preclinical experiments have revealed that CRISPR/Cas9 could effectively target lncRNAs in kidney disorders. However, its clinical translation in AKI and CKD conditions remains unclear, and various biological challenges remain to be addressed. This review aims to investigate advancements in CRISPR/Cas9 that target lncRNAs in the kidney, highlighting the limitations and future directions for advancing CRISPR/Cas9-based lncRNA therapy and translating these findings into clinical applications.},
}
@article {pmid41456843,
year = {2026},
author = {Chang, C and Yang, J and Liu, Z and Chen, J and Wang, B and Li, J and Liu, H},
title = {Layer-by-layer coated chitosan-CRISPR/Cas9 mTOR nanoparticles: A novel approach to inhibit lens epithelial cell proliferation and migration for preventing posterior capsule opacification.},
journal = {Experimental eye research},
volume = {264},
number = {},
pages = {110828},
doi = {10.1016/j.exer.2025.110828},
pmid = {41456843},
issn = {1096-0007},
mesh = {*Capsule Opacification/prevention &amp; control/pathology/metabolism ; *Chitosan/pharmacology/chemistry ; Cell Proliferation ; *TOR Serine-Threonine Kinases/antagonists &amp; inhibitors/genetics ; *Epithelial Cells/pathology/metabolism ; *Nanoparticles/chemistry ; Cell Movement ; *CRISPR-Cas Systems ; Animals ; Humans ; Rabbits ; *Lens, Crystalline/cytology ; Posterior Capsule of the Lens/pathology ; Epithelial-Mesenchymal Transition ; Coated Materials, Biocompatible ; Lenses, Intraocular ; },
abstract = {Posterior capsular opacification (PCO) is the most common complication following cataract surgery and a significant cause of vision impairment. PCO arises from the proliferation, migration, and epithelial-mesenchymal transition (EMT) of residual lens epithelial cells (LECs), driven by an activated mTOR signalling pathway. Previous research has demonstrated that inhibiting mTOR activity effectively reduces LEC proliferation and EMT in rabbit models. However, achieving sustained mTOR inhibition remains a challenge. In this study, we encapsulated the CRISPR/Cas9 system targeting mTOR into chitosan nanoparticles (Chi-gRNA) with an average size of 135 nm. These nanoparticles exhibited resistance to DNase I digestion. To prolong release duration, we incorporated these Chi-gRNA nanoparticles onto the surface of intraocular lenses (IOLs) via layer-by-layer (LbL) assembly. The LbL coatings consisted of alternating layers of positively charged polyethyleneimine (PEI) and negatively charged heparin, interspersed with Chi-gRNA nanoparticles over five consecutive cycles. Spectral analysis confirmed the successful integration and coating of nanoparticles, with characteristic peaks validating the electrostatic assembly of the layers. In vitro assays demonstrated that Chi-gRNA-coated IOLs significantly inhibited the proliferation, migration, and adhesion of human lens epithelial cells (hLECs). These findings highlight the potential of LbL-coated IOLs to deliver CRISPR/Cas9 system-targeting mTOR nanoparticles as a novel and effective strategy to prevent PCO in patients undergoing cataract surgery. This approach offers a promising avenue for the long-term management of this prevalent postoperative complication.},
}
@article {pmid41457318,
year = {2026},
author = {Brandt, D and Dörrich, AK and Persicke, M and Kemmler, A and Leonhard, T and Haak, M and Nölting, S and Ruwe, M and Schmid, N and Thormann, KM and Kalinowski, J},
title = {A pentose, as a cytosine nucleobase modification in Shewanella phage Thanatos genomic DNA, mediates enhanced resistance toward host restriction systems.},
journal = {Applied and environmental microbiology},
volume = {92},
number = {1},
pages = {e0133325},
pmid = {41457318},
issn = {1098-5336},
support = {34.EFRE-0300095/1703FI04//European Regional Development Fund/ ; },
mesh = {*Shewanella/virology ; *Cytosine/metabolism/chemistry ; *Bacteriophages/genetics/metabolism ; *DNA, Viral/genetics/metabolism/chemistry ; Genome, Viral ; },
abstract = {UNLABELLED: Co-evolution of bacterial defense systems and phage counter-defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. In order to evade nuclease-based mechanisms that target DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits the recognition and restriction by endonucleases. We found that Shewanella phage Thanatos DNA is insensitive to multiple restriction enzymes and also to Cas I-Fv and Cas9 cleavage. Furthermore, with nanopore sequencing, the phage DNA showed severely impaired basecalling. In addition to an adenine methylation, the data indicated an additional, much more substantial nucleobase modification. Using liquid chromatography-mass spectrometry (LC-MS), we identified an unknown configuration of a deoxypentose attached to cytosine as an undiscovered modification of phage DNA, which is present in Thanatos genomic DNA and likely mediates resistance to restriction endonucleases, as well as reducing Cas nuclease activity significantly. To elucidate the underlying enzyme functions, we identified structural homologs of Thanatos proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of the phage protein TH1_063 by in vitro. Inactivation of TH1_060 leads to an almost complete inhibition of phage propagation, indicating an important role of the cytosine modification in phage survival and/or proliferation.<br><br>IMPORTANCE: Several phages extensively decorate their DNA building blocks, providing an effective protection against various host and phage-produced restriction systems. These modifications allow the phages to distinguish between their own DNA and that of the host, significantly increasing the establishment of the phage chromosome upon entry into the host and subsequent phage proliferation. Several different modifications have been previously identified and characterized. Here, we describe a hitherto unknown cytosine modification, consisting of a deoxypentose-putatively xylose-that provides protection against various bacterial restriction systems, including DNA-targeting CRISPR/Cas systems. Our findings expand the range of DNA modifications that phages use for protection.},
}
@article {pmid41457547,
year = {2025},
author = {Song, S and Yao, D and Cai, Z and Yue, X and Qiao, C and Xue, C},
title = {[Construction of a CRISPR-Cas6-mediated lycopene synthase assembly regulation method].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {12},
pages = {4745-4758},
doi = {10.13345/j.cjb.250396},
pmid = {41457547},
issn = {1872-2075},
mesh = {Lycopene ; *CRISPR-Cas Systems/genetics ; *Escherichia coli/genetics/metabolism ; *Metabolic Engineering/methods ; *Intramolecular Lyases/genetics/metabolism ; Plasmids/genetics ; Carotenoids/metabolism ; },
abstract = {A CRISPR-Cas6-mediated lycopene synthase assembly regulation strategy was developed to optimize the metabolic pathway of lycopene biosynthesis in Escherichia coli and enhance production efficiency. Leveraging the orthologous properties of EcCas6e and Csy4 within the Cas6 protein family, along with RNA scaffolding, we constructed a protein-RNA complex for enzyme assembly. Sixteen plasmids (LYC-1 to LYC-16) were designed, and the assembly strategy was systematically optimized by varying the gene arrangement, linker length, and RNA scaffold expression. The performance of RNA scaffold-based enzyme assembly was compared with conventional protein linker-based approaches. Lycopene production was quantified via high-performance liquid chromatography (HPLC) to evaluate system performance. The recombinant strain LYC-3-4, which co-localized CrtB and CrtI via EcCas6e-Csy4 protein-RNA complexes, achieved the highest lycopene yield (4.02 mg/L), 58% higher than the control strain LYC-3-5 (2.55 mg/L) with mismatched RNA hybridization regions, and 41% higher than strain LYC-6 (2.86 mg/L), in which the enzymes were expressed separately. This result indicates that protein-RNA-mediated spatial co-localization significantly enhanced the substrate channeling effect, whereas other assembly configurations either failed to improve or even reduced lycopene production. In summary, we exploited the protein assembly capability of CRISPR-Cas6 proteins in combination with RNA scaffolds to achieve efficient enzyme co-localization within the lycopene biosynthetic pathway. This approach offers a convenient, flexible, and scalable tool for enzyme assembly regulation in metabolic engineering, with potential applications in microbial production of lycopene and other valuable metabolites.},
}
@article {pmid41457550,
year = {2025},
author = {Sun, Y and Li, W and Wang, P and Pei, J and Yang, L and Qiu, L and Liu, H},
title = {[CRISPRi-mediated regulation of nitrate metabolism genes in Chlamydomonas reinhardtii enhances lipid accumulation].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {12},
pages = {4794-4809},
doi = {10.13345/j.cjb.250363},
pmid = {41457550},
issn = {1872-2075},
mesh = {*Chlamydomonas reinhardtii/genetics/metabolism ; *Lipid Metabolism/genetics ; *Nitrates/metabolism ; Nitrate Reductase/genetics/metabolism ; Nitrogen/metabolism ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Nitrite Reductases/genetics ; },
abstract = {The global energy crisis and environmental pollution are becoming increasingly serious. The development of sustainable and clean renewable energy has become a key direction of scientific research. Microalgae are ideal raw materials for biodiesel production due to their efficient photosynthetic ability, fast growth rate, and rich lipid content. Chlamydomonas reinhardtii, as a model organism of unicellular eukaryotic green algae, has the advantages of a clear genetic background and convenient operation, which makes it an ideal target for the study of lipid metabolism in microalgae. Nitrogen stress can induce lipid accumulation in microalgae, while its molecular mechanism has not been fully elucidated. In this study, we used a CRISPR interference (CRISPRi) system to regulate key genes of nitrogen metabolism in a targeted manner and thus simulated the nitrogen stress environment to investigate its effect on lipid accumulation in C. reinhardtii, aiming to provide a new technological strategy for the efficient production of microalgal lipids. The CRISPRi system was constructed to inhibit the expression of the nitrate reductase gene (CrNIT1) and the nitrite reductase gene (CrNII1) in C. reinhardtii FACHB-2220. We evaluated the effects of nitrogen metabolism inhibition on lipid accumulation by measuring the cell growth, lipid content, and expression levels of key genes. The algal strain ΔNIT1-4 with inhibited CrNIT1 expression showed the CrNIT1 expression 10.27% that of the wild type (WT, and the strain ΔNII1-4 with inhibited CrNII1 expression showed the CrNII1 expression16.02% that of WT, indicating that the CRISPRi system effectively inhibited the transcription of the target genes. Under the condition of nitrogen abundance, the cell density of ΔNIT1-4 and ΔNII1-4 was only 33.7% and 40.2%, respectively, of that of WT. The total lipid content of ΔNIT1-4 and ΔNII1-4 was 34.41% and 33.45% of the dry weight, respectively, which was significantly higher than that of WT. In this study, we successfully simulated the nitrogen stress effect by suppressing the key genes of nitrogen metabolism through the CRISPRi system and significantly improved the lipid accumulation efficiency of C. reinhardtii. This study elucidates the regulatory relationship between nitrogen metabolism and lipid synthesis, providing a theoretical basis and technical support for the industrial application of microalgae in bioenergy production.},
}
@article {pmid41457595,
year = {2025},
author = {Chang, Y and Fan, S and Hao, T and Dai, J and He, W},
title = {[Characterization of cbm2813 encoding the cytochrome P450 enzyme in the biosynthetic gene cluster of carbomycin].},
journal = {Sheng wu gong cheng xue bao = Chinese journal of biotechnology},
volume = {41},
number = {11},
pages = {4125-4137},
doi = {10.13345/j.cjb.250364},
pmid = {41457595},
issn = {1872-2075},
mesh = {*Multigene Family/genetics ; *Streptomyces/genetics/metabolism/enzymology ; *Cytochrome P-450 Enzyme System/genetics/metabolism ; Escherichia coli/genetics/metabolism ; Macrolides/metabolism ; *Bacterial Proteins/genetics/metabolism ; CRISPR-Cas Systems ; *Anti-Bacterial Agents/biosynthesis ; },
abstract = {Carbomycin, a 16-membered macrolide antibiotic produced in Streptomyces thermotolerans, comprises two components, carbomycin A (CA) and carbomycin B (CB). CB is converted into CA through epoxidation of the C12-C13 double bond. The gene cbm2813, located in the biosynthetic gene cluster of carbomycin, encodes a cytochrome P450 enzyme considered to catalyze this epoxidation. In this study, the functional and enzymatic properties of the cytochrome P450 enzyme Cbm2813 in the carbomycin biosynthesis gene cluster were characterized by in vivo gene inactivation and in vitro enzymatic reactions. We employed the CRISPR-Cas9 system to delete cbm2813 and obtained the mutant Δcbm2813. The fermentation products of the mutant contained CB but not CA. Complementation of Δcbm2813 restored CA production. Cbm2813 was successfully expressed in Escherichia coli and then purified. In vitro enzyme assays confirmed that Cbm2813 specifically recognized CB but not structurally similar 16-membered macrolide antibiotics, such as josamycin, midecamycin, and isovalerylspiramycin I. Cbm2813 exhibited the maximal activity at pH 5.5 and 36 ℃, with the catalytic efficiency kcat/Km of 4.39×10[3] L/(mol·s). Molecular docking suggested that the C9 carbonyl group of CB coordinated with the heme iron in the active site of the enzyme, ensuring strict substrate specificity. This study expands the toolbox of characterized P450 enzymes and advances the understanding of carbomycin biosynthesis.},
}
@article {pmid41457810,
year = {2026},
author = {Zhao, J and Long, Y and Zhang, Y and Hou, C and Huo, D},
title = {Nano-Mechanical DNA Devices Coupled With CRISPR-Cas12a for CA15-3 Detection.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {10},
pages = {e11023},
doi = {10.1002/smll.202511023},
pmid = {41457810},
issn = {1613-6829},
support = {81772290//National Natural Science Foundation of China/ ; CYB240064//Graduate Scientific Research and Innovation Foundation of Chongqing, China/ ; 2023-K08//Open Research Fund of State Key Laboratory of Digital Medical Engineering/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *DNA/chemistry ; Humans ; Biosensing Techniques/methods ; *Nanotechnology/methods ; Electrochemical Techniques ; },
abstract = {Accurate monitoring of cancer markers is crucial for clinical treatment and prognosis. CA15-3 activity levels are strongly associated with clinical progression of breast cancer, but their monitoring often relies on large instruments and professionals, and the process is time-consuming and costly. To address these concerns, we proposed an electrochemical biosensing strategy that integrated nano-mechanical DNA devices coupled with the CRISPR-Cas12a to drive molecularly gated functionalized substrates for the ultrasensitive detection of CA15-3. Specifically, Triple helical molecular switch (THMS) as a signal input switch to ensure target recognition specificity and the diffusion-limited 3D DNA walking machine coupled with CRISPR-Cas12a technology as signal amplification means. Based on the bimolecular dynamics model, the rate constants k1 (1.40 × 10[5] M[-1]sec[-1]) and k2 (2.5 × 10[4] M[-1]sec[-1]) of the GNP-PEG(+)/T 3D orbitals modified with positively charged SH-PEG-NH2 are larger than those of unmodified orbitals, proving that nanointerface diffusion restriction effect can accelerate the toehold-mediated chain displacement reaction (TMDR). With the assistance of Co-N/C modified screen-printed electrode (SPE-Co-N/C) sensing interface, the calculated detection limit of CA15-3 is as low as 7.14 × 10[-6] U mL[-1]. The proposed assay, which demonstrated satisfactory selectivity and reproducibility, and correlated highly with ELISA kit results, offered a promising tool for breast cancer early detection and therapeutic monitoring.},
}
@article {pmid41458320,
year = {2025},
author = {Ghiotto, G and Francescato, L and Biancalani, MA and Treu, L and Campanaro, S},
title = {Hydrogen excess drives metabolic reprogramming and viral dynamics in syngas-converting microbiomes.},
journal = {Environmental science and ecotechnology},
volume = {28},
number = {},
pages = {100637},
pmid = {41458320},
issn = {2666-4984},
abstract = {Microbial communities drive essential bioprocesses, including the conversion of synthesis gas into biomethane, a sustainable energy source that supports circular carbon economies. In anaerobic environments, specialized consortia of bacteria and archaea facilitate syngas methanation through syntrophic interactions, where hydrogenotrophic methanogens play a central role in reducing carbon dioxide and monoxide with hydrogen. However, imbalances in gas ratios, particularly excess hydrogen, can disrupt these interactions and impair overall efficiency. Yet, the molecular mechanisms underlying microbial responses to such imbalances remain poorly understood. Here we show that hydrogen excess triggers profound metabolic and viral remodeling in a thermophilic anaerobic microbiome, leading to reduced methane yields and ecological instability. This reprogramming involves transcriptional downregulation of methanogenesis genes in the dominant archaeon Methanothermobacter thermautotrophicus, coupled with upregulation of CRISPR-Cas and restriction-modification systems that correlate with diminished activity of an associated phage, indicating activated host defenses against viral threats. Concurrently, bacterial species such as those from Tepidanaerobacteraceae enhance carbon fixation via the Wood-Ljungdahl pathway, serving as electron sinks to mitigate redox imbalance. These adaptive responses highlight the microbiome's resilience mechanisms under stress, revealing viruses as both stressors and selective forces in syntrophic systems. Such insights advance our understanding of microbiome dynamics in bioconversion processes and guide the engineering of more stable microbial consortia for optimized syngas-to-methane conversion amid variable feedstocks.},
}
@article {pmid41458398,
year = {2025},
author = {Kang, J and Koo, J and Oh, H and Bae, E},
title = {Structural characterization of anti-CRISPR protein AcrIE9.},
journal = {Structural dynamics (Melville, N.Y.)},
volume = {12},
number = {6},
pages = {064701},
pmid = {41458398},
issn = {2329-7778},
abstract = {The arms race between bacteria and bacteriophages has driven the evolution of both CRISPR-Cas systems and anti-CRISPR (Acr) proteins. AcrIE9, a type I-E Acr protein identified in Pseudomonas aeruginosa, inhibits Cascade-mediated DNA binding by interacting with the Cas7e subunit. However, its structural basis and precise inhibitory mechanism have remained unclear. Here, we report the crystal structure of AcrIE9 at 1.73 Å resolution, along with additional structural and biochemical analyses. AcrIE9 exists as both monomer and dimer in solution, while the crystal structure reveals a homodimeric assembly. Each protomer adopts a unique α/β architecture, and structural similarity searches indicate that AcrIE9 represents a previously uncharacterized protein fold. In vitro binding assays using individually purified type I-E Cas subunits from P. aeruginosa did not detect direct interaction with AcrIE9, including with Cas7e. These findings suggest that AcrIE9 may recognize a composite interface formed only within the intact Cascade complex, consistent with the AlphaFold3 prediction of multivalent interactions with Cas7e subunits. Taken together, this study provides the structural characterization of AcrIE9 and supports an inhibitory mechanism involving a multi-subunit binding surface on Cascade.},
}
@article {pmid41459216,
year = {2025},
author = {Sun, Y and Zhao, Q and Li, W and Kwok, LY and Zhang, H},
title = {Genomic diversity and functional adaptation of Limosilactobacillus reuteri isolated from diverse ecological niches.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1732127},
pmid = {41459216},
issn = {1664-302X},
abstract = {Limosilactobacillus reuteri is a widely utilized probiotic, however, the genomic diversity and evolutionary mechanisms underlying its adaptation to various hosts and environments remain incompletely understood. This study employed comparative genomics to analyze 176 L. reuteri genomes from animal (rodents, mammals, ruminants, and birds), human intestinal, and food sources (dairy products, fermented foods; 89 newly sequenced and 92 retrieved, 5 excluded by ANI < 95%). We assessed genomic features, average nucleotide identity, pan/core genomes, carbohydrate-active enzymes, bacteriocin production, CRISPR-Cas systems, and antibiotic resistance genes. The pan-genome consisted of 16,814 genes, while the core genome contained 553 genes. Core-gene phylogeny revealed seven clades, rodents isolates were positioned closer to the root. The clustering trend of fermented foods isolates in the phylogenetic tree may indicate that these strains have undergone convergent evolution or adaptive evolution in a specific environment. CAZymes varied across sources, and the predicted bacteriocin clusters were enriched in animal-derived, particularly in rodent isolates. CAZy functional composition in L. reuteri is shaped by the ecological niche and host environment, reflecting a pattern of host-driven evolutionary adaptation. CRISPR-Cas systems were present in 23.3% of genomes, predominantly in rodents isolates, indicating strong anti-phage capabilities. The heterogeneity of CRISPR-Cas systems among sources suggests that subpopulations of L. reuteri have been subjected to different evolutionary pressures. The predominance of Type II systems agrees with their widespread occurrence in lactobacilli. The presence of multiple probiotic function-related genes across all separation sources confirms the robust probiotic potential of L. reuteri. Antibiotic resistance genes, including tet, ermB, and vatE, were most prevalent among animal-derived isolates, with the highest numbers occurring in mammals and the lowest in rodents. Therefore, strain-specific safety assessments are necessary prior to clinical or food applications. The findings underscore the significance of host-specific adaptations in shaping the genetic and functional profiles of L. reuteri, offering valuable implications for its application in food-derived, human-derived, animal-derived and therapeutics.},
}
@article {pmid41459814,
year = {2026},
author = {Celle, M and Aniorte, S and Issa, AR and Falabregue, M and Jin, H and Sanchez-Mirasierra, I and Ding, S and Soukup, SF and Seugnet, L and Liao, L and Lesca, G and Walter, L and Mollereau, B},
title = {A dwdr45 knock-out drosophila model to decipher the role of autophagy in BPAN.},
journal = {Human molecular genetics},
volume = {35},
number = {3},
pages = {},
doi = {10.1093/hmg/ddaf198},
pmid = {41459814},
issn = {1460-2083},
support = {//French Ministry of Higher Education and Research/ ; 101067877//Marie Sklodowska-Curie Action fellowship/ ; //China Scholarship Council/ ; },
mesh = {Animals ; *Autophagy/genetics ; Disease Models, Animal ; *Drosophila Proteins/genetics/metabolism ; Gene Knockout Techniques ; Drosophila melanogaster/genetics ; CRISPR-Cas Systems ; *Neurodegenerative Diseases/genetics/pathology/metabolism ; *Carrier Proteins/genetics/metabolism ; Phenotype ; Drosophila/genetics ; Humans ; },
abstract = {Beta-propeller protein-associated neurodegeneration (BPAN) is a rare neurological disorder characterized by severe cognitive and motor impairments. BPAN is caused by de novo pathogenic variants in the WDR45 gene on the X chromosome. WDR45 gene encodes the protein WDR45/WIPI4, a known regulator of autophagy. A defective autophagy has been observed in cellular models of BPAN disease and is associated with neurological dysfunctions in wdr45 knockout (KO) mice. However, it remains unclear whether the autophagic defect directly contributes to all WDR45 loss-induced phenotypes or whether other WDR45-dependent cellular functions are involved. To investigate this, we generated a CRISPR/Cas9-mediated KO of CG11975 (dwdr45 KO), the Drosophila homolog of WDR45. Our analysis revealed that dwdr45 KO flies display BPAN-like phenotypes, including impaired locomotor function, seizure-like behavior, autophagy dysregulation and iron dyshomeostasis. Additionally, dwdr45 KO flies exhibit shortened lifespan compared to control flies. These findings demonstrate that dwdr45 KO fly is a relevant in-vivo model for investigating the key cellular and molecular mechanisms underlying BPAN-associated phenotypes. Here we showed that induction of autophagy in dwdr45 KO flies improved both the shortened lifespan and the seizure-like behavior, but did not restore locomotor function. This suggests that defective autophagy contributes to some, but not all, aspects of the phenotypes resulting from loss of dWdr45 function.},
}
@article {pmid41461227,
year = {2026},
author = {Su, S and Zhang, X and Wang, X and Qiu, C and Xu, Z and Piñero, JC and Peng, X and Li, F and Zuo, Y and Chen, M},
title = {CRISPR/Cas9-based evidence that overexpression of Gm-mGST1 mediates abamectin resistance in the oriental fruit moth, Grapholita molesta.},
journal = {Insect biochemistry and molecular biology},
volume = {187},
number = {},
pages = {104472},
doi = {10.1016/j.ibmb.2025.104472},
pmid = {41461227},
issn = {1879-0240},
mesh = {Animals ; *Ivermectin/analogs &amp; derivatives/pharmacology ; *Moths/genetics/drug effects/metabolism ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; *Insecticides/pharmacology ; *Glutathione Transferase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Chloride Channels/genetics/metabolism ; Larva/genetics/growth &amp; development/drug effects ; },
abstract = {Abamectin-based insecticides are widely used in integrated pest management and are particularly effective against fruit borers such as the oriental fruit moth, Grapholita molesta. However, rapid resistance evolution threatens their long-term efficacy. This study elucidates the role of the glutathione S-transferase gene Gm-mGST1 in abamectin resistance in G. molesta. A laboratory-selected resistant strain (AB-R) exhibited an 85.5-fold increase in resistance compared with a susceptible strain (AB-S). Sequencing of glutamate-gated chloride channel (GmGluCl) gene revealed no target-site mutations, implicating a metabolic resistance mechanism. In AB-R, GST enzymatic activity was significantly elevated. GST synergist diethyl maleate (DEM) increased the toxicity of abamectin more strongly in the abamectin-resistant G. molesta strain than in the susceptible strain, indicating that GSTs contribute to abamectin resistance. Gm-mGST1 showed strong and stage-specific overexpression under abamectin exposure. Functional analysis using CRISPR/Cas9 knockout of Gm-mGST1 in the AB-R strain reduced resistance 16.3-fold, providing the definitive evidence that a GST gene directly mediates abamectin resistance in G. molesta. The catalytic activity of recombinant Gm-mGST1 was verified in vitro using CDNB as the substrate. Additionally, abamectin exhibited a certain degree of inhibitory effect on the activity of Gm-mGST1. HPLC analysis further revealed that the peak area of abamectin significantly decreased in the presence of recombinant Gm-mGST1, while ectopic expression in Drosophila melanogaster increased abamectin tolerance by 1.97-fold. There is a significant positive correlation between the abamectin resistance levels and the expression levels of Gm-mGST1 in field populations of G. molesta. These findings identify Gm-mGST1 as a critical gene involved in abamectin resistance and establish it as a potential molecular marker for monitoring resistance in field populations. More broadly, this study sets a precedent for integrating CRISPR/Cas9 gene editing into insecticide resistance research, bridging the gap between correlative evidence and functional validation, and providing a framework for developing GST-targeted resistance management strategies in orchard pests. This study provides evidence using CRISPR/Cas9 to confirm the contribution of GST to abamectin resistance in insects.},
}
@article {pmid41461407,
year = {2026},
author = {Shahid, M},
title = {Molecular engineering and in-silico biotechnological innovations for microbial degradation of persistent pesticides.},
journal = {Pesticide biochemistry and physiology},
volume = {217},
number = {},
pages = {106833},
doi = {10.1016/j.pestbp.2025.106833},
pmid = {41461407},
issn = {1095-9939},
mesh = {*Pesticides/metabolism ; Biodegradation, Environmental ; *Biotechnology ; Metabolic Engineering ; Soil Microbiology ; Bacteria/metabolism/genetics ; Computer Simulation ; },
abstract = {The persistence of recalcitrant pesticides in agricultural soils poses a serious threat to environmental and public health. Conventional remediation methods often have limited efficiency and, sustainability. Whereas, microbial degradation provides an eco-friendly and attractive alternative. This review highlights advances in molecular and biotechnological tools driving microbial pesticide degradation. It also emphasizes key genes, enzymatic pathways, and resilient microbes driving recalcitrant pesticide degradation. This review discusses the integration of next-generation sequencing, multi-omics platforms, CRISPR-Cas editing, synthetic biology, and AI-driven metabolic engineering in advancing microbial pesticide degradation. It also highlights progress in rhizosphere microbiome research, bioinformatics pipelines, and field-scale validation. The transition from lab to field highlights precision bioremediations' potential for sustainable pesticide management.},
}
@article {pmid41461563,
year = {2026},
author = {Riley, SE and Noskova Fairley, M and Xia, S and Cunningham, R and Cholewa-Waclaw, J and Feng, Y and Hansen, CG},
title = {In vivo screen reveals specific roles of Hippo pathway components in development and regeneration.},
journal = {Life science alliance},
volume = {9},
number = {3},
pages = {},
pmid = {41461563},
issn = {2575-1077},
support = {/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Animals ; *Zebrafish/genetics/metabolism ; *Regeneration/genetics/physiology ; *Zebrafish Proteins/metabolism/genetics ; Signal Transduction/genetics ; Hippo Signaling Pathway ; *Protein Serine-Threonine Kinases/metabolism/genetics ; YAP-Signaling Proteins ; CRISPR-Cas Systems/genetics ; Macrophages/metabolism ; Larva ; Animal Fins/physiology ; Gene Expression Regulation, Developmental ; Transcription Factors/metabolism/genetics ; Transcriptional Coactivator with PDZ-Binding Motif Proteins ; },
abstract = {The Hippo signalling pathway is a major regulator of regeneration and development. However, the comparative importance and functional roles of individual Hippo pathway components in vivo are greatly unknown, particularly within the vertebrate lineage. To gain direct and comparable insights, we took advantage of the zebrafish larva model system. We generated individual and combined CRISPR/Cas9 F0 knockouts of a range of core Hippo pathway genes, including upstream regulators, the co-transcriptional regulators Yap1/Taz, and Yap1/Taz target genes. We analysed and compared the resulting developmental and regenerative phenotypes. Our findings highlight that paralogues of core components have distinct, but in some instances overlapping, functions. Intriguingly, we find that Yap1 and Taz have differential roles during development and regeneration. In addition, we characterise and compare two tail fin regenerative paradigms: after both severe and mild injury. These injury paradigms are drastically different and elicit diverse resolution processes. We confirm critical roles of the immune system in the regenerative process. Macrophage recruitment is reduced during severe tail fin regeneration after Yap1 and Taz loss, appearing earlier in yap1 than wwtr1 Crispants and correlating with defective regenerative function. This defective macrophage involvement might therefore be one of the mediators of the deficient regeneration in these two Crispants. Overall, our analysis emphasises distinct requirements and responses of the Hippo pathway during development and across different regenerative paradigms.},
}
@article {pmid41463573,
year = {2025},
author = {Li, Y and Gong, K and Wang, X and Sun, Z and Ding, F},
title = {Heat Shock Transcription Factors as Central Integrators of Plant Stress Responses: From Thermotolerance to Multi-Stress Resilience.},
journal = {Biology},
volume = {14},
number = {12},
pages = {},
pmid = {41463573},
issn = {2079-7737},
support = {ZR2025MS427//Shandong Provincial Natural Science Foundation/ ; 3247180862//National Natural Science Foundation of China/ ; },
abstract = {Heat shock transcription factors (HSFs) have long been recognized for their essential role in mediating thermotolerance via the activation of heat shock proteins (HSPs). Recent studies, however, have significantly broadened this view, revealing that HSFs function as versatile transcriptional regulators orchestrating plant adaptation to a wide range of abiotic and biotic stresses. This review synthesizes current knowledge of HSF structure, activation, and canonical roles in the heat shock response, while emphasizing emerging insights into their diverse functions beyond heat stress. Evidence from both model and crop species demonstrates that many HSFs confer tolerance to a broad range of stresses, including drought, cold, salinity, oxidative stress, and pathogen attack, through intricate crosstalk with hormonal (e.g., ABA, SA, JA) and redox signaling pathways, as well as MAPK-mediated phosphorylation. We also discuss biotechnological strategies such as CRISPR/Cas-mediated genome editing, stress-inducible promoter engineering, and synthetic transcriptional circuits that offer promising avenues for fine-tuning HSF expression and enhancing multi-stress resilience in crops. A deeper understanding of HSF multifunctionality not only advances our comprehension of plant stress biology but also provides a foundation for engineering resilient crops in the context of global climate change.},
}
@article {pmid41465342,
year = {2025},
author = {Sattarov, R and Kuznetsov, A and Klimko, V and Ignatyeva, E and Ivanov, R and Karabelsky, A and Fizikova, A},
title = {The Template-Jumping Editing Approach in F9-Associated Hemophilia B Gene Therapy.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465342},
issn = {1422-0067},
support = {Agreement No. 18-03 on 10 September 2024//"Sirius" Federal Territory "Scientific and technological development of the "Sirius" Federal Territory"/ ; },
mesh = {*Hemophilia B/therapy/genetics ; *Genetic Therapy/methods ; *Gene Editing/methods ; *Factor IX/genetics ; Humans ; CRISPR-Cas Systems ; Mutation ; Animals ; Genetic Vectors/genetics ; },
abstract = {Hemophilia B is a hereditary bleeding disorder caused by mutations localized throughout the F9 gene. Existing gene therapy products containing AAV vectors have significant limitations. Replacement therapy with coagulation factor FIX infusions is not an optimal way of treatment, as patients still have periodic bleeding and require frequent transfusions. Moreover, approximately 5% of adult patients with hemophilia B develop inhibitory antibodies to recombinant forms of FIX. Therefore, it is important to develop universal CRISPR/Cas gene therapy approaches for F9 editing using non-viral delivery systems to enable gene reversion to a functional sequence at an early stage of disease development and establishment of the patients' immune system. In this study, a unique approach of F9 prime-editing was tested for the first time. This method is estimated to edit 7.3% of pathogenic F9 mutation types. Specifically, it targets the gene region encoding amino acids 374 V to 408 Q, which accounts for approximately 9.35% of patients with hemophilia B. An advantage of this gene therapy approach is the absence of the need to change Primer Binding Site (PBS) or Reverse Transcriptase Template (RTT) sequences until going from preclinical to clinical trials, as well as the introduction of gain of function mutations in order to compensate for the low prime-editing frequencies and enhance the effect of treatment in vivo.},
}
@article {pmid41465565,
year = {2025},
author = {Mikhaylova, E and Khusnutdinov, E and Terekhov, M and Pozdeev, D and Gusev, O},
title = {Pig Genome Editing for Agriculture: Achievements and Challenges.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465565},
issn = {1422-0067},
support = {075-15-2025-014 (075-15-2024-666)//The Ministry of Science and Higher Education of Russian Federation/ ; },
mesh = {*Gene Editing/methods ; Animals ; Swine/genetics ; *Agriculture/methods ; CRISPR-Cas Systems ; Breeding/methods ; *Genome ; },
abstract = {The remaining problems in pig farming may no longer be solved with traditional methods. The search for genetic variants associated with desired characteristics and involvement of animals with superior genetics in breeding programs is rarely effective for polygenic traits and pleiotropic genes. The lack of diversity in the germplasm also limits the use of breeding, but some beneficial mutations that did not occur naturally can be introduced manually via genome editing methods. Mutations discovered in other species, such as cattle, can be reproduced in pigs. Traits that were previously pursued for centuries might be achieved by genome editing in a few years. Enormous progress has been made in producing pigs resistant to viruses and in increasing meat productivity and quality. But there are still pressing problems such as lameness and damaging behaviors that probably cannot be solved without genome editing techniques. Their wider application is complicated by the requirement for large amounts of biomaterial, surgical manipulations and cell culture, as well as by the shift towards biomedical research. This review concentrates on the main achievements and challenges in pig agricultural genetics that can be addressed by genome editing.},
}
@article {pmid41465590,
year = {2025},
author = {Nguyen, PD and Nakanishi, K and Nguyen, HP and Nguyen, HV and Kitao, M and Yoshimoto, M and Kamei, K},
title = {Characterisation of the Novel Cutibacterium acnes Phage KIT09 and First Report of CRISPR-Cas-Independent Bacteriophage Resistance in Phylotype IA1.},
journal = {International journal of molecular sciences},
volume = {26},
number = {24},
pages = {},
pmid = {41465590},
issn = {1422-0067},
support = {JPJSCCB20230005//Japan Society for the Promotion of Science/ ; },
mesh = {*Bacteriophages/genetics/isolation &amp; purification/physiology ; *Propionibacterium acnes/virology/genetics ; *CRISPR-Cas Systems ; Humans ; Mutation ; Genome, Viral ; *Propionibacteriaceae/virology ; },
abstract = {Despite being a commensal bacterium, Cutibacterium acnes has been widely considered a major opportunistic pathogen due to its capacity for biofilm production and inflammatory induction, causing device-related, post-implant infections, and skin inflammatory diseases. In this study, we isolated and characterised the novel bacteriophage Cutibacterium acnes phage KIT09 as a potential antimicrobial candidate for the treatment of Cutibacterium acnes-related infections such as acne vulgaris and postsurgical infections. Subsequently, phage-resistant bacterial mutants were generated through phage KIT09 exposure and characterised. Wastewater samples were collected for the isolation of C. acnes phages, followed by their characterisation using C. acnes National Institute of Technology and Evaluation (NITE) Biological Resources Center (NBRC) 107605 (phylotype IA1). Resistant mutants were isolated after prolonged exposure of the newly isolated phage to host bacteria and then characterised. A novel C. acnes phage, designated KIT09, was isolated, demonstrating prolonged bacteriolysis lasting up to 96 h at a multiplicity of infection of 10, and exhibiting high thermal and pH stability. Following sustained selective pressure by phage KIT09, three phage-resistant bacterial isolates were obtained, forming smaller colonies than the wild-type strain, but maintaining a high phage adsorption capacity (>90% after 20 min). Whole-genome sequencing revealed 12 nucleotide mutations across five genes, including six non-synonymous substitutions. Three genes encoding a two-component histidine kinase, DNA processing protein A (DprA), and a ThuA-containing domain protein were mutated in all resistant isolates. Characterisation of the novel phage KIT09 demonstrated its robust lytic activity and environmental stability against C. acnes phylotype IA1. Isolated resistant mutants retained high phage adsorption, accompanied by recurrent mutations in genes encoding a two-component histidine kinase, DprA, and a ThuA-domain protein, suggesting the presence of alternative, CRISPR-Cas-independent resistance mechanisms in C. acnes.},
}
@article {pmid41466059,
year = {2025},
author = {Chen, W and He, P and Ding, L and Lou, W and Wang, Y and Shi, W and Fan, Z and Sheng, Y and Luo, J and Tan, Z and Wang, Y and Di, W and Ke, X and Yu, B},
title = {EPI-SauriCas9-based mouse ovarian cancer models recapitulating pten deletion in patients.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {159},
pmid = {41466059},
issn = {2399-3642},
support = {82403651//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {Animals ; Female ; *PTEN Phosphohydrolase/genetics ; *Ovarian Neoplasms/genetics/pathology/drug therapy ; Mice ; Disease Models, Animal ; Humans ; Gene Deletion ; Tumor Suppressor Protein p53/genetics ; CRISPR-Cas Systems ; },
abstract = {Ovarian cancer remains a deadly gynecological malignancy, with PTEN loss and TP53 mutations frequently implicated in its progression. However, suitable models for studying ovarian cancers with PTEN and TP53 deletions are rare. Here we develop and validate the mouse ovarian epithelium with Pten and Trp53 deletions (MEPP) model using the EPI-SauriCas9 system. We demonstrate the role of Pten loss in promoting tumorigenicity and metastasis. Single-cell RNA sequencing reveals distinct epithelial subpopulations with varying metastatic potential. MEPP also recapitulates key features of human ovarian cancer, including its immune landscape and therapeutic responses. High-throughput drug screening identifies FK228 and thioguanine as promising therapeutic candidates, both of which show in vivo efficacy and are validated in PTEN-deleted organoids. Together, these results establish MEPP as a platform for studying PTEN-deleted ovarian cancer and provide a strategy for generating clinically relevant tumor models through targeted gene editing.},
}
@article {pmid41467478,
year = {2026},
author = {Niazian, M and de Ronne, M and Beauchamp, CJ and Belzile, F and Torkamaneh, D},
title = {CRISPR-induced knockouts reveal a dual role for the soybean NFR5α gene in symbiotic nitrogen fixation and root hair development.},
journal = {The plant genome},
volume = {19},
number = {1},
pages = {e70143},
pmid = {41467478},
issn = {1940-3372},
support = {6548//Genome Canada/ ; 337003//Fonds de recherche du Qu&#xe9;bec - Nature et technologies (FRQNT)/ ; },
mesh = {*Glycine max/genetics/growth &amp; development/microbiology ; *Nitrogen Fixation/genetics ; *Plant Roots/growth &amp; development/genetics ; *Symbiosis/genetics ; CRISPR-Cas Systems ; Gene Knockout Techniques ; *Plant Proteins/genetics/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats ; Plant Root Nodulation/genetics ; },
abstract = {Nitrogen fixation in soybeans, facilitated by symbiotic interactions with rhizobia, is a cornerstone of sustainable agriculture, reducing reliance on synthetic fertilizers. However, the efficiency of symbiotic nitrogen fixation (SNF) varies due to natural genetic variation in SNF-related genes. Our study underscores the pivotal role of the GmNFR5α gene not only in nodulation but also in root hair development, which is crucial for effective nutrient uptake and plant yield. Through detailed genetic analyses and clustered regularly interspaced short palindromic repeats (CRISPR)-based manipulations, we identified and characterized multiple knockout mutants, notably GmNFR5α-KO and combined GmNFR5α+GmROP6-KO, which exhibited significant reductions in root hair density and nodulation. These phenotypic changes correspond with the downregulation of key root hair development genes such as TTG, RHD1, RHD2, and KJK, establishing a clear link between GmNFR5α function and root hair formation. The potential of leveraging these genetic insights to improve nitrogen fixation in legumes and introduce SNF capabilities into cereal crops could revolutionize crop fertilization strategies, offering a sustainable solution to global agricultural challenges.},
}
@article {pmid41467797,
year = {2026},
author = {Ji, HJ and Jang, A-Y and Han, SH and Kim, M-K and Lamien, CE and Wijewardana, V and Ahn, KB and Kim, K-H and Song, JY and Seo, HS},
title = {Accurate serotype identification of Streptococcus pneumoniae using nanopore Cas9-targeted serotype identification (nCATSerotyping).},
journal = {Journal of clinical microbiology},
volume = {64},
number = {2},
pages = {e0098425},
pmid = {41467797},
issn = {1098-660X},
support = {22202MFDS171//Ministry of Food and Drug Safety/ ; 2018E240600//Korea Disease Control and Prevention Agency/ ; 523140-26//Korea Atomic Energy Research Institute/ ; RS-2022-00164721//National Research Foundation of Korea/ ; CRP D32039//International Atomic Energy Agency/ ; },
mesh = {*Streptococcus pneumoniae/classification/genetics/isolation &amp; purification ; Humans ; *Serotyping/methods ; *Pneumococcal Infections/microbiology ; Serogroup ; *Nanopore Sequencing/methods ; Republic of Korea ; CRISPR-Cas Systems ; Pneumococcal Vaccines ; },
abstract = {Streptococcus pneumoniae (pneumococcus) is a leading cause of community-acquired pneumonia and invasive diseases, particularly among children and the elderly. The introduction of pneumococcal conjugate vaccines has significantly reduced invasive pneumococcal disease, but the prevalence of non-vaccine serotypes and newly emerging serotypes is increasing globally. Thus, accurate serotyping is essential for epidemiological surveillance and the development of next-generation multivalent pneumococcal vaccines. Conventional serotyping methods, including multiplex polymerase chain reaction (mPCR), monoclonal antibody (mAb) assays, and Quellung reaction using rabbit antisera, are limited by serotype coverage and cross-reactivity, making the detection of new or emerging serotypes challenging. In this study, we developed a nanopore Cas9-targeted serotyping (nCATSerotyping) platform, which employs Cas9-mediated enrichment of the capsular polysaccharide synthesis locus followed by Oxford Nanopore sequencing. Applying this method to 276 clinical pneumococcal isolates collected in South Korea (2018-2020), we achieved a serotyping success rate of 97.10% (268/276), significantly outperforming conventional methods such as mAb and mPCR, which identified only 76.45% (211/276) of isolates. Whole-genome sequencing of the remaining eight non-typeable isolates revealed them to be non-pneumococcal (oral streptococci), confirming 100% accuracy for S. pneumoniae serotyping. Importantly, our method identified emerging and underrepresented serotypes, including serotype 13 and null capsule clade strains. nCATSerotyping offers a rapid, accurate, and comprehensive solution for pneumococcal serotyping, with significant advantages in identifying novel and non-typeable strains. This scalable platform will be a valuable tool for global serotype surveillance and next-generation multivalent pneumococcal vaccine development.IMPORTANCEAccurate pneumococcal serotyping is critical for vaccine development and epidemiological surveillance, particularly as non-vaccine serotypes emerge following widespread pneumococcal conjugate vaccine implementation. Current serotyping methods face significant limitations in coverage and accuracy, identifying around 76% of pneumococcal isolates and failing to detect emerging serotypes like serotype 13 and null capsule clades. The nanopore Cas9-targeted serotyping platform addresses these critical gaps by achieving 100% serotyping accuracy for confirmed Streptococcus pneumoniae isolates while identifying previously undetectable strains that conventional methods missed. This comprehensive approach is essential for monitoring vaccine effectiveness, understanding serotype replacement patterns, and informing next-generation vaccine development strategies. Furthermore, the identification of misclassified oral streptococci highlights the diagnostic precision needed for accurate pneumococcal surveillance, ensuring that epidemiological data accurately reflect true pneumococcal disease burden and serotype distribution patterns.},
}
@article {pmid41468058,
year = {2026},
author = {Vasileva, A and Abramova, M and Selkova, P and Arseniev, A and Musharova, O and Malysheva, P and Demkina, A and Khodorkovskii, M and Severinov, K},
title = {Streptococcus uberis Cas9-A Compact Type II-A Nuclease Recognizing a Unique PAM and Functional in Human Cells.},
journal = {The CRISPR journal},
volume = {9},
number = {1},
pages = {21-35},
doi = {10.1177/25731599251404417},
pmid = {41468058},
issn = {2573-1602},
mesh = {Humans ; *Streptococcus/genetics/enzymology ; *CRISPR-Cas Systems ; Gene Editing/methods ; *CRISPR-Associated Protein 9/metabolism/genetics ; Clustered Regularly Interspaced Short Palindromic Repeats ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Endonucleases/metabolism/genetics ; Animals ; *Bacterial Proteins/genetics/metabolism ; Cattle ; },
abstract = {Several type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 RNA-guided nucleases are commonly used for genome engineering. Their relatively large size and requirements for specific protospacer adjacent motif (PAM) sequences flanking their targets prompt continuous searches for additional more compact Cas9 enzymes with new PAM specificities. Here, we present SuCas9, a compact nuclease from Streptococcus uberis, a bacterium inhabiting the mammary glands of dairy cattle. SuCas9 recognizes a novel 5'-NNAAA-3' PAM, efficiently cleaves DNA in vitro, and is active in human cells. SuCas9 thus expands the available genome editing toolset and may find biotechnological and medicinal applications in the future.},
}
@article {pmid41468306,
year = {2026},
author = {Wang, Y and Li, Y and Li, J and Li, M and Qiu, X},
title = {Silencing of optogenetic and chemogenetic transgenes in human iPSCs involves promoter methylation and methylation-independent mechanisms.},
journal = {Epigenetics},
volume = {21},
number = {1},
pages = {2606983},
pmid = {41468306},
issn = {1559-2308},
mesh = {Humans ; *Promoter Regions, Genetic ; *Induced Pluripotent Stem Cells/metabolism/cytology ; HEK293 Cells ; *DNA Methylation ; Optogenetics ; *Transgenes ; *Gene Silencing ; CRISPR-Cas Systems ; DNA Transposable Elements ; Gene Editing ; Chemogenetics ; },
abstract = {The transplantation of neural progenitor cells derived from induced pluripotent stem cells (iPSCs) has therapeutic potential for the treatment of neurological diseases. However, the functional integration of transplanted iPSC-derived neurons into host neural networks remains controversial. Optogenetic and chemogenetic tools offer the means to assess such integration. However, constructing modifiable iPSC-derived neurons requires efficient gene editing. Here, we used CRISPR/Cas9 (targeting the AAVS1 safe harbor) and PiggyBac transposon systems to insert optogenetic and chemogenetic receptors (ChR2/hM4Di) into human iPSCs. While both systems successfully integrated genes into the genomes of HEK293T cells and iPSCs, receptor expression was detected only in HEK293T cells. Bisulfite sequencing revealed extensive methylation of the TRE3G BI promoter (95.3-98.2%) in iPSCs, in contrast to low methylation (5.9%) in HEK293T cells. For PiggyBac, the methylation of CMV/EF1α promoters in iPSCs exhibited integration site-dependent variability (0-95.2%). Notably, even hypomethylated clones failed to show gene expression, suggesting that additional regulatory mechanisms, such as histone modifications or chromatin remodeling, may contribute to transcriptional silencing. Differentiation into neural stem cells does not reverse methylation nor restore protein expression. Our findings demonstrate that the CRISPR/Cas9 and PiggyBac systems enable the integration of optochemical receptor genes into iPSCs. However, promoter methylation or other epigenetic and non-epigenetic gene-silencing mechanisms could pose barriers to efficient protein expression from the integrated transgene in iPSCs.},
}
@article {pmid41468840,
year = {2026},
author = {Shi, T and Jin, X},
title = {Probing neuropsychiatric disorders through in vivo CRISPR screening.},
journal = {Current opinion in genetics &amp; development},
volume = {96},
number = {},
pages = {102424},
pmid = {41468840},
issn = {1879-0380},
support = {R01 HG012819/HG/NHGRI NIH HHS/United States ; R01 MH137042/MH/NIMH NIH HHS/United States ; },
mesh = {Humans ; Animals ; *Mental Disorders/genetics/pathology ; Mice ; *CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Single-Cell Analysis/methods ; Genetic Predisposition to Disease ; Schizophrenia/genetics ; Brain/pathology/metabolism ; },
abstract = {Although there are many known risk alleles associated with adult-onset psychiatric disorders such as schizophrenia [1-4], bipolar disorder [5-7], and major depressive disorder [8-10], the mechanistic links between these risk alleles and disease pathology, especially on a circuit-level, remain unclear. In vivo pooled CRISPR screening with single‑cell readout (in vivo Perturb‑seq) has begun to fill this gap by mapping causal genes to defined cell states directly in animal tissues [11-14]. Here, we review recent developments and applications of in vivo Perturb-seq in the mouse brain and highlight the potential of utilizing human cellular systems to extend these approaches. Additionally, we discuss how in vivo Perturb-seq can couple genetic perturbation with physiological or environmental perturbations to better model psychiatric diseases with environmental triggers.},
}
@article {pmid41468873,
year = {2026},
author = {Zhang, J and Zhang, X and Xie, X and Han, B and Zhao, F and Yang, X and Zhang, W and Jiang, Y and Zhang, X},
title = {CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform based on a lightful copper nanocluster for highly sensitive detection of mycotoxin.},
journal = {Talanta},
volume = {301},
number = {},
pages = {129326},
doi = {10.1016/j.talanta.2025.129326},
pmid = {41468873},
issn = {1873-3573},
mesh = {*Copper/chemistry ; *Biosensing Techniques/methods ; *Aflatoxin B1/analysis ; *CRISPR-Cas Systems ; *Metal Nanoparticles/chemistry ; Limit of Detection ; Food Contamination/analysis ; Fluorescence ; *Mycotoxins/analysis ; },
abstract = {The problem of mycotoxin contamination in foodstuffs has attracted widespread attention and posed a great threat to human health. Therefore, the sensitive and effective detection of mycotoxins is of great importance for preserving public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform was constructed for highly sensitive and fast detection of aflatoxin B1. The copper nanoclusters were synthesized with marker-free DNA single strands within 5 min, showing the outstanding fluorescence properties. With the existence of aflatoxin B1, the released complementary DNA (cDNA) triggered multiple isothermal amplification reaction. Subsequently, the obtained amplification products triggered the trans-cleavage activity of CRISPR/Cas12a system, which degraded the DNA single strands for synthesis of copper nanoclusters, leading to a decreased fluorescent signal. Benefiting from good fluorescence properties of copper nanoclusters, the established biosensing platform for aflatoxin B1 detection had a high specificity and a limit of detection of 47.51 pg/mL in the linear range of 0.05-10 ng/mL. The proposed platform provided a new insight for the detection of non-nucleic acid targets.},
}
@article {pmid41469506,
year = {2025},
author = {Wang, T and Tian, Y and Yin, R and Li, M and Luo, J and Yang, Y and Zhang, C and Chen, H and Wang, Y and Lu, D},
title = {In vivo genome editing with a novel Cj4Cas9.},
journal = {Communications biology},
volume = {9},
number = {1},
pages = {152},
pmid = {41469506},
issn = {2399-3642},
support = {82070258//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Gene Editing/methods ; Animals ; Mice ; *CRISPR-Cas Systems ; Proprotein Convertase 9/genetics ; *CRISPR-Associated Protein 9/genetics/metabolism ; Humans ; },
abstract = {Natural CRISPR-Cas9 systems provides diverse properties for genome editing, yet finding compact variants remains a priority. In this study, we screened a panel of 11 CjCas9 orthologous using a GFP activation assay and identified seven active nucleases. Among these, Cj4Cas9 stood out as particularly noteworthy due to its compact genome size (985 amino acids) and unique PAM preference (5'-NNNGRY-3'). Cj4Cas9 demonstrates efficient disruption of the Tyr gene in mouse zygotes, resulting in an albino phenotype. Furthermore, when delivered via AAV8, Cj4Cas9 achieves efficient genome editing of the Pcsk9 gene in mouse liver, leading to reduced serum cholesterol and LDL-C levels. Seeking to further expand its utility, we engineered Cj4Cas9 for higher activity by introducing L58Y/D900K mutations, resulting in a variant termed enCj4Cas9. This variant exhibits a two-fold increase in nuclease activity compared to the wild-type Cj4Cas9 and recognizes a simplified N3GG PAM, considerably expanding its targeting scope. These findings establish Cj4Cas9 and its engineered variants for fundamental research and therapeutic applications.},
}
@article {pmid41471176,
year = {2025},
author = {Bhowmik, S and Rivu, S and Bari, ML and Ahmed, S},
title = {Genome Mining of Cronobacter sakazakii in Bangladesh Reveals the Occurrence of High-Risk ST83 and Rare ST789 Lineages.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
pmid = {41471176},
issn = {2076-0817},
support = {BIO-34//University Grant Commission, Bangladesh/ ; },
mesh = {Bangladesh/epidemiology ; Humans ; *Cronobacter sakazakii/genetics/isolation &amp; purification/classification/pathogenicity ; *Genome, Bacterial ; *Enterobacteriaceae Infections/microbiology/epidemiology ; Virulence Factors/genetics ; Infant ; Food Microbiology ; Plasmids/genetics ; Infant, Newborn ; Whole Genome Sequencing ; Phylogeny ; Infant Formula/microbiology ; },
abstract = {Cronobacter sakazakii is a foodborne pathogen of major concern due to its link with severe neonatal infections through powdered infant formula (PIF). However, its genomic epidemiology in Bangladesh remains uncharacterized. We report the first whole-genome analysis of three isolates from PIF. Two isolates (S41_PIFM and S44_RUTF) belonged to ST83, a lineage repeatedly associated with neonatal meningitis, septicemia, and persistence in PIF production environments, while the third (S43_TF) represented ST789, a recently described and rare lineage of unknown pathogenic potential. Pan-genome and comparative analyses identified 39 virulence determinants, 19 antimicrobial-resistance genes, and diverse mobile genetic elements. ST83 isolates harbored plasmid replicons IncFII(pCTU2) and pESA2, while the ST789 isolate carried insertion sequence ISKpn34, indicating horizontal gene transfer potential. All strains encoded I-E CRISPR-Cas systems. The detection of globally recognized high-risk ST83 clones alongside the novel ST789 lineage highlights emerging public health risks. This study provides the first genomic insights into C. sakazakii in Bangladesh and underscores the urgent need for genomic surveillance and strengthened food safety monitoring to protect infant health in low- and middle-income countries.},
}
@article {pmid41471222,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {From Methylomes to CRISPR Epigenetic Editing: New Paths in Antibiotic Resistance.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
pmid = {41471222},
issn = {2076-0817},
mesh = {*Gene Editing/methods ; *Epigenesis, Genetic ; *Bacteria/genetics/drug effects ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; DNA Methylation ; *Epigenome ; *Drug Resistance, Microbial/genetics ; Gene Expression Regulation, Bacterial ; Epigenome Editing ; },
abstract = {Antibiotic resistance (AR) has long been interpreted through the lens of genetic mutations and horizontal gene transfer. Yet, mounting evidence suggests that epigenetic regulation, including DNA and RNA methylation, histone-like proteins, and small non-coding RNAs, plays a similarly critical role in bacterial adaptability. These reversible modifications reshape gene expression without altering the DNA sequence, enabling transient resistance, phenotypic heterogeneity, and biofilm persistence under antimicrobial stress. Advances in single-molecule sequencing and methylome mapping have uncovered diverse DNA methyltransferase systems that coordinate virulence, efflux, and stress responses. Such epigenetic circuits allow pathogens to survive antibiotic exposure, then revert to susceptibility once pressure subsides, complicating clinical treatment. Parallel advances in CRISPR-based technologies now enable direct manipulation of these regulatory layers. CRISPR interference (CRISPRi) and catalytically inactive dCas9-fused methyltransferases can silence or reactivate genes in a programmable, non-mutational manner, offering a new route to reverse resistance or sensitize pathogens. Integrating methylomic data with transcriptomic and proteomic profiles further reveals how epigenetic plasticity sustains antimicrobial tolerance across environments. This review traces the continuum from natural bacterial methylomes to engineered CRISPR-mediated epigenetic editing, outlining how this emerging interface could redefine antibiotic stewardship. Understanding and targeting these reversible, heritable mechanisms opens the door to precision antimicrobial strategies that restore the effectiveness of existing drugs while curbing the evolution of resistance.},
}
@article {pmid41472024,
year = {2025},
author = {Yi, M and Hu, Y and Fan, B and Pan, Y and Pan, B and Wang, J and Liu, Z},
title = {Advances in Novel Detection Technologies for Occult Hepatitis B Virus Infection: Building an Ultra-Sensitive Barrier for Transfusion Safety.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472024},
issn = {2076-2607},
abstract = {Occult hepatitis B virus infection (OBI), characterized by extremely low viral loads and the persistent intrahepatic presence of cccDNA, poses a profound challenge to global public health security. With a prevalence ranging from 0.06% to over 15% in different donor populations, OBI maintains a risk of transmission and can progress to hepatocellular carcinoma. Its prevention and control have long been limited by the sensitivity constraints of conventional detection methods, highlighting the urgent need for more sensitive diagnostic innovations. Emerging technologies offer distinct breakthroughs: ddPCR facilitates absolute quantification; CRISPR-Cas systems coupled with isothermal amplification enable rapid, point-of-care testing; third-generation sequencing resolves viral integration and mutations; and nanomaterials enhance the signal detection. This review synthesises advancements in OBI diagnostic technologies and provides a comparative overview of their strengths, limitations, and transfusion safety implications, as well as their potential applications in blood transfusion. Recommendations are also proposed to inform the advancement of OBI risk control in blood transfusion and to guide the development of novel diagnostic technologies, particularly relevant to regions with high HBV endemicity, such as China.},
}
@article {pmid41472049,
year = {2025},
author = {Slukin, PV and Fursov, MV and Volkov, DV and Sizova, AA and Detushev, KV and Dyatlov, IA and Fursova, NK},
title = {Diversity of CRISPR-Cas Systems Identified in Urological Escherichia coli Strains.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472049},
issn = {2076-2607},
support = {075-15-2025-525 of 30.05.2025.//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Type I-E and I-F CRISPR-Cas systems were identified in 237 E. coli strains isolated from patients with urinary tract infections (UTIs) between 2004 and 2019. The strains were classified into nine distinct groups (I-IX) based on the presence or absence of cas genes and repeat regions (RRs). Within the type I-E systems, two sequence variants were identified, distinguished by polymorphisms in the casB, cas3, cas7, cas5, and cas6 genes. The direct repeats (DRs) also differed, with I-E-associated RRs ranging from 26 to 32 bp and I-F-associated RRs consistently being 28 bp. We identified 762 unique spacers (29-35 bp in length) across the strain collection, while the number of spacers per strain varied from 1 to 47, and potential DNA targets were determined for 65 spacers, targeting 38 bacteriophage genomes, 19 plasmids, and 8 cas genes of the I-F type CRISPR-Cas system. Multilocus sequence typing (MLST) revealed 68 sequence types and 24 clonal complexes (CCs), with the most prevalent being ST131, CC10, CC69, CC405, CC14, CC38, CC73, and CC648. Significant correlations were observed between specific phylogroups/CCs, the type of CRISPR-Cas system present, and distinct profiles of virulence and antibiotic resistance genes.},
}
@article {pmid41472204,
year = {2025},
author = {Dong, J and He, X and Bao, S and Wei, Z},
title = {Diagnostic Methods for Bovine Coronavirus: A Review of Recent Advancements and Challenges.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472204},
issn = {1999-4915},
support = {KJZC-2024-15//Gansu Provincial Department of Agriculture and Rural Affairs Science and Technology Support Project/ ; },
mesh = {Animals ; Cattle ; *Coronavirus, Bovine/genetics/isolation &amp; purification ; *Cattle Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Coronavirus Infections/diagnosis/veterinary/virology ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; CRISPR-Cas Systems ; },
abstract = {Bovine coronavirus(BCoV) is a significant pathogen causing substantial economic losses in the cattle industry through increased calf mortality, reduced growth performance, and decreased milk yield. Rapid and accurate diagnostic methods are therefore essential for controlling BCoV transmission. Current diagnostic methods comprise two primary categories: conventional techniques and cutting-edge innovations. Conventional approaches, including molecular methods like RT-PCR/qRT-PCR and immunological assays such as ELISA and neutralization tests, remain the main diagnostic methods. However, they are limited by laboratory dependency as well as the necessary balance between speed and sensitivity. These limitations have promoted the development of innovative methods, including isothermal amplification, CRISPR/Cas systems, droplet digital PCR, and integrated platforms. This review comprehensively analyzes the advantages, limitations, and applications of current diagnostic methods, highlighting integrated platforms such as RPA-CRISPR-LFA and microfluidics-based LFA. These innovations bridge critical performance gaps by enhancing sensitivity and specificity while enabling field application, demonstrating significant potential as next-generation point-of-care diagnostics for managing this economically critical pathogen.},
}
@article {pmid41472250,
year = {2025},
author = {Tan, C and Xing, S and Xie, X and Chen, X and Liu, X and Wang, W and Liu, L and Zhou, X and Wu, J and Li, C},
title = {Development and Application of a Rapid Field Detection Technology for DENV-2 Based on the HUDSON Nucleic Acid Extraction-Free/RT-RAA/CRISPR-Cas12a System.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472250},
issn = {1999-4915},
support = {2024YFC2607800//Chunxiao-Li/ ; },
mesh = {*Dengue Virus/genetics/isolation &amp; purification/classification ; *Dengue/diagnosis/virology ; *CRISPR-Cas Systems ; Humans ; Sensitivity and Specificity ; Animals ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Dengue fever has become a major global public health challenge due to its rapidly in-creasing incidence. Rapid on-site detection of dengue virus (DENV) is critical for early diagnosis, timely patient isolation, and outbreak control. In this study, dengue virus serotype 2 (DENV-2), the predominant strain circulating in tropical and subtropical regions, was selected as the target pathogen. We established a one-tube rapid detection assay that integrates the HUDSON nucleic acid extraction-free protocol, reverse transcription recombinase-aided amplification (RT-RAA), and CRISPR/Cas12a-mediated trans cleavage activity. The method achieved a detection limit of 1 × 10[2] copies/μL for simulated infected samples and exhibited no cross-reactivity with other DENV serotypes (DENV-1, DENV-3, DENV-4) or with other arboviruses, including Zika, Japanese encephalitis, yellow fever, and chikungunya viruses. The assay demonstrated high sensitivity and specificity across various sample types, including mosquitoes, rodents, blood, and cultured cells, with results consistent with quantitative PCR (qPCR). Requiring only basic equipment such as a water bath, the system enables on-site detection of DENV-2 within 1 h. This simple, cost-effective, and reliable assay provides a practical tool for field-based DENV-2 surveillance and supports effective public health responses in resource-limited settings.},
}
@article {pmid41472258,
year = {2025},
author = {Gladue, DP and O'Mahony, A},
title = {CRISPR Treatments for AI-Designed Synthetic Viruses: Rapid Programmable Countermeasures for Emerging and Engineered Viruses.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472258},
issn = {1999-4915},
mesh = {Humans ; *CRISPR-Cas Systems ; Genome, Viral ; *Artificial Intelligence ; *Synthetic Biology/methods ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Viruses/genetics ; Animals ; Genetic Engineering/methods ; },
abstract = {The convergence of artificial intelligence and synthetic biology is innovating and accelerating the design of novel viral genomes, expanding both therapeutic opportunities and dual-use risk. This review articulates a countermeasure strategy for emerging and engineered viruses leveraging the programmable CRISPR modality. Building on mounting in vitro and in vivo evidence that Cas9 degrades DNA viruses (e.g., Orthopoxviruses, HSV-1, ASFV), while Cas13 targets RNA viral genomes (e.g., Influenza A, Dengue, RSV), both leading to reduced viremia, diminished disease burden, and alleviated symptoms. Here, we outline a rapid-response pipeline to position CRISPR-based countermeasures in translational and pandemic-response frameworks, linking real-time sequencing to AI-assisted gRNA selection and multiplexed cassette design to achieve viral targeting efficacy. To minimize resistance and off-target risk, we emphasize multi-gRNA cocktails, continuous genomic surveillance, and adaptive gRNA rotation. We also propose governance mechanisms, such as pre-cleared gRNA repositories, transparent design logs, standardized off-target/safety screening, and alignment with evolving nucleic-acid-synthesis screening frameworks to enable emergency deployment while preserving security. Furthermore, compressing the time from sequence to treatment and complementary to vaccines and small-molecule antivirals, CRISPR represents a technologically agile and strategically essential capability to combat both natural outbreaks and AI-enabled biothreats. Collectively, programmable CRISPR antivirals represent an auditable, rapidly adaptable foundation for next-generation biodefense preparedness.},
}
@article {pmid41472363,
year = {2026},
author = {Ajibaye, O and Dada, IS and Mbye, H and Nwankwo, IC and Oriero, E and Amambua-Ngwa, A},
title = {Innovative approach in malaria research: Harnessing CRISPR-Cas9 for antimalarial drug-resistance studies in Africa.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {4},
pages = {1909-1920},
pmid = {41472363},
issn = {1525-0024},
mesh = {*CRISPR-Cas Systems ; Humans ; *Antimalarials/therapeutic use/pharmacology ; *Drug Resistance/genetics ; Gene Editing/methods ; Africa ; *Plasmodium falciparum/genetics/drug effects ; *Malaria/drug therapy/parasitology/epidemiology/genetics ; Animals ; Malaria, Falciparum/drug therapy/parasitology ; },
abstract = {Malaria remains a major global health challenge, particularly in sub-Saharan Africa, where Plasmodium falciparum is increasingly resistant to frontline antimalarial therapies. CRISPR-Cas9, a groundbreaking genome editing tool, has become a transformative force in biomedical research, offering valuable insights into the genetic mechanisms underlying drug resistance. This review provides a comprehensive overview of the CRISPR-Cas9 system, its origin, evolution, and application in antimalarial drug-resistance research, with particular emphasis on African studies. We examine the role of CRISPR in elucidating resistance pathways, validating molecular markers, and enhancing diagnostic strategies. Additionally, a systematic scoping review highlights the geographic scope and focus of CRISPR-related malaria research conducted across Africa. Significantly low application and adoption were observed. Despite existing technological and regulatory barriers, CRISPR holds strong potential for accelerating antimalarial drug discovery and advancing precision medicine. Its strategic application in malaria-endemic regions could significantly bolster efforts toward disease control and eventual elimination.},
}
@article {pmid41474015,
year = {2026},
author = {Cheng, X and Dong, J and Jain, P and Qin, S and Miao, Y and Liu, K and Theja, MLV and Butch, CJ and Wang, Y and Lane, LA},
title = {Ultrasound Activated Hybrid-Biomimetic Nanocarriers That Combine Tumor-Confined CRISPR/Cas9 Metabolic Reprogramming and Cuproptosis With Anticancer Macrophage Polarization.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {10},
pages = {e10436},
doi = {10.1002/smll.202510436},
pmid = {41474015},
issn = {1613-6829},
support = {113-2221-E-038-004//National Science and Technology Council/ ; 114-2221-E-038-019//National Science and Technology Council/ ; TMU111-AE1-B12//Taipei Medical University/ ; 113FRP-28//Taipei Medical University Shuang Ho Hospital Special Research Plan/ ; 114FRP-26//Taipei Medical University Shuang Ho Hospital Special Research Plan/ ; 82127806//National Natural Science Foundation of China/ ; 2022300326//Fundamental Research Funds for the Central Universities/ ; 0213-14380238//Fundamental Research Funds for the Central Universities/ ; 202205033//Nanjing Life and Health Science and Technology Special Project/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *Macrophages/metabolism/drug effects ; Humans ; Animals ; *Nanoparticles/chemistry ; Metal-Organic Frameworks/chemistry ; Cell Line, Tumor ; *Copper/chemistry ; Mice ; *Biomimetic Materials/chemistry ; *Ultrasonic Waves ; *Neoplasms/pathology ; *Drug Carriers/chemistry ; *Biomimetics/methods ; Metabolic Reprogramming ; },
abstract = {Nanomedicine aims to develop nanocarriers that provide strong cell selectivity and efficient intracellular delivery. Additionally, therapeutic strategies are expanding to include metabolic pathways to trigger apoptosis and reduce tumor growth, especially in cases resistant to conventional chemotherapy. Here, we have created nanocarriers with hybrid-biomimetic coatings that, upon ultrasound activation, release encapsulated copper-based metal-organic frameworks (MOFs) and COP1 gene knockout Cas9 ribonucleoproteins (RNPs). This hybrid-membrane coating, which combines tumor and immune cell membranes with perfluorocarbons, enhances tumor-to-normal cell uptake and allows for controlled release and cytolytic entry of the nanocarrier contents. We observe that the RNPs efficiently knockout the COP1 gene, thereby arresting the cancer cell cycle in the G0/G1 phase and promoting mitochondrial respiration over anaerobic glycolysis. This increased respiration makes cancer cells more susceptible to cuproptosis triggered by the MOFs and decreases tumor lactate levels, preventing lactate-driven M2 polarization of tumor-infiltrating macrophages. Furthermore, the nanocarriers' cellular selectivity leaves macrophages unharmed. These effects enable infiltrating macrophages to retain an anticancer M1 polarization and continue to foster a more active immune response. The combination of tumor-specific genetic metabolic reprogramming and enhanced cuproptosis activity, along with increased immune activity, results in significant tumor growth suppression and improved survival rates.},
}
@article {pmid41474367,
year = {2026},
author = {Rasool, HMH and Gong, X and Jin, Y and Liu, M and Yanyan, W and Qu, M and Anxiang, Z and Lingling, X and Li, Z and Zhou, J and Chen, Q},
title = {Development of Visual and Fluorescence Detection Method of Brucella by RPA-CRISPR/Cas12a Assay.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {1},
pages = {e71397},
doi = {10.1096/fj.202503610R},
pmid = {41474367},
issn = {1530-6860},
support = {2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; 2024BEG-02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2024312885//Comprehensive Control Technology For Brucellosis and Hydatic Disease in Xinjiang/ ; CAAS-ZDRW202410//The Agricultural Science and Technology Innovation Program (ASTIP)/ ; },
mesh = {*Brucella/genetics/isolation &amp; purification ; Animals ; *Brucellosis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; Cattle ; Fluorescence ; Limit of Detection ; Humans ; },
abstract = {Brucella is a significant pathogen in the livestock industry, causing Brucellosis, a zoonotic disease that leads to considerable health and economic losses in both humans and animals. Current diagnostic methods for Brucella, including culture, serological assays, and PCR/qPCR, are valuable tools but have inherent limitations. These include the requirement for BSL-3 laboratories, trained personnel, complex procedures, expensive equipment, issues with sensitivity and specificity, and the time-consuming nature of assays, making them unsuitable for large-scale epidemiological screening. Therefore, there is a critical need to develop a rapid, portable, and cost-effective diagnostic method with high specificity and sensitivity. In this study, we established a rapid, portable, reliable, and inexpensive detection method for Brucella genus identification based on RPA-CRISPR/Cas12a technology. Specific RPA primers and crRNA sequences were designed targeting the bcsp31 gene of Brucella. Subsequently, both a fluorescence assay and a lateral flow strip (LFS) assay were developed after optimizing the conditions using the RPA-CRISPR/Cas12a system. The limit of detection (LoD) was 1 copy/μL for RPA-CRISPR/Cas12a-F and 10 copies/μL for RPA-CRISPR/Cas12a-LFS and the entire assay was completed in less than 30 min. This method demonstrated excellent specificity in distinguishing Brucella from other closely related pathogens. Moreover, the RPA-CRISPR/Cas12a assay showed high concordance with classical quantitative real-time PCR when testing diverse clinical samples (blood, serum, milk, semen, vaginal secretions). Together, these findings make this method a promising tool for Brucella detection, with potential applications in both field surveillance and clinical diagnostics.},
}
@article {pmid41474623,
year = {2026},
author = {Banh, DV and Goldberg, GW and Marraffini, LA},
title = {Phage induction of Staphylococcus aureus pathogenicity islands promotes the CRISPR-Cas adaptive immune response.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116776},
pmid = {41474623},
issn = {2211-1247},
support = {R01 GM149834/GM/NIGMS NIH HHS/United States ; },
mesh = {*Staphylococcus aureus/virology/genetics/immunology/pathogenicity ; *CRISPR-Cas Systems/genetics/immunology ; *Genomic Islands/genetics ; *Adaptive Immunity ; *Staphylococcus Phages/genetics ; Animals ; *Bacteriophages ; Mice ; },
abstract = {Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements carrying virulence genes that spread upon infection by helper phages that induce their transfer. Staphylococci also carry type II and III CRISPR-Cas systems that mount an adaptive immune response against phages through the acquisition of spacer sequences from viral genomes, directing Cas nucleases to their targets. Whether and how SaPIs and CRISPR interact with each other during helper phage infection is not known. Here we report that, as a result of the packaging of incomplete helper phage genomes into SaPI particles, defective viral DNA delivered into new hosts stimulates spacer acquisition in both CRISPR types. Once immunized, staphylococci target the helper phage and prevent SaPI mobilization. Our work reveals an unexpected synergy between CRISPR-Cas systems and SaPIs that enhances antiphage immunity and could favor the retention of beneficial elements within the population.},
}
@article {pmid41474879,
year = {2026},
author = {Cheng, M and Wang, Y and Lin, W and Ye, J and Wu, M and Xiang, B and Liu, L and Sun, B},
title = {A Universal Light-Activated CRISPR-RNA Based on Split Direct Repeat for One-Pot Cas12a Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {98},
number = {1},
pages = {706-716},
doi = {10.1021/acs.analchem.5c05722},
pmid = {41474879},
issn = {1520-6882},
mesh = {*CRISPR-Cas Systems ; *Light ; *CRISPR-Associated Proteins/genetics/metabolism ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; *RNA/genetics ; },
abstract = {Spatiotemporal regulation of CRISPR-Cas systems holds significant promise for precision gene editing and molecular diagnostics. While photochemical strategies for CRISPR activity control have advanced, a universal regulatory approach remains elusive. Here, we report a modular light-activated CRISPR-RNA design through splitting conventional crRNA within the direct repeat (DR) into two functional domains: a conserved 5' split direct repeat (5' SDR) and a variable 3' split direct repeat (SDR) + spacer (3' SDR-Spacer) module. Double-stranded extensions were introduced at the cleavage site to preserve functional integrity. Through screening of light-sensitive caging group modification sites in the universal 5' SDR, a novel light-activated CRISPR-RNA system was developed. This system only requires spacer redesign of the 3' SDR-Spacer for new targets, while the caged 5' SDR is universal. Thereupon, we established a universal light-activated CRISPR-RNA assisted one-pot RAA-Cas12a detection system (UniLight-CRISPR). When applied to Mycoplasma pneumoniae detection using qPCR-validated clinical samples, UniLight-CRISPR demonstrated 95.45% sensitivity and 100% specificity, matching the performance of conventional two-step Cas12a assays. This universal photo regulation strategy not only addresses current limitations in CRISPR diagnostics but also provides a blueprint for adapting other Cas enzymes. We anticipate broad applications of our universal light-activated CRISPR-RNA system, extending from molecular diagnostics to gene-editing research.},
}
@article {pmid41475278,
year = {2026},
author = {Kolanchi, P and Saminathan, N and Selvaraj, D and Krishnamoorthy, A and Palanivelu, K and Aruchalam, A},
title = {Bioprocess and genetic advances enhancing Beauveria bassiana biocontrol efficacy.},
journal = {Microbial pathogenesis},
volume = {211},
number = {},
pages = {108272},
doi = {10.1016/j.micpath.2025.108272},
pmid = {41475278},
issn = {1096-1208},
mesh = {*Beauveria/genetics/pathogenicity/metabolism/physiology ; *Pest Control, Biological/methods ; Virulence ; Animals ; Insecta/microbiology ; *Biological Control Agents ; Gene Editing ; Secondary Metabolism ; },
abstract = {Beauveria bassiana is a widely exploited entomopathogenic fungus that has emerged as a central component of ecologically sustainable pest management. Recent years have witnessed rapid progress across its biological understanding, technological development, and application potential. This review synthesizes contemporary advances spanning infection biology, secondary metabolite biosynthesis, strain development, bioprocess engineering, formulation science, and genetic improvement. At the molecular level, multi-omics studies have elucidated the coordinated regulation of surface adhesion, cuticular penetration, host immune modulation, dimorphic transitions, and toxin production, revealing gene networks that govern virulence, stress tolerance, and ecological adaptation. These insights have informed improved strategies for strain isolation and high-throughput phenotypic screening, enabling the selection of isolates with enhanced pathogenicity, environmental robustness, and endophytic competence. Parallel advances in solid-state and submerged fermentation, supported by agro-industrial substrates and data-driven optimization, have strengthened large-scale production of infective propagules with consistent quality. Such gains are further reinforced by modern formulation approaches, including oil-based dispersions, encapsulation systems, nanoemulsions, and seed-coating technologies, which collectively improve spore stability, persistence, and delivery under heterogeneous field conditions. More recently, CRISPR/Cas-based genome editing and pathway engineering have opened new avenues for precision enhancement of virulence traits, metabolic output, and abiotic stress resilience. Despite these achievements, the broader adoption of B. bassiana remains constrained by variable field performance, slower speed of action relative to chemical insecticides, strain-dependent efficacy, and regulatory and quality-control challenges. By integrating fundamental biology with technological innovation and practical limitations, this review provides a coherent framework for advancing B. bassiana from laboratory optimization to reliable field implementation, underscoring its promise as a next-generation, environmentally aligned biocontrol agent in modern agriculture.},
}
@article {pmid41475346,
year = {2026},
author = {Behera, AK and Kim, JJ and Kordale, S and Pekovic, F and Damodaran, AP and Kumari, B and Vidak, S and Dickson, E and Xiao, MS and Duncan, G and Andresson, T and Misteli, T and Valkov, E and Gonatopoulos-Pournatzis, T},
title = {RNA-coupled CRISPR screens reveal ZNF207 as a regulator of LMNA aberrant splicing in progeria.},
journal = {Molecular cell},
volume = {86},
number = {1},
pages = {41-59.e15},
pmid = {41475346},
issn = {1097-4164},
support = {ZIA BC012019/ImNIH/Intramural NIH HHS/United States ; },
mesh = {Humans ; *Progeria/genetics/metabolism/pathology ; *Lamin Type A/genetics/metabolism ; *Alternative Splicing ; *CRISPR-Cas Systems ; Ribonucleoprotein, U1 Small Nuclear/metabolism/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA Precursors/genetics/metabolism ; Zinc Fingers ; },
abstract = {Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-seq (CRISPR-based identification of regulators of alternative splicing with phenotypic sequencing), a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitatively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-seq identified both known and untested regulators, enriched for proteins involved in RNA splicing and metabolism. As a proof-of-concept, CRASP-seq analysis of the LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin protein levels in patient-derived cells. We further show that ZNF207's zinc-finger domain broadly impacts alternative splicing through direct interactions with U1 small nuclear ribonucleoprotein (snRNP) components. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-seq to uncover key regulators and domains of alternative splicing.},
}
@article {pmid41475348,
year = {2026},
author = {Gao, X and Zhu, K and Zhang, W and Wang, L and Wang, L and Hua, L and Niu, T and Qin, B and Yu, X and Zhu, H and Cui, S},
title = {RNA anti-CRISPRs deplete Cas proteins to inhibit the CRISPR-Cas system.},
journal = {Molecular cell},
volume = {86},
number = {2},
pages = {317-331.e5},
doi = {10.1016/j.molcel.2025.12.005},
pmid = {41475348},
issn = {1097-4164},
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/genetics/metabolism/chemistry ; Cryoelectron Microscopy ; *Pectobacterium/genetics/metabolism/enzymology ; *Bacterial Proteins/genetics/metabolism/chemistry ; *RNA, Bacterial/genetics/metabolism/chemistry ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Protein Binding ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Models, Molecular ; Nucleic Acid Conformation ; Protein Conformation ; },
abstract = {RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.},
}
@article {pmid41475352,
year = {2026},
author = {Martinho, C and Hoshino, M and Raphalen, M and Bukhanets, V and Kerur, A and Bogaert, KA and Luthringer, R and Coelho, SM},
title = {Efficient CRISPR-Cas genome editing in brown algae.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101273},
pmid = {41475352},
issn = {2667-2375},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Phaeophyceae/genetics ; Polyethylene Glycols/chemistry ; },
abstract = {Brown algae represent the third most complex lineage to have independently evolved multicellularity, distinct from plants and animals. Yet, functional studies of their development and evolution have been limited by the absence of efficient genome editing tools. Here, we present a robust, high-efficiency, and transgene-free CRISPR-based genome editing platform applicable across four ecologically and biotechnologically important brown algal species. Using Ectocarpus as a model, we optimized a polyethylene glycol (PEG)-mediated ribonucleoprotein (RNP) delivery system that achieves reproducible editing across multiple loci without cloning or specialized equipment. As proof of concept, we recreated the hallmark imm mutant phenotype by precisely editing the IMMEDIATE UPRIGHT (IMM) locus. APT/2-fluoroadenine (2-FA) selection further enhanced specificity with minimal false positives. The method was easily transferable to other species, including kelps. This platform now enables functional genomics in brown algae, providing powerful tools for investigating development, life cycle regulation, and the independent evolution of complex multicellularity.},
}
@article {pmid41475353,
year = {2026},
author = {Marks, D and Garcia, E and Kumar, S and Tyson, K and Koch, C and Ivanov, AP and Edel, JB and Mirza, HB and Flanagan, W and Dunsby, C and French, PMW and McNeish, IA},
title = {Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor.},
journal = {Cell reports methods},
volume = {6},
number = {1},
pages = {101270},
pmid = {41475353},
issn = {2667-2375},
support = {FC001999/WT_/Wellcome Trust/United Kingdom ; },
mesh = {Female ; Humans ; *Ovarian Neoplasms/drug therapy/pathology/metabolism/genetics ; *Fluorescence Resonance Energy Transfer/methods ; *Biosensing Techniques/methods ; Poly(ADP-ribose) Polymerase Inhibitors/pharmacology/therapeutic use ; Cell Line, Tumor ; *CRISPR-Cas Systems/genetics ; Animals ; Mice ; *Poly(ADP-ribose) Polymerases/metabolism ; *Poly (ADP-Ribose) Polymerase-1/metabolism/genetics ; },
abstract = {Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionized the treatment of ovarian high-grade serous carcinoma (HGSC), particularly in homologous recombination-deficient tumors. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within 3 years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood, as current experimental methodologies lack resolution and throughput. To address this, we develop an intramolecular fluorescence resonance energy transfer (FRET)-based biosensor by CRISPR-Cas9 dual labeling of endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells. This biosensor enables real-time, single-cell analysis of PARP trapping dynamics. Using fluorescence lifetime imaging microscopy (FLIM), we reveal dose-dependent PARP trapping, differentiate the trapping efficiencies of four clinically approved PARPi, and observe reduced trapping in PARPi-resistant models in vitro and in vivo. This biosensor provides critical insights into PARPi resistance mechanisms, with implications for developing more effective therapies and advancing personalized treatment for ovarian cancer patients.},
}
@article {pmid41475537,
year = {2026},
author = {Tang, W and Ma, M and Song, W and Kou, M and Wang, X and Yan, H and Li, C and Zhang, A and Gao, T and Gao, R and Zhang, Y and Li, Q},
title = {An efficient CRISPR/Cas9-mediated editing of phytoene desaturase in hexaploid sweetpotato.},
journal = {Plant science : an international journal of experimental plant biology},
volume = {364},
number = {},
pages = {112967},
doi = {10.1016/j.plantsci.2025.112967},
pmid = {41475537},
issn = {1873-2259},
mesh = {*Ipomoea batatas/genetics/enzymology ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; *Oxidoreductases/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Polyploidy ; Chlorophyll/metabolism ; },
abstract = {CRISPR/Cas9-mediated gene editing has emerged as a pivotal tool for functional genomics and crop improvement. For the first time, we applied CRISPR/Cas9-mediated editing to the IbPDS gene in the purple-fleshed cultivar 'XZS-8', achieving mutation efficiencies up to 98.18 %. Loss-of-function mutations in IbPDS induced visually discernible albino phenotypes. Hi-TOM sequencing confirmed deletion mutations within the target locus, with editing efficiencies ranging from 17.77 % to 65.90 % (gRNA1) and 87.87-98.18 % (gRNA2). Knockout lines showed significant reductions in chlorophyll a and b content, confirming functional disruption of IbPDS. Collectively, our results demonstrate efficient CRISPR/Cas9-mediated genome editing for generating mutants in the hexaploid sweetpotato.},
}
@article {pmid41476041,
year = {2025},
author = {Xi, W and Xu, Y and Bao, W and Ding, Z and Xiao, H and Yang, J and Yan, X and Ping, Y},
title = {In vivo chemogenetic RNA editing of macrophages by bioengineered viruses for sepsis treatment.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {927},
pmid = {41476041},
issn = {2041-1723},
support = {82425055//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32261143727//National Natural Science Foundation of China (National Science Foundation of China)/ ; 82504685//National Natural Science Foundation of China (National Science Foundation of China)/ ; 2024M762890//China Postdoctoral Science Foundation/ ; 2025T180980//China Postdoctoral Science Foundation/ ; GZB20240672//China Postdoctoral Science Foundation/ ; LMS25H300002//Natural Science Foundation of Zhejiang Province (Zhejiang Provincial Natural Science Foundation)/ ; },
mesh = {*Sepsis/therapy/genetics/immunology ; Animals ; *Macrophages/metabolism ; Mice ; *RNA Editing/genetics ; *Lentivirus/genetics ; Disease Models, Animal ; NLR Family, Pyrin Domain-Containing 3 Protein/genetics/metabolism ; Mice, Inbred C57BL ; Genetic Vectors/genetics ; Humans ; Genetic Therapy/methods ; Bioengineering ; CRISPR-Cas Systems ; Male ; Chemogenetics ; },
abstract = {Sepsis, a life-threatening condition arising from a dysregulated host response to infection, remains a significant clinical challenge with limited therapeutic options. RNA editing presents a promising avenue for modulating gene expression to attenuate the inflammatory cascade characteristic of sepsis. Here, we introduce an approach utilizing chemogenetic activation of CasRx-based RNA editing via bioengineered lentiviruses for the treatment of sepsis. Our strategy involves the targeted delivery of biomineralized lentiviral vectors encoding RNA-editing enzymes and chemogenetic switches to M1 macrophage populations implicated in sepsis pathogenesis. Upon the administration of a small molecule ligand, the chemogenetic switches activate the RNA-editing tool, CasRx, thereby enabling precise and repeated downregulation of NLRP3 mRNA. We demonstrate the efficacy and repeatability of this viral-based approach in mouse models of sepsis, highlighting its potential as a versatile therapeutic strategy for mitigating sepsis-induced inflammation. This study underscores the utility of chemogenetic technologies in harnessing the power of RNA editing for the treatment of sepsis and other inflammatory disorders.},
}
@article {pmid41476394,
year = {2026},
author = {Zhuang, M and Song, J and Hu, X and Wang, X},
title = {Metabolic Engineering of Escherichia coli Nissle 1917 for Efficient Production of p-Coumaric Acid.},
journal = {ACS synthetic biology},
volume = {15},
number = {1},
pages = {210-222},
doi = {10.1021/acssynbio.5c00639},
pmid = {41476394},
issn = {2161-5063},
mesh = {*Escherichia coli/metabolism/genetics ; *Metabolic Engineering/methods ; *Coumaric Acids/metabolism ; *Propionates/metabolism ; Ammonia-Lyases/genetics/metabolism ; Tyrosine/biosynthesis/metabolism ; CRISPR-Cas Systems/genetics ; Plasmids/genetics/metabolism ; Escherichia coli Proteins/genetics/metabolism ; Quorum Sensing/genetics ; Phenylalanine/biosynthesis ; },
abstract = {p-Coumaric acid is a valuable phytochemical with significant roles in anticancer cell proliferation, antianxiety, and neuroprotection and as a key precursor for various flavonoids. However, the production of p-coumaric acid in microorganisms is often limited by enzyme compatibility and its antimicrobial effects. In this study, a p-coumaric acid producing Escherichia coli strain was constructed. First, the cryptic plasmids pMUT1 and pMUT2 were eliminated from E. coli Nissle 1917 by using the CRISPR/Cas9 method to mitigate their interference with heterologous gene expression, and the resulting strain WEN01 was used to screen for the genes encoding for tyrosine ammonia-lyase with superior host compatibility. Next, the gene tyrR encoding a global regulator was knocked out to alleviate the repression of l-tyrosine production. The key genes pheL and pheA involved in phenylalanine biosynthesis were knocked out to reduce byproduct formation, resulting in the strain WEN06. Finally, the quorum sensing system was used to overexpress the key genes aroG[fbr] and tyrA[fbr] in the l-tyrosine biosynthetic pathway, and the resulting strain WEN06/pWT101-AT, pWT104F could produce 462.6 mg/L p-coumaric acid in shake flask fermentation. In fed-batch fermentation, the engineered strain WEN06/pWT101-AT, pWT104F could produce 10.3 g/L p-coumaric acid with a glucose conversion yield of 0.13 g/g and a productivity of 0.14 g/L/h. This work provides a novel strategy for the efficient production of p-coumaric acid and lays a foundation for the efficient production of antimicrobial natural products in bacteria.},
}
@article {pmid41477711,
year = {2025},
author = {Rozov, SM and Deineko, EV},
title = {[Recombinase-Based Engineering of Plant Genomes in the Era of Genome Editing].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {6},
pages = {873-890},
doi = {10.7868/S3034555325060016},
pmid = {41477711},
issn = {0026-8984},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *CRISPR-Cas Systems ; *DNA Nucleotidyltransferases/genetics/metabolism ; *Recombinases/genetics/metabolism ; *Plants/genetics ; DNA Transposable Elements ; },
abstract = {The rapidly evolving CRISPR/Cas-based genome editing technologies, which have dominated nearly all areas of molecular biology over the past decade, still face several unresolved challenges. One of the major limitations of current genome editing tools is the low efficiency of targeted long-sequence insertions. This issue is particularly critical in plant systems, where genome editing efficiency is hindered by specific cellular characteristics. Site-specific recombinases (SSRs), which have long been employed in genetic engineering to mediate various genomic rearrangements-including deletions, duplications, insertions, and inversions-are limited in their application by the requirement for preexisting recombination recognition sites in the genome. However, CRISPR/Cas and recombinase tools complement each other, and their combined use offers a powerful strategy to overcome key limitations of genome editing. The discovery of CRISPR-associated transposons such as CAST and OMEGA, which naturally utilize their own recombinases, marks a significant advance in genome engineering, providing an elegant example of the natural convergence between CRISPR and recombinase technologies.},
}
@article {pmid41477825,
year = {2026},
author = {Song, LCT and Alker, ATP and Oromí-Bosch, A and Swartz, SE and Martinson, JNV and Arora, J and Wang, AM and Rovinsky, R and Smith, SJ and Pierce, EC and Deutschbauer, AM and Doudna, JA and Cress, BF and Rubin, BE},
title = {Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing.},
journal = {Science advances},
volume = {12},
number = {1},
pages = {eaea1429},
pmid = {41477825},
issn = {2375-2548},
mesh = {*Gene Editing/methods ; *Transposases/genetics/metabolism ; Escherichia coli/genetics ; Vibrio cholerae/genetics ; *CRISPR-Cas Systems ; *Genome, Bacterial ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {CRISPR-associated transposases (CASTs) hold tremendous potential for microbial genome editing because of their ability to integrate large DNA cargos in a programmable, site-specific manner. However, their widespread application has been hindered by poorly understood host factor requirements for transposition. To address this gap, we conducted the first genome-wide screen for host factors affecting Vibrio cholerae CAST (VchCAST) activity using an Escherichia coli RB-TnSeq library and identified 15 genes affecting VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity, and two were inhibitory. Guided by the identification of homologous recombination effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors and increased editing efficiency by 55.2-fold in E. coli, 5.6-fold in Pseudomonas putida, and 10.8-fold in Klebsiella michiganensis while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting VchCAST activity and enhances its efficiency as a bacterial genome editor.},
}
@article {pmid41477891,
year = {2026},
author = {Luo, G and Song, J and Fu, Y and Jiang, Y and Gao, Y and Zhong, Z and Li, L and Wei, Y and Jia, HR and Guo, L and Fu, T and Wu, Q and Tan, W},
title = {SPARK-seq: A high-throughput platform for aptamer discovery and kinetic profiling.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6780},
pages = {eadv6127},
doi = {10.1126/science.adv6127},
pmid = {41477891},
issn = {1095-9203},
mesh = {*Aptamers, Nucleotide/chemistry/genetics ; Kinetics ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; *Single-Cell Analysis/methods ; *Membrane Proteins/metabolism/genetics/chemistry ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; },
abstract = {Cell surface proteins are key disease biomarkers and therapeutic targets, yet high-throughput methods for aptamer discovery targeting these proteins in situ remain limited. We introduce single-cell perturbation-driven aptamer recognition and kinetics sequencing (SPARK-seq), a high-throughput platform integrating single-cell messenger RNA and aptamer sequencing with CRISPR-based surface protein perturbation. In a single experiment, SPARK-seq simultaneously mapped 5535 distinct aptamers to eight surface proteins, capturing interactions across more than two orders of magnitude in protein abundance and spanning diverse biophysical classes. The method discriminated closely related paralogous proteins with no detectable cross-reactivity and provided kinetic information that enabled the prioritization of aptamers with slow dissociation rates. Leveraging this kinetic diversity, we engineered variants with improved off-rate properties. SPARK-seq establishes a platform for high-efficiency discovery and rational variant design of aptamers and functional nucleic acids, unlocking possibilities in diagnostics and therapeutics.},
}
@article {pmid41477961,
year = {2026},
author = {Gong, P and Tao, D and Chen, Q and Yang, Y and Xie, S and Chen, X and Shi, S and Wang, Y and Wang, L and Qian, Y and Ye, S},
title = {A rapid, visual, ultrasensitive and highly specific method for detecting adeno-associated virus 2020 based on the LAMP-CRISPR/Cas12a system.},
journal = {Poultry science},
volume = {105},
number = {2},
pages = {106344},
pmid = {41477961},
issn = {1525-3171},
mesh = {Animals ; *Poultry Diseases/diagnosis/virology ; *Parvoviridae Infections/veterinary/diagnosis/virology ; *Nucleic Acid Amplification Techniques/veterinary/methods ; *CRISPR-Cas Systems ; *Dependovirus/isolation &amp; purification/genetics ; *Ducks ; *Molecular Diagnostic Techniques/veterinary/methods ; Sensitivity and Specificity ; Phylogeny ; },
abstract = {Avian parvovirus infection would lead to growth retardation, weight loss, physical deformities and increased mortality in poultry, causing substantial economic losses to the poultry industry. Therefore, the development of a rapid, visual, ultrasensitive and highly specific method is essential for timely diagnosis and effective control of the avian parvovirus infection. In this study, we developed a detection platform based on loop-mediated isothermal amplification (LAMP) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 12a (Cas12a) system. Firstly, we have identified a novel avian parvovirus strain from diseased Muscovy ducks. Through genome sequencing, sequence assembly and phylogenetic tree analysis, we have identified this novel avian parvovirus as an adeno-associated virus (AAA) belonging to the family Parvoviridae, subfamily Parvovirinae and genus Dependoparvovirus. So, the novel virus strain was named AAV-2020. Next, specific sgRNAs and LAMP primers targeting the 3 capsid proteins (Cap) genes of AAV-2020 were designed and optimized. Moreover, the CRISPR/Cas12a-based system demonstrated a limit of detection as low as 2 copies/μL for AAV-2020. Importantly, the system could effectively distinguish AAV-2020 from 3 closely related AAV strains with high sequence similarity, indicating excellent specificity. In summary, we developed a novel, rapid, visual, ultrasensitive and highly specific detection system for AAV-2020, offering a reliable tool for early diagnosis and on-site detection of avian parvovirus infections, which would aid in the prevention and control of avian parvovirus infection in poultry industry.},
}
@article {pmid41478231,
year = {2026},
author = {Liu, Y and Mei, H and Gao, C and Yang, Y},
title = {Rapid detection of respiratory syncytial virus using RT-LAMP-CRISPR/Cas12b on a gravity-driven microfluidic chip.},
journal = {Diagnostic microbiology and infectious disease},
volume = {114},
number = {3},
pages = {117244},
doi = {10.1016/j.diagmicrobio.2025.117244},
pmid = {41478231},
issn = {1879-0070},
mesh = {Humans ; *Respiratory Syncytial Virus Infections/diagnosis/virology ; *Molecular Diagnostic Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; *Respiratory Syncytial Virus, Human/isolation &amp; purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; *Lab-On-A-Chip Devices ; RNA, Viral/genetics ; Limit of Detection ; Point-of-Care Systems ; },
abstract = {AIMS: Respiratory syncytial virus (RSV) is the most significant pathogen causing acute lower respiratory tract infections in children. Early detection of RSV can help control disease progression and reduce complications. However, RT-qPCR-based detection methods cannot provide accurate results within one hour and are not suitable for use in resource-limited settings. Therefore, there is a pressing need to develop a rapid and precise bedside RSV detection assay to meet clinical demands.<br><br>METHODS AND RESULTS: Firstly, we developed a RT-LAMP-assisted CRISPR/Cas12b method for detecting RSV M gene, capable of identifying target RNA with a limit of detection as low as 100 copies/&#x3bc;L within 40 min. Secondly, we created and tested a sampling lysis reagent, demonstrating its effectiveness in enabling direct detection without the need for nucleic acid extraction, thus improving bedside detection efficiency. Lastly, to facilitate use in resource-limited areas, we designed and developed a gravity-driven microfluidic chip that simplifies the stepwise process of RT-LAMP amplification and CRISPR/Cas12b detection. This chip allows for visual recognition of results without the need for an external power source when used in point-of-care (POC) settings. This assay showed 99% agreement with RT-qPCR, highlighting its potential for practical application. Additionally, no cross-reactivity was observed with other respiratory pathogens infection, demonstrating good clinical specificity.<br><br>CONCLUSIONS: In summary, the platform we developed is faster and more user-friendly than RT-qPCR, while achieving comparable sensitivity.<br><br>IMPACT STATEMENT: Our finding fills the gap in the inability to detect RSV in POC setting, safeguarding the health of children, and offers new insights for the innovation of CRISPR diagnostics.},
}
@article {pmid41478283,
year = {2026},
author = {Zhang, X and Zhang, Y and Liu, X and Liu, C and Liu, Y and He, Y and Qiu, Y and Sun, L and Hu, J and Gao, Y and Wei, W and Liu, J},
title = {FOCAS: Transcriptome-wide screening of individual m[6]A sites functionally dissects epitranscriptomic control of gene expression in cancer.},
journal = {Cell},
volume = {189},
number = {3},
pages = {922-938.e23},
doi = {10.1016/j.cell.2025.11.037},
pmid = {41478283},
issn = {1097-4172},
mesh = {Humans ; *Adenosine/analogs &amp; derivatives/metabolism/genetics ; *Neoplasms/genetics/metabolism ; *Transcriptome/genetics ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; CRISPR-Cas Systems/genetics ; RNA, Messenger/metabolism/genetics ; RNA, Untranslated/metabolism/genetics ; Epigenesis, Genetic ; },
abstract = {Although N[6]-methyladenosine (m[6]A) is a pervasive RNA modification essential for gene regulation, dissecting the functions of individual m[6]A sites remains technically challenging. To overcome this, we developed functional m[6]A sites detection by CRISPR-dCas13b-FTO screening (FOCAS), a CRISPR-dCas13b-based platform enabling high-throughput, site-specific functional screening of m[6]A. Applying FOCAS to four human cancer cell lines identified 4,475 m[6]A-regulated genes influencing cell fitness via both mRNAs and non-coding RNAs (ncRNAs), many of which are newly linked to cancer and exhibit dynamic developmental expression. FOCAS uncovered context-dependent and reader-specific effects of m[6]A within the same gene, revealing its intricate regulatory logic. We further uncovered universal and cell-type-specific m[6]A patterns, with unique sites enriched in ncRNAs and universal ones in transcription-related genes. In SMMC-7721 cells, we identified m[6]A-regulated transcriptional networks that demonstrated extensive epitranscriptome-transcriptome crosstalk. Overall, this study established a powerful, unbiased approach for the functional dissection of m[6]A, advancing the understanding of its complexity and therapeutic relevance in cancers.},
}
@article {pmid41478438,
year = {2026},
author = {Ma, Q and Wang, N and Qiao, K and Luo, K and Zhao, C and Yan, J and Fan, S and Rong, J and Ma, Q},
title = {The R2R3-MYB transcription factor GhMYB35 governs anther development and pollen viability in upland cotton.},
journal = {Gene},
volume = {984},
number = {},
pages = {149990},
doi = {10.1016/j.gene.2025.149990},
pmid = {41478438},
issn = {1879-0038},
mesh = {*Gossypium/genetics/growth &amp; development/metabolism ; *Pollen/genetics/growth &amp; development ; *Transcription Factors/genetics/metabolism ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; *Flowers/genetics/growth &amp; development ; Plant Infertility/genetics ; CRISPR-Cas Systems ; },
abstract = {Male reproductive development is fundamental to the life cycle of flowering plants, culminating in seed production. Aberrations in anther development frequently lead to male sterility, yet the underlying molecular mechanisms in upland cotton (Gossypium hirsutum) remain largely uncharacterized. The R2R3-MYB family of transcription factors are known key regulators of diverse developmental processes, including male fertility in several model species. Here, we identify and functionally characterize GhMYB35, an R2R3-MYB transcription factor that plays an essential role in cotton anther development. CRISPR/Cas9-mediated knockout of GhMYB35 resulted in complete male sterility, with mutants (ghmyb35) exhibiting indehiscent anthers, shorter filaments, and a total absence of viable pollen. Expression analyses reveal that GhMYB35 is predominantly expressed in anthers, with peak expression of its A- and D-subgenome homoeologs occurring at developmental stage 7. Subcellular localization results show that bothGhMYB35_AandGhMYB35_Dare nuclear-localized transcription factors. Furthermore, the total absence of GhMYB35 leads to pollen abortion and subsequent anther collapse without dehiscence. Collectively, our findings establish GhMYB35 as a critical regulator of anther maturation, thereby elucidating a key component of the molecular network governing male fertility in cotton.},
}
@article {pmid41478695,
year = {2026},
author = {Zhang, H and Han, M and Pang, Z and Li, W and Li, X and Sun, B},
title = {The lldD lactate dehydrogenase is a determinant of lactic acid tolerance in Pichia kudriavzevii by pyruvate metabolism pathway.},
journal = {Food microbiology},
volume = {136},
number = {},
pages = {104989},
doi = {10.1016/j.fm.2025.104989},
pmid = {41478695},
issn = {1095-9998},
mesh = {*Pichia/genetics/metabolism/enzymology/drug effects ; *Lactic Acid/metabolism/pharmacology ; Fermentation ; *L-Lactate Dehydrogenase/genetics/metabolism ; Metabolic Networks and Pathways ; *Pyruvic Acid/metabolism ; *Fungal Proteins/genetics/metabolism ; Ethanol/metabolism ; CRISPR-Cas Systems ; Glucose/metabolism ; Gene Knockout Techniques ; },
abstract = {Lactic acid stress is common in traditional fermented foods. Pichia kudriavzevii owes its industrial prevalence to its superior acid tolerance, making deciphering its response mechanism imperative for sustainable fermentation processes. This work explored the remarkable acid tolerance of P. kudriavzevii, which can tolerate 80 g/L lactic acid. The key lactic acid tolerance gene lldD of P. kudriavzevii was determined through transcriptomic analysis. Importantly, this work knocked out the key gene lldD related to lactic acid tolerance for the first time by using the CRISPR-Cas9 technology. Meanwhile, the lldD knockout strain (pk-ΔlldD) was successfully constructed. There were significant differences between pk-ΔlldD and P. kudriavzevii in terms of tolerance to lactic acid, metabolism of lactic acid, utilization of glucose and ethanol production. In addition, the deletion of the gene lldD has a significant impact on the ABC transporter and metabolites of the amino acid metabolic pathway in P. kudriavzevii. In conclusion, this work provides a theoretical basis for engineering high acid-tolerant industrial yeast strains through targeted genetic modification. It helps enhance the stability of fermentation processes under lactic acid stress and ultimately lays a foundation for promoting efficient and low-loss production in the fermentation industry.},
}
@article {pmid41478989,
year = {2026},
author = {Huo, Z and Tu, J and Shoemaker, R and Lee, DF and Zhao, R},
title = {Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2983},
number = {},
pages = {343-359},
pmid = {41478989},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Cell Line ; *Tumor Suppressor Protein p53/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Human Embryonic Stem Cells/metabolism/cytology ; Exons ; Gene Knock-In Techniques/methods ; *Genetic Engineering/methods ; },
abstract = {CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system, has been widely accepted and employed in gene editing of a vast range of cell types. Compared to zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), CRISPR shows a less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome wide screening. Here we will describe the procedure of generating a stable cell line with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.},
}
@article {pmid41478990,
year = {2026},
author = {Hu, O and Provvido, A and Zhu, Y},
title = {Generation of IL17RB Knockout Cell Lines Using CRISPR/Cas9-Based Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2983},
number = {},
pages = {361-370},
pmid = {41478990},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Gene Knockout Techniques/methods ; *Receptors, Interleukin-17/genetics ; Cell Line ; HEK293 Cells ; },
abstract = {CRISPR/Cas9-based genome editing is an inexpensive and efficient tool for genetic modification. Here, we present a methodological approach for establishing interleukin-17 receptor B (IL17RB) knockout cell lines using CRISPR/Cas9-mediated genomic deletion. The IL17RB gene encodes for a cytokine receptor that specifically binds to IL17B and IL17E and is overexpressed in various cancers. The method involves CRISPR design, CRISPR cloning, delivery of the CRISPR clone into cells, and verification of IL17RB gene deletion by deletion screening primer design, genomic DNA extraction, and polymerase chain reaction (PCR). A similar approach can be used for generating mammalian cell lines with gene knockout for other genes of interest.},
}
@article {pmid41479062,
year = {2026},
author = {Lee, NJ and Matsuoka, RL},
title = {CRISPR/Cas9-Based Mutagenesis Strategies for Efficient Biallelic Gene Inactivation and Consistent Phenotypic Detection in F0 Zebrafish.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2989},
number = {},
pages = {319-332},
pmid = {41479062},
issn = {1940-6029},
support = {R01 NS117510/NS/NINDS NIH HHS/United States ; },
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; Phenotype ; *Mutagenesis ; Alleles ; *Gene Silencing ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The zebrafish is a valuable animal model for investigating the genetic basis of vertebrate evolution, development, behavior, and regeneration. However, the existence of numerous gene paralogs in the zebrafish genome represents a major challenge, complicating functional genomic research using reverse-genetics approaches. To facilitate reverse genetics-based phenotypic screens, we recently presented simple methods that enable efficient induction of biallelic gene disruptions in F0 zebrafish, providing a rapid avenue for screening potential gene functions through consistent phenotypic detection. Here, we describe detailed protocols for these CRISPR/Cas9-based mutagenesis strategies to achieve highly effective biallelic gene inactivation in F0 zebrafish. The high consistency of these strategies, combined with a streamlined workflow, offers a robust phenotypic screening platform for a quick and reliable functional assessment of genes of interest, both individually and in a scalable manner. These strategies enhance the efficacy of successful F0 zebrafish phenotypic screening, thereby accelerating functional genetic studies using this powerful model organism.},
}
@article {pmid41479230,
year = {2026},
author = {Zhang, J and Liu, Y and Cao, W and Ruan, R and Wang, M},
title = {Establishment of a Novel CRISPR/Cas9-Based Multiplex Editing System in the Citrus Postharvest Pathogen Penicillium digitatum.},
journal = {Journal of agricultural and food chemistry},
volume = {74},
number = {1},
pages = {1167-1174},
doi = {10.1021/acs.jafc.5c11144},
pmid = {41479230},
issn = {1520-5118},
mesh = {*Penicillium/genetics/pathogenicity/metabolism ; *Citrus/microbiology ; *CRISPR-Cas Systems ; *Gene Editing/methods ; *Plant Diseases/microbiology ; Fungal Proteins/genetics/metabolism ; },
abstract = {Penicillium digitatum, the causal fungus of the citrus green mold, leads to substantial postharvest losses in the citrus industry. In this study, we engineered a versatile CRISPR/Cas9-mediated gene editing platform capable of generating multiple sgRNAs from a single polycistronic transcript. By incorporating endogenous tRNAs and the strong promoter PdMLE1 into the CRISPR/Cas9 system, the efficiency of single gene editing can reach up to 94.2%. By taking advantage of the efficient shearing and processing capabilities of tRNA, the platform enabled multiplex editing with efficiencies of 44.4% for two-gene and 33.3% for three-gene modifications, respectively. After two rounds of three-gene editing, we were able to successfully obtain hextuple-gene mutants. Finally, functional characterization revealed that the target polysaccharide-lyase-encoding genes play limited roles in pathogenicity in P. digitatum. Taken together, our results represent a powerful tool for genome engineering in P. digitatum, facilitating research into its pathogenesis.},
}
@article {pmid41479566,
year = {2025},
author = {Antipenko, ID and Venedyukhina, SA and Sorokina, NP and Kucherenko, IV and Smirnova, TS and Rogov, GN and Shkurnikov, MY},
title = {Whole-Genome Sequencing Uncovers Metabolic and Immune System Variations in Propionibacterium freudenreichii Isolates.},
journal = {Acta naturae},
volume = {17},
number = {4},
pages = {72-82},
pmid = {41479566},
issn = {2075-8251},
abstract = {Propionibacterium freudenreichii plays a crucial role in the production of Swiss-type cheeses; however, genomic variability among strains, which affects their technological traits, remains insufficiently explored. In this study, whole-genome sequencing and comparative analysis were performed on five industrial P. freudenreichii strains. Despite their overall high genomic similarity, the strains proved different in gas production and substrate metabolism. Phylogenetic analysis revealed a close relationship between strain FNCPS 828 and P. freudenreichii subsp. shermanii (z-score = 0.99948), with the latter being unable to reduce nitrates but being able to metabolize lactose. The narG gene encoding the nitrate reductase alpha subunit was detected in only one of the five analyzed strains - FNCPS 828 - and in 39% of previously described P. freudenreichii genomes, suggesting its potential as a marker of nitrate-reducing capability. Analysis of 112 genomes showed that the I-G CRISPR-Cas system was present in more than 90% of the strains, whereas the type I-E system was found in approximately 25%. All the five study strains harbored the type I-G system; strain FNCPS 3 additionally contained a complete type I-E system with the highest number of CRISPR spacers, some of which matched previously published bacteriophage sequences. The most prevalent anti-phage defense systems included RM I, RM IV, AbiE, PD-T4-6, HEC-06, and ietAS. These findings highlight the genetic diversity of P. freudenreichii strains, which is of great importance in their industrial applications. The identification of narG as a potential marker of nitrate-reducing activity, along with detailed mapping of CRISPR- Cas systems, boosts opportunities for the rational selection and engineering of starter cultures with tailored metabolic properties and increased resistance to bacteriophages.},
}
@article {pmid41481232,
year = {2026},
author = {Li, Y and Zhang, W and Wei, Z and Li, H and Liu, X and Zheng, T and Aziz, T and Xing, C and Meng, A and Wu, X},
title = {Stage- and tissue-specific gene editing using 4-OHT-inducible Cas9 in whole organism.},
journal = {The Journal of cell biology},
volume = {225},
number = {4},
pages = {},
pmid = {41481232},
issn = {1540-8140},
support = {#3258820001//National Natural Science Foundation of China/ ; #2023YFA1800300//National Key Research and Development Program of China/ ; 2018YFC1003304//National Key Research and Development Program of China/ ; #202302AO370011//Southwest United Graduate School/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Zebrafish/genetics/embryology ; *Tamoxifen/analogs &amp; derivatives/pharmacology ; *CRISPR-Cas Systems/genetics ; Mice ; Female ; *CRISPR-Associated Protein 9/genetics/metabolism ; Germ Cells/metabolism ; Organ Specificity ; Humans ; Receptors, Estrogen/genetics/metabolism ; Animals, Genetically Modified ; },
abstract = {Vertebrate genes function in specific tissues and stages, so their functional studies require conditional knockout or editing. In zebrafish, spatiotemporally inducible genome editing, particularly during early embryogenesis, remains challenging. Here, we establish inducible Cas9-based editing in defined cell types and stages. The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos. As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction. The system also works in early mouse embryos. Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.},
}
@article {pmid41481737,
year = {2026},
author = {Qi, S and Wei, L and Ding, Z and Zhong, F and Yang, S and Wu, L and Yang, X and Kang, B and Dan, M and Gan, J and Li, C and Su, X},
title = {Targeting transthyretin by one Cas9 variant with superfidelity and broad compatibility.},
journal = {Science advances},
volume = {12},
number = {1},
pages = {eadu6505},
pmid = {41481737},
issn = {2375-2548},
mesh = {*Prealbumin/genetics/metabolism ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Amyloid Neuropathies, Familial/genetics/therapy ; HEK293 Cells ; },
abstract = {Amyloid transthyretin (ATTR) amyloidosis is a fatal disease caused by the accumulation of misfolded transthyretin proteins. Although knocking down the TTR gene by CRISPR-Cas9 represents a promising strategy for treating ATTR amyloidosis, its efficiency and safety remain to be further investigated. Here, we report a systematic investigation of SpCas9-based TTR editing. Besides the target site, wild-type SpCas9 and the reported variants induced extensive off-target edits. To improve the fidelity, we performed structural analysis and designed a series of SpCas9 variants. Studies demonstrated that SpCas9-Mut5 is an ultrahigh-fidelity variant, which induces extremely low levels of off-target edits and translocations without substantial impairment of on-target editing activity. SpCas9-Mut5 is compatible with the adenine base editor (ABE) system, markedly reducing off-target edits and narrowing the editing window. In conclusion, our study suggests that SpCas9-Mut5 is an excellent candidate for TTR gene editing. Besides ATTR amyloidosis, SpCas9-Mut5 and its derivative ABE could be widely used in the treatment of other diseases.},
}
@article {pmid40670899,
year = {2026},
author = {Alam, T},
title = {Advances in Tissue Culture-Free Genetic Engineering and Genome Editing of Peanut.},
journal = {Molecular biotechnology},
volume = {68},
number = {4},
pages = {1684-1698},
pmid = {40670899},
issn = {1559-0305},
mesh = {*Arachis/genetics/growth &amp; development ; *Gene Editing/methods ; Plants, Genetically Modified/genetics ; *Genetic Engineering/methods ; CRISPR-Cas Systems ; Genome, Plant ; Tissue Culture Techniques ; },
abstract = {Plant transformation and genome editing are pivotal in advancing peanut biotechnology, yet traditional tissue culture-dependent methods are hindered by lengthy protocols, genotype dependency, and somaclonal variation. CRISPR/Cas technologies have revolutionized breeding by enabling precise, multiplex genome editing to improve traits such as disease resistance, allergen reduction, seed quality, and yield. However, variable transformation efficiencies and chimerism remain challenges. This review examines emerging tissue culture-independent techniques such as nanoparticle-based delivery, viral vectors, pollen magnetofection, pollen tube injection, node injection, and vacuum infiltration that offer rapid, cost-effective gene transfer. It also highlights the integration of high-throughput screening, robust selection markers, and automation, including robotics and advanced imaging, to refine transformation pipelines. These methodological breakthroughs promise to overcome current limitations and accelerate the development of improved peanut cultivars for sustainable agriculture.},
}
@article {pmid40671314,
year = {2025},
author = {Kumar, P and Bisht, NC},
title = {High-level production of health-beneficial glucoraphanin by multiplex editing of AOP2 gene family in mustard.},
journal = {Plant biotechnology journal},
volume = {23},
number = {10},
pages = {4668-4680},
pmid = {40671314},
issn = {1467-7652},
support = {BT/PR23893/GET/119/81/2017//Department of Biotechnology, Ministry of Science and Technology, India/ ; BT/PR53959/PBN/18/17/2024//Department of Biotechnology, Ministry of Science and Technology, India/ ; },
mesh = {Sulfoxides ; *Glucosinolates/metabolism/biosynthesis ; Oximes ; *Imidoesters/metabolism ; *Mustard Plant/genetics/metabolism ; *Gene Editing/methods ; CRISPR-Cas Systems ; Plants, Genetically Modified/metabolism ; *Plant Proteins/genetics/metabolism ; *Isothiocyanates/metabolism ; },
abstract = {Intake of glucosinolates through the consumption of cruciferous vegetables has been associated with numerous health benefits. In recent decades, glucosinolate glucoraphanin has gained a lot of attention, as its hydrolysis product (sulforaphane) is known to possess numerous health-promoting benefits, including anti-cancer and chemopreventive activities. However, due to the low availability of glucoraphanin in most of the cultivated Brassica crops (except broccoli), there is an increasing interest in many laboratories around the world to manipulate the glucosinolate profile for human benefit. Here, we report the high-level production of health-beneficial glucoraphanin by CRISPR/Cas9 editing of the ALKENYL HYDROXALKYL PRODUCING 2 (BjuAOP2) gene family, displaying distinct expression profiles in the allotetraploid mustard, Brassica juncea. Multiplex editing of five BjuAOP2 homologues, using four gRNAs, provided glucoraphanin accumulation up to 41.60, 75.10, 59.21 and 27.64 μmoles/g dry weight in sprouts, microgreens, seeds and leaves, respectively, of the transgene-free BjuAOP2-edited lines, while providing a significant reduction of the anti-nutritional and goitrogenic alkenyl glucosinolates including progoitrin. The glucoraphanin enhancement in BjuAOP2-edited lines was found to be dose-dependent, wherein loss-of-function mutations in BjuAOP2.A09 and BjuAOP2.B01 homologues had a more prominent effect. The transgene-free BjuAOP2-edited lines were stable for high glucoraphanin and performed at par with the wild-type plants for various seed quality and yield parameters when tested under containment conditions in the field. The development of high-glucoraphanin mustard will help its adoption as a global superfood with health-promoting benefits and as a bioactive source of high-value sulforaphane for industrial production.},
}
@article {pmid40671523,
year = {2025},
author = {Ma, E and Chen, K and Shi, H and Wasko, KM and Esain-Garcia, I and Trinidad, MI and Zhou, K and Ye, J and Doudna, JA},
title = {Directed evolution expands CRISPR-Cas12a genome-editing capacity.},
journal = {Nucleic acids research},
volume = {53},
number = {13},
pages = {},
pmid = {40671523},
issn = {1362-4962},
support = {//Life Sciences Research Foundation/ ; U01AI142817/GF/NIH HHS/United States ; R21HL173710/HB/NHLBI NIH HHS/United States ; R21 HL173710/HL/NHLBI NIH HHS/United States ; B656358//DOE/ ; //Mr Li Ka Shing/ ; /HHMI/Howard Hughes Medical Institute/United States ; U54AI170792/AI/NIAID NIH HHS/United States ; //Emerson Collective/ ; UH3AI150552/AI/NIAID NIH HHS/United States ; U19AI171110/AI/NIAID NIH HHS/United States ; U19NS132303/AI/NIAID NIH HHS/United States ; //Innovative Genomics Institute/ ; U19 AI135990/AI/NIAID NIH HHS/United States ; //CB-Hampton University Summer Program/ ; 24180//Lawrence Livermore National Laboratory/ ; U19 NS132303/NS/NINDS NIH HHS/United States ; //Rett Syndrome Research Trust/ ; UH3 AI150552/AI/NIAID NIH HHS/United States ; U01 AI142817/AI/NIAID NIH HHS/United States ; DE-AC02-05CH11231//DOE/ ; //The Jane Coffin Childs Fund for Medical Research/ ; U19 AI171110/AI/NIAID NIH HHS/United States ; //Koret-Berkeley-TAU/ ; U54 AI170792/AI/NIAID NIH HHS/United States ; U19AI135990/AI/NIAID NIH HHS/United States ; 2553571//DOE/ ; 2334028//NSF/ ; },
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *CRISPR-Associated Proteins/genetics/metabolism ; *Directed Molecular Evolution/methods ; *Endodeoxyribonucleases/genetics/metabolism ; *Bacterial Proteins/genetics/metabolism ; Genome, Human ; },
abstract = {CRISPR-Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5'-TTTV-3' protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a's gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5'-TTTV-3' PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5'-NYHV-3' PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.},
}
@article {pmid40672171,
year = {2025},
author = {Sinnott, RW and Solanki, A and Govind, AP and Green, WN and Dickinson, BC},
title = {Engineering a human-based translational activator for targeted protein expression restoration.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40672171},
issn = {2692-8205},
support = {R01 EB035016/EB/NIBIB NIH HHS/United States ; },
abstract = {Therapeutic modalities to programmably increase protein production are in critical need to address diseases caused by deficient gene expression via haploinsufficiency. Restoring physiological protein levels by increasing translation of their cognate mRNA would be an advantageous approach to correct gene expression, but has not been evaluated in an in vivo disease model. Here, we investigated if a translational activator could improve phenotype in a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCN1a haploinsufficiency, by increasing translation of the SCN1a mRNA. We identifiy and engineere human proteins capable of increasing mRNA translation using the CRISPR-Cas Inspired RNA-targeting System (CIRTS) platform to enable programmable, guide RNA (gRNA)-directed translational activation with entirely engineered human proteins. We identify a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3), that can drive targeted, sustained translation increases up to 100% from three endogenous transcripts relevant to epilepsy and neurodevelopmental disorders. AAV-delivery of CIRTS-4GT3 targeting SCN1a mRNA to a Dravet syndrome mouse model led to increased SCN1a translation and improved survivability and seizure threshold - key phenotypic indicators of Dravet syndrome. This work validates a new strategy to address SCN1a haploinsufficiency and emphasizes the preclinical potential translational activation has to address neurological haploinsufficiency.},
}
@article {pmid40672203,
year = {2025},
author = {Hodge, CA and Donegan, NP and Armstrong, DA and Hayden, MS and Howell, AL},
title = {ENHANCED CLEAVAGE OF GENOMIC CCR5 USING CASX2[Max].},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {40672203},
issn = {2692-8205},
support = {I01 BX005248/BX/BLRD VA/United States ; P30 CA023108/CA/NCI NIH HHS/United States ; },
abstract = {Development of novel CRISPR/Cas systems enhances opportunities for gene editing to treat infectious diseases, cancer, and genetic disorders. We evaluated CasX2 (PlmCas12e), a class II CRISPR system derived from Planctomycetes, a non-pathogenic bacterium present in aquatic and terrestrial soils. CasX2 offers several advantages over Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus Cas9 (SaCas9), including its smaller size, distinct protospacer adjacent motif (PAM) requirements, staggered cleavage cuts that promote homology-directed repair, and no known pre-existing immunity in humans. A recent study reported that a three amino acid substitution in CasX2 significantly enhanced cleavage activity (1). Therefore, we compared cleavage efficiency and double-stranded break repair characteristics between the native CasX2 and the variant, CasX2[Max], for cleavage of CCR5, a gene that encodes the CCR5 receptor important for HIV-1 infection. Two CasX2 single guide RNAs (sgRNAs) were designed that flanked the 32 bases deleted in the natural CCR5 Δ32 mutation. Nanopore sequencing demonstrated that CasX2 using sgRNAs with spacers of 17 nucleotides (nt), 20 nt or 23 nt in length were ineffective at cleaving genomic CCR5. In contrast, CasX2[Max] using sgRNAs with 20 nt and 23 nt spacer lengths, enabled robust genomic cleavage of CCR5. Structural modeling indicated that two of the CasX2[Max] substitutions enhanced sgRNA-DNA duplex stability, while the third improved DNA strand alignment within the catalytic site. These structural changes likely underlie the increased activity of CasX2[Max] in cellular gene excision. In sum, CasX2[Max] consistently outperformed native CasX2 across all assays and represents a superior gene-editing platform for therapeutic applications.},
}
@article {pmid40673390,
year = {2026},
author = {Guerra, F and De Rouck, S and Verhulst, EC},
title = {SYNCAS-mediated CRISPR-Cas9 genome editing in the Jewel wasp, Nasonia vitripennis.},
journal = {Insect molecular biology},
volume = {35},
number = {1},
pages = {48-55},
pmid = {40673390},
issn = {1365-2583},
support = {OCENW.M.22.140//Dutch Research Council (NWO)/ ; G035420N//Research Foundation Flanders (FWO)/ ; 101123162/ERC_/European Research Council/International ; },
mesh = {Animals ; *Wasps/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Female ; Gene Knockout Techniques ; Insect Proteins/genetics ; },
abstract = {Genetic engineering is a formidable approach to studying biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis, an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to 10 times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques.},
}
@article {pmid40673518,
year = {2025},
author = {Wan, W and Cui, C and Zhou, Y and Wang, J and Zhao, X and Cui, X and Sun, J and Yu, P and Feng, J and Wang, T and Wang, L and Xu, J},
title = {Targeting Mettl14 Using an RNA-Targeting Clustered Regularly Interspaced Short Palindromic Repeat-High-Fidelity Cas13x System Attenuates Doxorubicin-Induced Cardiotoxicity.},
journal = {Journal of the American Heart Association},
volume = {14},
number = {15},
pages = {e040700},
pmid = {40673518},
issn = {2047-9980},
mesh = {Animals ; *Doxorubicin/toxicity/adverse effects ; *Methyltransferases/genetics/metabolism ; Male ; Cardiotoxicity ; *CRISPR-Cas Systems ; Mice ; Disease Models, Animal ; Mice, Inbred C57BL ; *Cardiomegaly/chemically induced/genetics/prevention &amp; control ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Physical Conditioning, Animal ; Myocytes, Cardiac/pathology/drug effects ; },
abstract = {BACKGROUND: Doxorubicin is an effective chemotherapy drug used to treat various types of cancer. However, doxorubicin treatment is associated with cardiotoxicity, which limits its clinical use. Exercise can benefit both cancer and cardiovascular disease. Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas13 (CRISPR-associated protein 13) platforms have emerged as effective technologies for targeting the expression of RNA in transcript levels. To develop exercise mimetics that can mimic the beneficial effects of exercise training to attenuate doxorubicin-induced cardiotoxicity, we are using the CRISPR-hf (high-fidelity)Cas13x system.<br><br>METHODS: Adult male mice were swim-trained twice a day for 4&#x2009;weeks to induce exercise-induced physiological cardiac hypertrophy. Adeno-associated virus 9-mediated METTL14 (methyltransferase-like 14) overexpression under the cardiac-specific ctnt promoter was used to overexpression METTL14 in&#xa0;vivo. RNA N[6]-methyladenosine inhibitor STM2457 was used to modulate global total RNA m[6]A levels in&#xa0;vivo. CRISPR-cr3-4/hfCas13x system was generated by hfCas13x guided crRNA3 and crRNA4 targeting the Mettl14 expressed under ctnt promoter and packaged in an adeno-associated virus 9.<br><br>RESULTS: Swimming exercise alleviated doxorubicin-induced cardiotoxicity. METTL14 was increased in doxorubicin-treated hearts but decreased in exercised hearts. METTL14 overexpression inhibited exercise-induced physiological cardiac hypertrophy. Conversely, STM2457 treatment reversed the suppressive effects of METTL14 overexpression on the physiological cardiac hypertrophy induced by exercise. Treatment with CRISPR-cr3-4/hfCas13x effectively inhibiting the expression of METTL14 in the heart, alleviating doxorubicin treatment-induced cardiac dysfunction and cardiac fibrosis.<br><br>CONCLUSIONS: Our results suggest that the CRISPR-hfCas13x system has the potential for generating exercise mimetics. Mimicking exercise by RNA-targeting Mettl14 suppression could be a therapeutic strategy for doxorubicin-induced cardiotoxicity.},
}
@article {pmid40673793,
year = {2025},
author = {Pan, Y and Yang, X and Zeng, Z and Liu, F and Luo, J and Shen, M and Zhou, W and Li, J and Jiang, G and Sun, L and Huang, H and Pan, R},
title = {[124]I-labelled BMSC-Derived Extracellular Vesicles Deliver CRISPR/Cas9 Ribonucleoproteins With a GFP-Reporter System to Inhibit Osteosarcoma Proliferation and Metastasis.},
journal = {Journal of extracellular vesicles},
volume = {14},
number = {7},
pages = {e70130},
pmid = {40673793},
issn = {2001-3078},
support = {82060491//the National Natural Science Foundation of China/ ; 82160568//the National Natural Science Foundation of China/ ; [2024]168//the Guizhou Provincial Science and Technology Projects/ ; [2021]395//the Guizhou Provincial Science and Technology Projects/ ; [2024]481//the Guizhou Provincial Science and Technology Projects/ ; },
mesh = {*Osteosarcoma/pathology/therapy/metabolism/genetics ; *CRISPR-Cas Systems ; Humans ; *Extracellular Vesicles/metabolism ; Cell Proliferation ; Animals ; *Mesenchymal Stem Cells/metabolism ; Mice ; Cell Line, Tumor ; *Ribonucleoproteins/metabolism/genetics ; *Bone Neoplasms/pathology/therapy/metabolism/genetics ; Iodine Radioisotopes ; Gene Editing ; Green Fluorescent Proteins/metabolism/genetics ; Mice, Nude ; Neoplasm Metastasis ; },
abstract = {Metastasis constitutes the principal factor leading to the unfavourable prognosis of osteosarcoma patients. Hypoxia, as the inherent microenvironment of osteosarcoma, can upregulate HIF-1α via multiple pathways, thereby facilitating osteosarcoma proliferation and metastasis. Our previous research indicated that the inwardly rectifying potassium channel subfamily J member 2 (KCNJ2) inhibits the degradation of HIF-1α in osteosarcoma. Concurrently, HIF-1α upregulates the expression of KCNJ2 through a positive feedback regulatory mechanism. This positive regulatory mechanism significantly promotes the proliferation and metastasis of osteosarcoma. Therefore, the development of a KCNJ2-targeted therapeutic strategy capable of disrupting this reciprocal regulatory loop represents a crucial intervention for impeding osteosarcoma progression. The CRISPR/Cas9 targeted gene editing technology has garnered extensive attention in the field of tumour treatment due to its high efficiency and low off-target rate. Nevertheless, the relative lag of the delivery systems has restricted its application. The extracellular vesicles (EVs) secreted by bone marrow mesenchymal stem cells (BMSCs) have a natural targeting specificity for osteosarcoma and possess superior biocompatibility, making them ideal carriers for in vivo delivery. However, it is essential to confirm whether the CRISPR/Cas9 system mediated by EVs can accurately function intracellularly. Hence, we developed a fluorescence-based Cas9 editing efficiency reporter system. When CRISPR/Cas9 system induces double-strand breaks at specific target sites and results in frameshift mutations, osteosarcoma cells will stably express GFP. This system enables the transformation of gene editing events into quantifiable fluorescence signals. Furthermore, we engineered radiolabelled EVs derived from BMSCs to deliver the CRISPR/Cas9 system targeting KCNJ2. Using this reporter system, we confirmed their efficient gene-editing capabilities in vitro. Additionally, leveraging their radiolabelling properties, we validated their targeted distribution in vivo. Subsequent investigations revealed that our constructed [124]I@EVs-Cas9 effectively suppresses the proliferation and metastasis of osteosarcoma by targeting the inhibition of KCNJ2 expression and promoting HIF-1α ubiquitin-dependent degradation (as depicted in Graphical Abstract).},
}
@article {pmid40673842,
year = {2025},
author = {Johnson, MJ and Sumstad, D and Folsom, TD and Slipek, NJ and DeFeo, AP and Growe, M and Kadidlo, D and Thyagarajan, B and Starr, TK and Lou, E and Choudhry, M and Moriarity, BS and Webber, BR and McKenna, DH},
title = {Clinical manufacture of CRISPR/Cas9-based cytokine-induced SH2 protein knock-out tumor-infiltrating lymphocytes for gastrointestinal cancers.},
journal = {Cytotherapy},
volume = {27},
number = {10},
pages = {1229-1239},
doi = {10.1016/j.jcyt.2025.06.007},
pmid = {40673842},
issn = {1477-2566},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Cytokines ; *Gastrointestinal Neoplasms/immunology/therapy/pathology/genetics ; Gene Knockout Techniques ; *Lymphocytes, Tumor-Infiltrating/immunology/metabolism ; },
abstract = {INTRODUCTION: The prognosis of stage IV gastrointestinal (GI) carcinomas is poor with a 15% five-year survival rate for colorectal carcinomas. To improve efficacy of tumor infiltrating lymphocytes (TIL), we isolated mutation-reactive autologous TIL and employed CRISPR/Cas9 to knockout (KO) the intracellular checkpoint protein CISH, which has been shown to enhance T cell expansion, functional avidity, and cytokine polyfunctionality, with consequent durable regression of established tumors in an animal model.<br><br>MATERIALS &amp; METHODS: TIL cultures were initiated from resected tumor fragments and maintained for six weeks before harvest and cryopreservation. Candidate neoantigens were nominated by exome sequencing and peptides were used to identify mutation reactive (MR) TIL. Selected MR TIL were thawed and allowed to recover for 24-36 h in media with 10% AB serum, 6000 IU/mL IL-2, and 5 ng/mL IL-7 and IL-15 followed by stimulation with plate-bound anti-CD3/soluble anti-CD28 for 4 days. CISH KO was performed by electroporation of Cas9 mRNA and chemically modified single guide RNA. Between 5 -7.5 million viable cells were added to each 100 cm[2] G-Rex vessel containing 600 mL expansion media (with allogeneic feeder MNC:TIL = 100:1) and incubated for 6-8 days. Cultures were evaluated and split according to cell concentration criteria (and dose cohort) and incubated for an additional 6-8 days. On day 14, all of the cells were harvested, washed with buffer and cryopreserved (5% DMSO). Lot release testing included: viability, %CD3+, cytology review, Gram stain, sterility, endotoxin, mycoplasma, and interferon gamma (IFN-&#x3b3;) production. Additional testing included DNA sequencing to determine genomic CISH editing efficiency and a Western blot for determination of CISH protein loss.<br><br>RESULTS: Patients with GI cancers (colon [10], rectal [8], pancreatic [1], and esophageal [1]) underwent tumor collection. Nineteen of 22 tumor biopsies sampled from 20 patients total proceeded to KO/expansion. Final TIL product results (mean [SD], median [range]) were: viable count (x 10[10]) -3.25 (3.67), 1.95 (0.018-12.40); viable TIL fold expansion -327.1 (364.8), 153.1 (8-1454); % viability - 76 (13), 78 (43-92); % CD3 -94.4 (5.4), 95.8 (78.6-99.4); % CISH KO efficiency - 75 (29), 87 (0-96); % editing efficiency - 59.9 (24.8), 66.9 (0.4-86). Viability fell below 70% for five TIL products. All other lot release testing has met specification. Thirteen patients have received TIL; six patients were not treated due to disease progression prior to anticipated infusion.<br><br>CONCLUSION: The translation of CRISPR/Cas9-based CISH KO MR TIL from the basic research lab to current good manufacturing practices The (cGMP) facility was successful, allowing for optimized, large-scale expansion in support of a first-in-human clinical trial to treat patients with metastatic GI cancers (ClinicalTrials.gov Identifier: NCT04426669).},
}
@article {pmid40673874,
year = {2025},
author = {Zhang, XM and Qiu, MY and Liao, L and Han, B and Deng, YD and Zhang, N and Hai, LT and Liang, L and Liu, MJ},
title = {Insights Into Coat Color Variation: Unraveling the Role of ASIP by Gene Editing in Fine-Wool Sheep.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {39},
number = {14},
pages = {e70807},
doi = {10.1096/fj.202500355R},
pmid = {40673874},
issn = {1530-6860},
support = {32160777//the National Natural Science Foundation of China/ ; Ky202471//the Public Welfare Research Institutes' Basic Research Fund Project in Autonomous Region/ ; 2023TSYCTD0007//the Scientific and Technological Innovation Team Project of Xinjiang Uygur Autonomous Region/ ; },
mesh = {Animals ; *Agouti Signaling Protein/genetics/metabolism ; Sheep/genetics ; *Gene Editing/methods ; *Hair Color/genetics ; *Pigmentation/genetics ; Female ; Phenotype ; CRISPR-Cas Systems ; Male ; },
abstract = {Sheep coat color is an important phenotypic trait that influences consumer preferences and breeding. Although the Agouti Signaling Protein (ASIP) gene has been linked to pigmentation, the mechanisms underlying its function remain unclear. In a previous study, we generated ASIP gene-edited fine-wool sheep (F0) with diverse coat colors using CRISPR/Cas9. Building on this foundation, the present study investigates coat color variation in 33 F1 offspring produced by mating F0 gene-edited rams with either F0 gene-edited ewes or wild-type white ewes. Comprehensive genotyping identified two inherited ASIP mutations-a 4-bp deletion and a 27-bp deletion with a 1-bp insertion-alongside natural variants (D5, D9) and copy number variation. These genetic differences were associated with a wide range of coat color phenotypes in the F1 population. To explore the underlying mechanisms, we conducted histological analyses (H&amp;E staining), immunohistochemistry, melanin content quantification, qRT-PCR, and Western blotting. Results showed that dark-coated individuals exhibited reduced ASIP expression and elevated levels of TYR and TYRP1, consistent with increased eumelanin synthesis. In contrast, white-coated sheep showed higher ASIP expression and lower levels of melanogenic enzymes. Our findings provide functional validation that ASIP is a central regulator of pigmentation in sheep and reveal how the interplay between gene editing, natural mutations, and gene dosage contributes to diverse coat color outcomes. This work establishes a valuable model for dissecting coat color genetics and offers a molecular framework for future breeding strategies aimed at wool color diversification and economic trait improvement in fine-wool sheep.},
}
@article {pmid40674211,
year = {2025},
author = {Li, J and Xie, L and Shi, C and Zhang, T and Lin, X and Da, H and Sun, K and Han, C and Zhu, B and Chen, X and Deng, H and Xu, J},
title = {CRISPR-based screening identifies the role of KRAB-containing transcription factors ZIM3 and ZNF394 in human major zygotic genome activation.},
journal = {Cell reports},
volume = {44},
number = {8},
pages = {116015},
doi = {10.1016/j.celrep.2025.116015},
pmid = {40674211},
issn = {2211-1247},
mesh = {Humans ; *Zygote/metabolism ; *Transcription Factors/metabolism/genetics ; *Genome, Human/genetics ; *CRISPR-Cas Systems/genetics ; Pluripotent Stem Cells/metabolism ; Gene Expression Regulation, Developmental ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Embryonic Development/genetics ; *Repressor Proteins/metabolism/genetics ; },
abstract = {The initiation of major zygotic genome activation (ZGA) is crucial for human early embryogenesis. However, the transcription factors (TFs) regulating major ZGA in humans remain largely unknown. Here, we performed a CRISPR-based activation screen of 1,603 human TFs in human extended pluripotent stem cells (hEPSCs), which identified 132 candidates as potential regulators of major ZGA. Further evaluation of these candidates revealed that the KRAB-containing TFs ZIM3 and ZNF394 activated totipotent features in hEPSCs upon overexpression. Importantly, simultaneous knockdown of these two TFs arrested human embryo development prior to the eight-cell embryo stage. Mechanistically, the KRAB domains contributed to ZIM3- and ZNF394-mediated totipotency induction in vitro, accompanied by the suppression of a set of four-cell embryo enriched genes. Our study provided valuable resources for totipotency and major ZGA regulation, suggesting an un-reported role of KRAB-containing TFs in major ZGA in humans.},
}
@article {pmid40674218,
year = {2025},
author = {Wilbie, D and Mastrobattista, E and de Jong, OG},
title = {Protocol to rapidly screen CRISPR-Cas9 gene editing outcomes in a cell population by mutating eGFP to a blue or non-fluorescent phenotype.},
journal = {STAR protocols},
volume = {6},
number = {3},
pages = {103950},
pmid = {40674218},
issn = {2666-1667},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Green Fluorescent Proteins/genetics ; Humans ; Mutation/genetics ; Phenotype ; },
abstract = {When designing genome editing therapy, it is crucial to measure outcomes of DNA damage repair. Here, we present a protocol to distinguish the outcome of targeted DNA damage repair from the bottom up, through a previously established readout of enhanced green fluorescent protein (eGFP) to blue fluorescent protein (BFP) mutations. We describe steps for producing eGFP-positive cells and differentiating between non-homologous end joining-induced gene knockout and homology-directed repair-induced-directed mutation in these cells. This protocol has potential for high-throughput and scalable assessment of gene editing techniques. For complete details on the use and execution of this protocol, please refer to Walther et al.[1] and Wilbie et al.[2].},
}
@article {pmid40674406,
year = {2025},
author = {Dinesh, RK and Wang, C and Qu, Y and Rustagi, A and Cousins, H and Zengel, J and Wang, X and Barnard, TR and Johnson, WA and Xu, G and Zhang, T and Magazine, N and Beck, A and Heilbroner, LM and Peters-Schulze, G and Wilk, AJ and Wang, M and Huang, W and Howitt, BE and Carette, J and Altman, R and Blish, CA and Cong, L},
title = {Membrane-wide screening identifies potential tissue-specific determinants of SARS-CoV-2 tropism.},
journal = {PLoS pathogens},
volume = {21},
number = {7},
pages = {e1013157},
pmid = {40674406},
issn = {1553-7374},
support = {K08 AI163369/AI/NIAID NIH HHS/United States ; R01 GM141627/GM/NIGMS NIH HHS/United States ; R35 GM153195/GM/NIGMS NIH HHS/United States ; R35 HG011316/HG/NHGRI NIH HHS/United States ; },
mesh = {Humans ; *SARS-CoV-2/physiology ; *COVID-19/virology/metabolism ; *Virus Internalization ; *Viral Tropism/physiology ; Angiotensin-Converting Enzyme 2/metabolism/genetics ; HEK293 Cells ; Organ Specificity ; CRISPR-Cas Systems ; Antigens, Differentiation ; },
abstract = {While SARS-CoV-2 primarily infects the respiratory tract, clinical evidence indicates that cells from diverse cell types and organs are also susceptible to infection. Using the CRISPR activation (CRISPRa) approach, we systematically targeted human membrane proteins in cells with and without overexpression of ACE2, thus identifying unrecognized host factors that may facilitate viral entry. Validation experiments with replication-competent SARS-CoV-2 confirmed the role of newly identified host factors, particularly the endo-lysosomal protease legumain (LGMN) and the potassium channel KCNA6, upon exogenous overexpression. In orthogonal experiments, we show that disruption of endogenous LGMN or KCNA6 decreases viral infection and that inhibitors of candidate factors can reduce viral entry. Additionally, using clinical data, we find possible associations between expression of either LGMN or KCNA6 and SARS-CoV-2 infection in human tissues. Our results identify potentially druggable host factors involved in SARS-CoV-2 entry, and demonstrate the utility of focused, membrane-wide CRISPRa screens in uncovering tissue-specific entry factors of emerging pathogens.},
}
@article {pmid40674424,
year = {2025},
author = {Kaur, K and Zaheer, J and Lang, F and Ribeiro, DL and Zhang, M and Song, H and Zhang, W and Chari, R and Alkaissi, H and Yang, C},
title = {mtKO: A dedicated guide RNA library for mitochondria research.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {122},
number = {29},
pages = {e2502285122},
pmid = {40674424},
issn = {1091-6490},
support = {HHSN261201500003C/CA/NCI NIH HHS/United States ; HHSN261201500003I/CA/NCI NIH HHS/United States ; },
mesh = {*Mitochondria/genetics/metabolism ; Humans ; *Superoxide Dismutase/genetics/metabolism ; Isocitrate Dehydrogenase/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics ; Gene Knockout Techniques/methods ; CRISPR-Cas Systems ; Mutation ; Gene Library ; Cell Line, Tumor ; Neoplasms/genetics/metabolism/pathology ; Animals ; Superoxide Dismutase 2 ; },
abstract = {Mitochondria are multifunctional organelles central to both physiological and pathological processes. In malignant cancer cells, mitochondrial reprogramming establishes the metabolic foundation to meet cellular demands, which is particularly important in tumor cells with existing metabolic perturbations. To identify key mitochondrial pathways supporting cancer development, we developed mitochondria Knockout (mtKO), a robust and unbiased CRISPR screening platform to pinpoint critical mitochondria-associated pathways. The mtKO screen revealed that the mitochondrial antioxidant enzyme SOD2 is essential for cells harboring IDH1 mutations. Mechanistically, SOD2 activity determines the disease manifestation of IDH1-mutated cancers, through maintaining redox homeostasis and mitochondrial fitness. This study introduces a powerful functional genomic tool to identify mitochondrial-centered pathways and reveals the selective mitochondrial vulnerability in Krebs cycle-deficient cancers for future therapeutic intervention.},
}
@article {pmid40674699,
year = {2025},
author = {Xin, C and Lu, Y and Gul, SL and Sun, W and Cao, Z and Kong, X and Fan, K and Li, S and Liu, X and Wang, XC and Chen, QJ},
title = {Never-germinating Arabidopsis seeds with LbCas12a-induced mutations in 6 clade A type 2C protein phosphatase genes.},
journal = {Plant physiology},
volume = {198},
number = {3},
pages = {},
pmid = {40674699},
issn = {1532-2548},
support = {2023YFD1202905//National Key Research and Development Program of China/ ; },
mesh = {*Arabidopsis/genetics/growth &amp; development ; *Seeds/genetics/growth &amp; development ; *Mutation/genetics ; *Germination/genetics ; *Arabidopsis Proteins/genetics/metabolism ; Gene Editing/methods ; *Phosphoprotein Phosphatases/genetics/metabolism ; CRISPR-Cas Systems/genetics ; },
abstract = {Cas12 nucleases, such as Cas12a, Cas12i, and Cas12f, are genome-editing tools that possess several unique attributes. However, the potential of various Cas12 variants for multiplex genome editing in Arabidopsis (Arabidopsis thaliana) remains insufficiently characterized. In this report, we systematically evaluated 18 additional targets and demonstrated that the LbCas12a variant carrying D156R and E795L mutations exhibits minimal target bias. Furthermore, we achieved an editing efficiency of at least 73.8% (45/61) in generating T1 homozygous sextuple mutants, with more than half of these mutants exhibiting a complete seed germination arrest phenotype. Comparative analysis of 7 LbCas12a variants revealed that the optimization of nuclear localization sequences, rather than codon usage, is fundamental for improved editing efficiency, and that the E795L mutation had synergistic effects with other mutations in highly efficient LbCas12a variants. Further investigation into 1 Cas12i3 and 2 AsCas12f variants showed that the Cas12i3 variant also exhibits sufficiently high editing efficiency in Arabidopsis, although additional refinements were required to mitigate its target bias. Collectively, in this study, we developed the most efficient clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) tool for multiplex genome editing in Arabidopsis, as demonstrated by the highly efficient generation of never-germinating seeds harboring mutations in 6 clade A type 2C protein phosphatase genes.},
}
@article {pmid40674704,
year = {2025},
author = {Yamaguchi, K and Koya, J and Mizuno, K and Mizukami, Y and Yoshifuji, K and Saito, Y and Tabata, M and Shingaki, S and Yuasa, M and Ito, Y and Nakashima, K and Dreval, K and Morin, RD and Chiba, K and Okada, A and Shiraishi, Y and Murakami, K and Kogure, Y and Ohshima, K and Kataoka, K},
title = {In vivo CRISPR screening reveals cooperation of KMT2D and TP53 deficiencies in B-cell lymphomagenesis.},
journal = {Blood advances},
volume = {9},
number = {19},
pages = {5040-5055},
pmid = {40674704},
issn = {2473-9537},
mesh = {*Tumor Suppressor Protein p53/genetics/deficiency ; Humans ; Animals ; Mice ; *Lymphoma, Large B-Cell, Diffuse/genetics/pathology ; *DNA-Binding Proteins/genetics/deficiency ; *CRISPR-Cas Systems ; *Lymphoma, B-Cell/genetics ; *Neoplasm Proteins/genetics/deficiency ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Line, Tumor ; },
abstract = {Although recent genetic studies have identified numerous genetic alterations in diffuse large B-cell lymphoma (DLBCL), their biological relevance remains elusive. Here, we performed in vivo CRISPR loss-of-function screening targeting 86 genes recurrently altered in DLBCL to examine oncogenicity of single-guide RNA (sgRNA)-targeted genes, association between genotype and lineage, occurrence of second-hit alterations, and cooperability among sgRNA-targeted genes and second-hit alterations. Transplantation of the CRISPR library-transduced hematopoietic stem/progenitor cells induces various hematologic malignancies, including B-cell lymphomas in mice. Enrichment analysis of sgRNA-targeted genes demonstrates significant overrepresentation of Kmt2d, Pax5, and Trp53 in B-cell lymphomas. Whole-exome sequencing identifies recurrent second-hit driver alterations, showing significant enrichment of Trp53 alterations in sgKmt2d-targeted B-cell lymphomas. Importantly, KMT2D and TP53 mutations are found to be the most prevalent co-occurring combination in human DLBCL, which is more prominent in relapsed/refractory DLBCL. Moreover, this combination confers significantly worse prognosis independent of clinical factors. Transcriptomic sequencing identifies overexpression of Yap1, the Hippo pathway component, in double sgKmt2d-targeted/Trp53-altered B-cell lymphomas. Furthermore, chromatin accessibility analysis demonstrates enrichment of transcriptional enhanced associate domain 1 binding motifs in regions that gained accessibility and increased expression of their nearest genes in these B-cell lymphomas. Most importantly, genetic and pharmacological inhibition of YAP1 suppresses in vitro cell proliferation and in vivo tumor growth of a human KMT2D/TP53-altered DLBCL cell line and prolongs survival of mice transplanted with double sgKmt2d-targeted/Trp53-altered B-cell lymphoma cells. Our findings demonstrate the utility of in vivo CRISPR screening to integrate human cancer genomics with mouse modeling and highlight the functional interplay between KMT2D and TP53 aberrations, providing insights into therapeutic strategies in DLBCL.},
}
@article {pmid40674851,
year = {2025},
author = {Li, D and Hu, J and Hu, J and Zhang, CY},
title = {Integration of AND logic circuit with CRISPR/Cas12a system for sensitive detection of biomarkers and accurate discrimination of breast cancer cells.},
journal = {Biosensors &amp; bioelectronics},
volume = {288},
number = {},
pages = {117790},
doi = {10.1016/j.bios.2025.117790},
pmid = {40674851},
issn = {1873-4235},
mesh = {Humans ; *CRISPR-Cas Systems ; *Breast Neoplasms/diagnosis/genetics ; *Biomarkers, Tumor/analysis/genetics ; Cell Line, Tumor ; Flap Endonucleases/analysis/genetics ; Single-Cell Analysis/methods ; *Biosensing Techniques/methods ; Limit of Detection ; },
abstract = {Flap endonuclease 1 (FEN1) is a structure-selective nuclease that is of great significance in maintaining genomic stability. FEN1 is up-regulation in various cancers and is regarded as a new biomarker for cancer diagnosis. Herein, we integrate DNA logic circuit with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system for sensitive detection of FEN1 at the cellular level and accurate discrimination of different cell types. In this design, FEN1 cleaves the flap-containing synthetic substrates, and subsequently magnetic separation separates the primers from the cleaved substrate fragments, inducing the release of the primers. The released primers can initiate strand displacement amplification (SDA) reaction to generate abundant activators that can activate the trans-cleavage activity of the CRISPR/Cas12a system to cleave the signal probes for the recovery of Cy5 fluorescence signal. This circuit displays superior sensitivity with a detection limit of 5.19 × 10[-5] U/μL, and it is capable of screening the FEN1 inhibitors, quantifying the activity of FEN1 with sensitivity at the single-cell level, and discriminating diverse FEN1 levels in clinical breast cancer tissues. We further construct an AND logic circuit based on miR-31 and FEN1 to simultaneously monitor the biomarkers at the cellular level and achieves accurate discrimination of different breast cancer cell types with a P-value of less than 0.05. The integration of DNA logic circuit with CRISPR/Cas12a system provides a new approach for biomarker-related biomedical research and clinical diagnostics.},
}
@article {pmid40675140,
year = {2026},
author = {Karimi, MA and Paryan, M and Behrouzian Fard, G and Sadeghian, H and Zarrinfar, H and Hosseini Bafghi, M},
title = {Challenges and Opportunities in the Application of CRISPR-Cas9: A Review on Genomic Editing and Therapeutic Potentials.},
journal = {Medical principles and practice : international journal of the Kuwait University, Health Science Centre},
volume = {35},
number = {1},
pages = {1-17},
pmid = {40675140},
issn = {1423-0151},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Humans ; *Genetic Therapy/methods ; },
abstract = {<p>Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) constitute a revolutionary gene-editing technology, allowing precise DNA modifications with vast potential for disease treatment and the creation of genetically modified organisms. This system consists of various components designed to target specific genes, requiring efficient nuclear access within target cells through diverse delivery methods, including physical techniques and carrier-based approaches. Despite its transformative promise, CRISPR faces several challenges, including efficient cellular delivery, off-target effects, immune responses, optimizing editing efficiency, and ethical concerns. Overcoming these hurdles is crucial for fully harnessing its applications. However, CRISPR-Cas9 offers remarkable opportunities for pioneering gene therapies across various disorders, including cancer, and could revolutionize agriculture by engineering disease-resistant crops. A key strength of CRIScPR lies in its adaptability to a broad spectrum of genes, significantly enhancing its versatility. The technology's potential further expands through emerging interdisciplinary integrations, such as artificial intelligence, machine learning, and biological imaging. These advancements can refine CRISPR's precision, improve efficiency, and mitigate existing limitations, positioning it as an indispensable tool in future genetic research. Overall, CRISPR-Cas9 promises transformative progress in healthcare and agriculture, solidifying its role as a cornerstone in the evolution of genetic engineering. </p>.},
}
@article {pmid40675426,
year = {2025},
author = {Li, W and Tang, Z and Zhu, X and Wang, B and Zhang, X and Ji, Z and Cheng, S},
title = {Ultrasensitive detection of MMP-2 via T7 RNA polymerase and CRISPR/Cas13a-Enhanced electrochemiluminescence biosensor for COPD diagnosis.},
journal = {Methods (San Diego, Calif.)},
volume = {242},
number = {},
pages = {80-88},
doi = {10.1016/j.ymeth.2025.07.005},
pmid = {40675426},
issn = {1095-9130},
mesh = {*Matrix Metalloproteinase 2/analysis/blood/genetics ; *Biosensing Techniques/methods ; Humans ; CRISPR-Cas Systems/genetics ; Limit of Detection ; Luminescent Measurements/methods ; *Electrochemical Techniques/methods ; *Pulmonary Disease, Chronic Obstructive/diagnosis/blood ; *DNA-Directed RNA Polymerases/genetics/metabolism ; *Viral Proteins/genetics/metabolism ; Metal Nanoparticles/chemistry ; Gold/chemistry ; Biomarkers ; Nucleic Acid Amplification Techniques/methods ; },
abstract = {In this work, an electrochemiluminescence (ECL) biosensor integrating T7 RNA polymerase amplification and CRISPR/Cas13a-mediated signal enhancement was developed for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2), a key biomarker associated with chronic inflammatory diseases such as COPD. A peptide nucleic acid (PNA) probe was designed to respond specifically to MMP-2 cleavage, enabling the release of DNA templates for subsequent T7 RNA polymerase-driven transcription amplification. The generated RNA triggers the collateral cleavage activity of CRISPR/Cas13a, resulting in a significant amplification of the ECL signal. The biosensor's surface was constructed using a AuNPs/Ti3C2Tx/Ru(II)-PEI nanocomposite, which enhanced signal transduction and stability. Under optimized conditions, the proposed biosensor achieved a detection limit as low as 62.05 fM, demonstrating superior sensitivity compared to conventional methods, as summarized in Table 1. The platform also exhibited excellent specificity and anti-interference capability, ensuring reliable detection of MMP-2 in complex biological samples. This study provides a simple yet highly efficient strategy for enzymatic biomarker detection, offering great potential for clinical applications in early disease diagnosis and monitoring.},
}
@article {pmid40675779,
year = {2025},
author = {Sherkow, JS},
title = {A "Bare Hope of A Result": The Second CRISPR Patent Appeal.},
journal = {The CRISPR journal},
volume = {8},
number = {5},
pages = {317-320},
doi = {10.1177/25731599251361362},
pmid = {40675779},
issn = {2573-1602},
mesh = {*Patents as Topic/legislation &amp; jurisprudence ; Humans ; *Gene Editing/legislation &amp; jurisprudence ; *CRISPR-Cas Systems ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; United States ; Dissent and Disputes/legislation &amp; jurisprudence ; },
abstract = {On May 12, 2025, the US Court of Appeals for the Federal Circuit issued its second decision in the long-running CRISPR patent dispute between the Regents of the University of California and related institutions (CVC) and the Broad Institute. This Perspective recounts the principal dispute to date, reviews the Federal Circuit's recent opinion, and provides a critique of its analysis. In particular, this Perspective highlights how the decision is self-contradictory and in tension with patent law's conception doctrine-when an inventor has formed a "definite and permanent" idea of an invention in the mind or whether the invention was little more than a "bare hope" of a result. This Perspective briefly concludes with the implications of this recent decision and where the underlying dispute is likely headed.},
}
@article {pmid40675807,
year = {2025},
author = {Bykonya, AG and Guschin, DY and Barlev, NA},
title = {Modern approaches to engineering human reporter cell lines using CRISPR within Safe Harbor loci and endogenous genes.},
journal = {Critical reviews in biotechnology},
volume = {45},
number = {8},
pages = {1816-1832},
doi = {10.1080/07388551.2025.2515834},
pmid = {40675807},
issn = {1549-7801},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Genes, Reporter/genetics ; Cell Line ; *Gene Editing/methods ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {Reporter systems are gaining increasing popularity in modern molecular biology as they provide reliable and clear readouts for various types of assays, both in cellulo and in vivo. The generation of reporter cell lines is instrumental for screening activators and inhibitors of signaling pathways to develop new therapeutic approaches. Reporter cell lines are those with stably integrated reporter constructs containing signaling genes (often luciferase or fluorescent proteins), enabling the visualization and tracking of protein expression. Although seemingly harmless and straightforward, untargeted genomic integration of reporter genes may severely affect the expression of neighboring genes, causing unwanted and unpredictable effects. Unlike the untargeted approach, the CRISPR/Cas9 system provides a more precise method of reporter integration, especially when reporters are integrated into Safe Harbor loci. This ensures minimal influence on neighboring genomic regions. This review discusses recent advancements in creating reporter lines using the CRISPR/Cas9 system and experimental approaches for identifying suitable Safe Harbor loci.},
}
@article {pmid40675820,
year = {2025},
author = {Turn, RE and Hilgendorf, KI and Johnson, CT and Han, K and Aziz-Zanjani, MO and Swails Bollinger, S and Domizi, P and Cheng, R and Rabiee, A and Zhu, Y and Jiang, Z and Asthana, A and Demeter, J and Svensson, KJ and Bassik, MC and Jackson, PK},
title = {A genome-wide, CRISPR-based screen reveals new requirements for translation initiation and ubiquitination in driving adipogenic fate change.},
journal = {Genes &amp; development},
volume = {39},
number = {19-20},
pages = {1241-1264},
pmid = {40675820},
issn = {1549-5477},
support = {P30 DK116074/DK/NIDDK NIH HHS/United States ; R01 DK125260/DK/NIDDK NIH HHS/United States ; T32 AG000266/AG/NIA NIH HHS/United States ; },
mesh = {Animals ; Mice ; *Ubiquitination/genetics ; *Adipogenesis/genetics ; 3T3-L1 Cells ; Lipogenesis/genetics ; Adipocytes/cytology/metabolism ; *Peptide Chain Initiation, Translational/genetics ; CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Mitosis/genetics ; },
abstract = {In response to excess nutrients, white adipose tissue expands by both generating new adipocytes and upregulating lipogenesis in existing adipocytes. Here, we performed a genome-wide functional CRISPR screen to identify regulators of adipogenesis in the mouse 3T3-L1 preadipocyte model. In this pooled screening strategy, we used FACS to isolate populations based on lipid content, gating for fluorescence intensity of lipophilic fluorescent BODIPY dye. Additionally, we categorized whether the gene functions primarily during mitotic clonal expansion, lipogenesis, or both. We found that translation initiation and ubiquitin-dependent protein stability regulators drive both adipogenic fate change and lipogenesis. We further supported these findings with proteomics, demonstrating that essential changes in protein reprogramming can drive or inhibit 3T3-L1 adipogenesis independent of transcription. Furthermore, we demonstrated that specific branches of the hypusination pathway, a conserved regulator of translation initiation, are critical for translating adipogenic inducers of mitotic clonal expansion and that the neddylation/ubiquitin pathway modulates insulin sensitivity during lipogenesis.},
}
@article {pmid40676076,
year = {2025},
author = {Borrelli, MJ and Buensuceso, A and Ramos Valdes, Y and Johnston, TPA and Haagsma, J and Shepherd, TG},
title = {CAMKKβ supports growth and viability of epithelial ovarian cancer in vitro and in vivo.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {25913},
pmid = {40676076},
issn = {2045-2322},
support = {PGSD3 - 560096 - 2021//Natural Sciences and Engineering Research Council of Canada/ ; 451405/CAPMC/CIHR/Canada ; },
mesh = {Female ; Humans ; *Calcium-Calmodulin-Dependent Protein Kinase Kinase/metabolism/genetics ; Animals ; *Carcinoma, Ovarian Epithelial/pathology/genetics/metabolism ; Cell Line, Tumor ; *Ovarian Neoplasms/pathology/genetics/metabolism ; Mice ; Cell Survival ; Spheroids, Cellular/metabolism/pathology ; Cell Proliferation ; AMP-Activated Protein Kinases/metabolism ; Autophagy ; Gene Knockout Techniques ; CRISPR-Cas Systems ; Xenograft Model Antitumor Assays ; },
abstract = {Epithelial ovarian cancer (EOC) metastasizes predominantly through multicellular aggregates known as spheroids, which disseminate within the peritoneal cavity and initiate secondary disease upon reattachment at distant sites. EOC spheroids resist detachment-induced cell death by upregulating stress responses including AMP-activated protein kinase (AMPK) signaling and AMPK-dependent macroautophagy (autophagy), highlighting these pathways as potential therapeutic targets. Previously, we used a pharmacological approach to putatively identify Ca[2+]/calmodulin-dependent protein kinase kinase 2 (CAMKKβ, encoded by CAMKK2) as the primary activator of AMPK in EOC spheroids. Herein we have generated CAMKK2 knockout EOC cell lines via CRISPR-Cas9 genome editing to confirm this function of CAMKKβ and explore the impacts of its loss using in vitro and in vivo models of metastatic EOC. CAMKK2 knockout spheroids exhibited decreased AMPK activation, autophagic flux, cell viability, and metastatic potential relative to parental spheroids, and intraperitoneal xenograft tumours lacking CAMKKβ grew slower than their CAMKKβ-intact counterparts. Effect magnitudes varied between cell line models, suggesting context-dependent roles for CAMKKβ in EOC and rationalizing further studies to characterize the underlying mechanisms. Altogether, our findings highlight CAMKKβ as an important contributor to metabolic reprogramming in EOC spheroids and as a potential therapeutic target in the setting of advanced disease.},
}
@article {pmid40676141,
year = {2025},
author = {Adnani, M and Hong, SH and Galli, S and Mahajan, A and Lu, C and Abualsaud, N and Biermann, T and Li, Y and Rivera, A and Sebsebie, BS and Caprio, L and Kuwahara, L and Krawczyk, E and Tilan, JU and Lee, Y and Rodriguez, O and Wang, H and Jin, L and Regan, M and de Assis, S and Albanese, C and Pack, SD and Cavalli, LR and Kitlinska, J},
title = {Targeted CRISPR approach reveals an essential role for neuropeptide Y receptor Y5 in Ewing sarcoma extrapulmonary metastasis.},
journal = {Oncogene},
volume = {44},
number = {36},
pages = {3350-3363},
pmid = {40676141},
issn = {1476-5594},
support = {1R03CA178809//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 ES031611/ES/NIEHS NIH HHS/United States ; P30-CA051008//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R21 CA198698/CA/NCI NIH HHS/United States ; R01 CA197964/CA/NCI NIH HHS/United States ; 1R01CA197964//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; S10 OD025153-01//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30 CA051008/CA/NCI NIH HHS/United States ; 1R21CA198698//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 1RO1CA123211//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 CA123211/CA/NCI NIH HHS/United States ; S10 OD025153/OD/NIH HHS/United States ; R03 CA178809/CA/NCI NIH HHS/United States ; },
mesh = {*Sarcoma, Ewing/genetics/pathology/metabolism ; *Receptors, Neuropeptide Y/genetics/metabolism ; Animals ; Humans ; Mice ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Neuropeptide Y/metabolism/genetics ; Neoplasm Metastasis ; *Bone Neoplasms/genetics/pathology ; Cell Movement/genetics ; },
abstract = {Ewing sarcoma (ES) is a pediatric malignancy that lacks adequate therapies for its metastatic form. These tumors constitutively express neuropeptide Y (NPY) and its Y5 receptor (Y5R), which leads to elevated levels of the peptide in patients' serum. In animal models, xenografts secreting NPY metastasize to extrapulmonary niches, including bone; the phenotype associated with adverse prognosis in ES patients. To determine the role of the NPY/Y5R axis in ES extrapulmonary dissemination, we used a doxycycline-inducible CRISPR/Cas9 system to knockout Y5R in SK-ES-1 xenografts that metastasize to these niches. We have shown that metastases developing from heterogenous SK-ES-1/Y5R-sgRNA primary tumors in doxycycline-treated mice were initiated exclusively by SK-ES-1 clones with a functional NPY5R gene. Similarly, metastasis from wild type SK-ES-1 xenografts was associated with a selection of clones with NPY5R gene gain. In vitro assays identified Y5R-dependent ES cell motility driven by RhoA activation as the mechanism underlying the metastatic effects of NPY. In ES cell lines that secrete NPY, the autocrine NPY/Y5R loop was responsible for maintaining basal cell motility, while ES cells that do not release the peptide responded to the exogenous NPY. These data provide evidence for the crucial role of the NPY/Y5R axis in ES metastasis.},
}
@article {pmid40676395,
year = {2026},
author = {Fereydani, NM and Galehdari, H and Hoveizi, E and Alghasi, A and Ajami, M and Andashti, B and Malayeri, A},
title = {Investigating the Efficacy of the CRISPR/Cas9 Gene-Editing System for Targeting the HBB FSC 36-37 (-T) Mutation Locus in Hematopoietic Stem Cells.},
journal = {Molecular biotechnology},
volume = {68},
number = {4},
pages = {1851-1866},
pmid = {40676395},
issn = {1559-0305},
support = {EE/1401.2.24.118699/SCU.ac.ir//Shahid Chamran University of Ahvaz/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Gene Editing/methods ; HEK293 Cells ; *Hematopoietic Stem Cells/metabolism ; RNA, Guide, CRISPR-Cas Systems/genetics ; beta-Thalassemia/genetics/therapy ; Mutation ; *beta-Globins/genetics ; Iran ; },
abstract = {The emergence of genome editing using the CRISPR/Cas9 system has opened up new possibilities and significantly improved the potential for long-term gene therapy of beta-thalassemia. In Iran, FSC 36/37 (-T) is one of the most common mutations among affected individuals, with the highest frequency in the West region (20.8%) and the South-West region (14%). In the context of a proof-of-concept investigation, we present a comprehensive design and assess the efficacy of a CRISPR construct specifically engineered to employ homology-directed repair for targeting the FSC 36/37 (-T) mutation in the HBB gene. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The guide RNAs were transferred to HEK293 cells. T7EI analysis and a non-denaturing PAGE system were employed to assess the effectiveness of the guide RNAs. Hematopoietic stem cells were isolated using the MACS system, and transfection was performed with the Lonza Nucleofector device. The edited cells were monitored using TaqMan-qPCR and RFLP-PCR techniques. Furthermore, the Cas9 enzyme's off-target cleavage sites were meticulously examined to ensure the specificity of the editing process. This research revealed successful genome editing in 30% of the clones analyzed in HEK293 cells and 23.91% of the examined clones in HSCs. Our findings demonstrate the potential of CRISPR/Cas9-mediated genome editing as a promising strategy for addressing genetic mutations associated with thalassemia and other monogenic diseases.},
}
@article {pmid40676485,
year = {2025},
author = {Ran, F and Huang, H and Shang, B and Peng, W and Wu, L and Ling, K and Xie, X},
title = {An ultrasensitive sensing strategy based on CRISPR/Cas13a and T7 RNA polymerase amplification for detection of extracellular vesicles.},
journal = {Analytical sciences : the international journal of the Japan Society for Analytical Chemistry},
volume = {41},
number = {10},
pages = {1627-1636},
pmid = {40676485},
issn = {1348-2246},
support = {82272960//the National Science Foundation of China/ ; HBCH2024005//the Open Project of Hubei Clinical Research Center of Hypertension/ ; 24Y142//and the Science and Technology Key Program of Shiyan/ ; 24Y154//and the Science and Technology Key Program of Shiyan/ ; 24Y161//and the Science and Technology Key Program of Shiyan/ ; Y202522//the Young Talent Project of Affiliated Dongfeng Hospital/ ; Y202512//the Young Talent Project of Affiliated Dongfeng Hospital/ ; Y202518//the Young Talent Project of Affiliated Dongfeng Hospital/ ; },
mesh = {*Extracellular Vesicles/chemistry/metabolism ; *DNA-Directed RNA Polymerases/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; Humans ; *Viral Proteins/metabolism/genetics ; *Nucleic Acid Amplification Techniques ; *Biosensing Techniques/methods ; Limit of Detection ; Aptamers, Nucleotide/chemistry ; },
abstract = {Extracellular vesicles (EVs) are important biomarkers for an early diagnosis of lung cancer. Herein, we proposed an ultrasensitive fluorescent sensing platform for EVs detection, which involves aptamer and streptavidin-modified magnetic nanoparticles (SA-MB) magnetic separation technology as well as T7 RNA polymerase-assisted CRISPR/Cas13a system, which can achieve target recycling signal amplification. In this detection method, biotin-modified CD63 aptamer hybridizes first with the aptamer Blocker (T7 promoter) and then binds to SA-MB. When adding EVs, the CD63 aptamer in CD63 aptamer/Blocker/SA-MB complex captures EVs causing the release of Blocker single chain. Subsequently, large amounts of ssRNAs, which are generated with the assistance of Blocker-initiated T7 RNA polymerase, were recognized by CRISPR/Cas13a and trigger its trans-cleavage report probe (F-Q). Eventually, the report probe labeled with fluorescent dye (FAM) and quench group (BHQ) at both ends was cut to produce fluorescent signal. The designed sensor combined this with a signal amplification strategy based on T7 RNA polymerase and CRISPR/Cas13a to significantly enhance the sensitivity and specificity of EVs detection. The use of magnetic separation technology eliminates interference from complex matrices and improves EVs detection efficiency, while the introduction of T7 RNA polymerase and CRISPR/Cas13a enables multiple amplifications of the sensor signals, and enhancing the accuracy and sensitivity of the method. Ultimately, the combination of multiple amplification reactions resulted in a detection limit (LOD) for EVs as low as 60 particles/mL (approximately 1 zmol/L). In addition, this detection method can specifically distinguish EVs from other confounding substances and efficiently detect plasma EVs from lung cancer and healthy individuals in actual samples. Indicating this sensing platform is a valuable tool for early lung cancer detection.},
}
@article {pmid40677232,
year = {2025},
author = {Chen, Z and Zhu, D and Lai, KS and Chen, Y and Hu, Y and Fang, Y and Yan, Z and Hu, B and Zhang, Z and Zhang, M and Li, F},
title = {VEGFA Stop-Gained Variant Deteriorates Cardiac Remodeling in Myocardial Infarction.},
journal = {Circulation. Genomic and precision medicine},
volume = {18},
number = {4},
pages = {e004879},
doi = {10.1161/CIRCGEN.124.004879},
pmid = {40677232},
issn = {2574-8300},
mesh = {Animals ; *Myocardial Infarction/genetics/pathology/metabolism/physiopathology ; *Vascular Endothelial Growth Factor A/genetics/metabolism ; Mice ; *Ventricular Remodeling/genetics ; Humans ; Disease Models, Animal ; Male ; CRISPR-Cas Systems ; },
abstract = {BACKGROUND: A sustained dosage of VEGFA (vascular endothelial growth factor A) is crucial for angiogenesis in both homeostasis and cardiovascular diseases. Start codon CUG-initiated alternative translation is a conserved mechanism for producing mature VEGFA. Genetic surveys have identified stop-gained variants predicted to prematurely terminate CUG-initiated translation without affecting start codon ATG-initiated translation. However, the impacts of these variants on the vasculature in steady-state and disease conditions remain unknown.<br><br>METHODS: Using CRISPR/Cas9 genome editing, we established the Vegfa[Q150X/Q150] allele (Q150X), a mouse genetic model that mimics the human VEGFA stop-gained variant. The effects of this variant were tested in both adult homeostatic conditions and the acute myocardial infarction (MI) model. We analyzed and quantified cardiac vasculature structure using immunofluorescence and light-sheet imaging. Furthermore, we characterized cellular heterogeneity, cell-cell interactions, and gene regulation using single-nucleus RNA sequencing, as well as cell type-specific transcriptomics and epigenomics.<br><br>RESULTS: Homozygous mice carrying the stop-gained variant were viable. VEGFA dosage was reduced to 70% in the Q150X homeostatic heart, with no significant alteration in cardiac function or vasculature. In the MI model, VEGFA dosage in Q150X was reduced to about 40% within the first week post-infarction, leading to functional deterioration in the post-MI hearts. Significant changes in cellular composition were observed 3 days post-MI. In particular, endothelial cells in Q150X diverged into a state that showed a higher level of hypoxia stress, an elevated inflammatory response, and increased extracellular matrix secretion. In addition, we observed an increase in Nppb[+] stressed cardiomyocytes in both 3 days post-MI and homeostasis. Finally, proinflammatory macrophages, neutrophils, and Cd8[+]T cells were enriched in the ischemic zone of Q150X hearts.<br><br>CONCLUSIONS: CUG-initiated translation contributes significantly to the production of mature VEGFA in ischemic hearts. VEGFA dosage is critical in determining the cellular microenvironment during ischemic injury.},
}
@article {pmid40678258,
year = {2025},
author = {Zhang, J and Li, J and Zhou, J and Zhong, J and Xu, Y and Mao, X and Xu, M and Luo, S and Yang, Y and Hu, R and Liu, DA and Chen, S and Qiu, Y and Chen, K and Yuan, J and Zhang, X and Li, X},
title = {A Critical Review of the CRISPR-Cas Technology in the Detection of SARS-CoV-2 Variants.},
journal = {The Canadian journal of infectious diseases &amp; medical microbiology = Journal canadien des maladies infectieuses et de la microbiologie medicale},
volume = {2025},
number = {},
pages = {9107724},
pmid = {40678258},
issn = {1712-9532},
abstract = {Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still going on, and as the epidemic situation continues, the genome of SARS-CoV-2 is also mutating and evolving, resulting in more and more SARS-CoV-2 mutant strains, which have brought serious pressure on the prevention and control of COVID-19. Given that the COVID-19 is still spreading, it is extremely important to rapidly identify SARS-CoV-2 variants by nucleic acid assays. Thus, developing highly sensitive and specific assays that are suitable for field testing, high-throughput, and automation, as well as other diagnostic applications for SARS-CoV-2 variants, is urgently needed. This paper reviews the research progress of novel CRISPR-based diagnostic methods for SARS-CoV-2 variants.},
}
@article {pmid40678944,
year = {2025},
author = {Zhu, C and Huang, Q and Fu, R and Xun, Z and Ou, Q and Xianyu, Y and Liu, C},
title = {A Triple-Modal Biosensing Strategy for Hepatitis B Virus Based on Mg[2+]-Mediated Modulation of CRISPR/Cas12a and Au@Pt Nanoparticles.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {21},
number = {36},
pages = {e05341},
doi = {10.1002/smll.202505341},
pmid = {40678944},
issn = {1613-6829},
support = {82030063 82372317 82372316//National Natural Science Foundation of China/ ; 2023Y4004//University-Industry Research Cooperation Project of Science and Technology of Fujian Province/ ; 2025C02124//"Pioneer" and "Leading Goose" R&amp;D Program of Zhejiang/ ; 2023QH1102//Startup Fund for Scientific Research of Fujian Medical University/ ; 2024QH2043//Startup Fund for Scientific Research of Fujian Medical University/ ; },
mesh = {*Hepatitis B virus/genetics/isolation &amp; purification ; *Biosensing Techniques/methods ; *Metal Nanoparticles/chemistry/ultrastructure ; *Gold/chemistry ; *CRISPR-Cas Systems/genetics ; *Magnesium/chemistry ; DNA, Viral ; *Platinum/chemistry ; Nucleic Acid Amplification Techniques ; Humans ; },
abstract = {Hepatitis B virus (HBV) infection remains a significant global public health issue, and rapid detection of HBV DNA is crucial for disease prevention and control. However, traditional methods for HBV DNA detection are limited by their reliance on precise instruments and single readout, which can hardly meet the requirements of on-site detection. In this study, the Mg[2+]-enhanced trans-cleavage activity of clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) is reported and coupled with loop-mediated isothermal amplification (LAMP) and Au@Pt nanoparticles as a signaling reporter for on-site detection of HBV DNA. This triple-modal biosensing strategy enables multiple signal readouts including UV-vis spectrum, RGB value, and temperature with high sensitivity and accuracy. The linear detection ranges using UV-vis spectroscopy, RGB color recognition, and photothermal modes are from 50 to 10,000 copies µL[-1], with the limitation of detection of 24.07, 39.65, and 23.33 copies µL[-1], respectively. This biosensing strategy is further employed for the qualitative detection of HBV DNA in 48 serum samples, achieving sensitivities of 100%, 100%, and 95.24% for triple modes. This work offers a promising tool of next-generation LAMP-CRISPR/Cas12a for the rapid and portable detection of nucleic acids.},
}
@article {pmid40680043,
year = {2025},
author = {Kurniawan, C and Itoh, T},
title = {Off-target sequence variations driven by the intrinsic properties of the Cas-sgRNA-DNA complex in genome editing.},
journal = {PloS one},
volume = {20},
number = {7},
pages = {e0328905},
pmid = {40680043},
issn = {1932-6203},
mesh = {*Gene Editing/methods ; *DNA/genetics/metabolism ; Humans ; *CRISPR-Cas Systems/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; Mutation ; },
abstract = {Genome-editing technologies hold significant potential across various biotechnological fields, yet concerns about possible risks, including off-target mutations, remain. To ensure safe and effective application, these unintended mutations must be rigorously examined and minimized. Computational approaches are anticipated to streamline the detection of off-target mutations; however, the performance of current prediction tools is limited, likely owing to insufficient knowledge of off-target mutation characteristics. In this study, we collected experimentally validated off-target mutation data and conducted a large-scale analysis of 177 nonredundant datasets obtained from six studies. We developed a method to assess the statistical significance of sequence pattern similarity and diversity between off-target sites. This method is based on a comparison of ordered relative entropy values for aligned target sequences, and it was compared with two other methods on the basis of Euclidean distance and the Pearson correlation coefficient. The three methods demonstrated clear correlations, indicating their validity. These methods were applied to 238 dataset pairs for the same target site, and it was revealed that off-target sequence patterns were quite similar across different experimental conditions, such as varying cell lines and independent experiments, suggesting that the intrinsic properties of the Cas-sgRNA-DNA complex play a key role in determining cleavage sites. However, newly engineered enzymes and those from different bacterial sources occasionally display unique off-target patterns, indicating the need for comprehensive evaluation of each new enzyme to develop reliable prediction tools. The insights gained from this study are expected to contribute to a better understanding of off-target mutation characteristics and support the development of more accurate computational prediction methods.},
}
@article {pmid40680452,
year = {2025},
author = {Yang, T and Zhang, S and Nie, K and Cheng, C and Peng, X and Huo, J and Zhang, Y},
title = {ZNF207-driven PRDX1 lactylation and NRF2 activation in regorafenib resistance and ferroptosis evasion.},
journal = {Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy},
volume = {82},
number = {},
pages = {101274},
doi = {10.1016/j.drup.2025.101274},
pmid = {40680452},
issn = {1532-2084},
mesh = {Humans ; *NF-E2-Related Factor 2/metabolism/genetics ; *Ferroptosis/drug effects ; *Drug Resistance, Neoplasm/genetics/drug effects ; *Peroxiredoxins/metabolism/genetics ; *Liver Neoplasms/drug therapy/pathology/genetics ; *Carcinoma, Hepatocellular/drug therapy/pathology/genetics ; *Phenylurea Compounds/pharmacology ; *Pyridines/pharmacology ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology ; CRISPR-Cas Systems ; },
abstract = {Regorafenib (RGF) is a critical second-line therapy for advanced hepatocellular carcinoma (HCC) following disease progression on sorafenib; however, the rapid onset of RGF resistance poses a significant barrier to enhancing patient outcomes. In this study, CRISPR/Cas9 screening in RGF-treated HCC cells identified Zinc Finger Protein 207 (ZNF207) as a primary driver of resistance. Further analysis revealed that ZNF207 promotes resistance by inducing antioxidant responses that inhibit ferroptosis, a form of iron-dependent cell death. Mechanistically, ZNF207 facilitates the lactylation of peroxiredoxin 1 (PRDX1) at lysine 67, enhancing nuclear translocation and activation of nuclear factor erythroid 2-related factor 2 (NRF2), a master regulator of antioxidant pathways. This ZNF207-PRDX1-NRF2 pathway creates a ferroptosis-resistant, pro-survival environment under RGF treatment, enabling HCC cells to evade cell death. Functional assays demonstrated that ZNF207 knockdown significantly enhances RGF sensitivity by restoring ferroptosis, with additional findings showing that disrupting PRDX1 lactylation or NRF2 activity similarly reverses resistance. Together, these findings establish a critical link between protein lactylation and RGF resistance, positioning the ZNF207-PRDX1-NRF2 axis as a promising therapeutic target to enhance treatment efficacy in HCC. The implications of this research extend beyond HCC, indicating that targeting ferroptosis-suppressive pathways may offer a broader approach to overcoming resistance in various cancers.},
}
@article {pmid40680607,
year = {2025},
author = {Eladl, O},
title = {CRISPR interference-mediated silencing of pro-inflammatory genes in autoimmune models: functional and biophysical evaluation with sustained effects in primary human PBMCs.},
journal = {International immunopharmacology},
volume = {163},
number = {},
pages = {115257},
doi = {10.1016/j.intimp.2025.115257},
pmid = {40680607},
issn = {1878-1705},
mesh = {Humans ; *Leukocytes, Mononuclear/immunology ; *Gene Silencing ; CRISPR-Cas Systems ; *Autoimmune Diseases/genetics/immunology/therapy ; CD40 Antigens/genetics ; Interleukin-6/genetics ; Interferon-gamma/genetics ; *Inflammation/genetics ; Cells, Cultured ; Cytokines/genetics ; },
abstract = {Autoimmune diseases are caused by unregulated immune stimulation and overproduction of inflammatory cytokines, leading to chronic tissue injury and disability. Current treatments are broad-acting and short-lived, highlighting the need for targeted molecular interventions with prolonged immunomodulatory effects. Here, we demonstrate CRISPR interference (CRISPRi), employing a catalytically inactive Cas9 fused to a KRAB repression domain, to potently and selectively silence major inflammatory genes-IL-6, CD40, and IFN-γ-in human immune cell lines. Utilizing biophysical methods such as surface plasmon resonance and fluorescence anisotropy, complemented by flow cytometric analysis, we validated functional interference with inflammatory signaling cascades and significant downregulation of immune activation markers. These findings indicate that CRISPRi not only represses gene expression but also significantly affects downstream cytokine-receptor interactions and immune cell activation. Compared to other gene silencing approaches, like siRNA and antisense oligonucleotides, CRISPRi showed greater specificity and longer duration, with sustained repression lasting up to 72 h post-treatment. Importantly, we confirmed these effects in primary human peripheral blood mononuclear cells (PBMCs), where CRISPRi significantly suppressed pro-inflammatory cytokine release and modulated immune cell phenotypes. These results highlight the potent, programmable, and sustained immunomodulatory potential of CRISPRi as a therapeutic for autoimmune diseases characterized by inflammation and immune dysregulation.},
}
@article {pmid40680677,
year = {2025},
author = {Li, X and Liu, J and Wang, R and Fu, H and Kim, M and Li, X and Peng, W and Man, S and Gao, Z and Ma, L},
title = {CRISPR-based one-pot detection: A game-changer in nucleic acid analysis.},
journal = {Biosensors &amp; bioelectronics},
volume = {288},
number = {},
pages = {117786},
doi = {10.1016/j.bios.2025.117786},
pmid = {40680677},
issn = {1873-4235},
mesh = {*CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods/instrumentation ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *Nucleic Acids/genetics/analysis/isolation &amp; purification ; Gene Editing/methods ; Point-of-Care Systems ; },
abstract = {The CRISPR/Cas system, originally developed as a gene-editing tool, has rapidly emerged as a powerful platform for nucleic acid detection due to its remarkable specificity, programmability, and robust trans-cleavage activity. While conventional CRISPR-based assays typically require separate amplification and detection steps, introducing complexity, manual intervention, and contamination risks. Recent innovations have led to the development of one-pot detection strategies that integrate target amplification with Cas-mediated signal generation within a single reaction vessel. These streamlined systems not only reduce handling steps and turnaround time but also enhance sensitivity and enable multiplexed detection of low-abundance nucleic acid targets. As such, one-pot CRISPR diagnostics are poised to transform point-of-care testing, pathogen surveillance, and clinical diagnostics. In this review, we summarize the latest advances in one-pot CRISPR-based detection technologies, evaluate their current limitations, and discuss strategies for optimization. Future efforts should focus on refining reaction kinetics, improving reagent stability, reducing costs, and expanding the repertoire of detectable targets. Furthermore, integration with portable platforms, AI-assisted signal interpretation, and the discovery of novel Cas effectors will be pivotal in realizing the full potential of CRISPR-based one-pot systems in diverse real-world applications.},
}
@article {pmid40680868,
year = {2025},
author = {Su, S and Xu, Z and Suo, J and Zhou, Y and Zhang, X and Ignatus, AD and Zuo, Y and Peng, X and Li, F and Chen, M},
title = {Function of the zasp52 gene detected by CRISPR/Cas9 in the global fruit borer Grapholita molesta.},
journal = {Insect biochemistry and molecular biology},
volume = {183},
number = {},
pages = {104363},
doi = {10.1016/j.ibmb.2025.104363},
pmid = {40680868},
issn = {1879-0240},
mesh = {Animals ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; *Moths/genetics/growth &amp; development/metabolism ; Larva/growth &amp; development/genetics/metabolism ; Pupa/growth &amp; development/genetics/metabolism ; Gene Editing ; },
abstract = {Zasp (Z band alternatively spliced PDZ-motif protein) is the core component of the Z-disc in muscle tissue and plays a vital role in the assembly and maintenance of myofibrils. The zasp has been studied in vertebrates, but it has only been reported in model organisms (e.g. Drosophila melanogaster) among insect species. Grapholita molesta is an important fruit pest with weak flight capacity. Flight capacity is important for the seasonal host switch of this pest. So far, the genes involved in the flight of G. molesta have not been analyzed. In this study, we identified and cloned the Gmzasp52 gene from G. molesta. This gene was expressed in different tissues and developmental stages of G. molesta. Using CRISPR/Cas9 gene editing technology, a homozygous Gmzasp52 gene knockout strain of G. molesta was successfully constructed. Knockout of the Gmzasp52 gene significantly prolonged the larval and preoviposition periods of G. molesta, and shortened the pupal period; the larval weight, pupal weight and fecundity decreased significantly, and the pupal mortality increased significantly; the Z-disc structure of the flight muscle was destroyed, and the myofibrils were damaged, resulting in a significant decrease in flight capacity. These results indicate that the Gmzasp52 plays an important role in the growth and development, reproduction, flight muscle structure and flight capacity of G. molesta. Our study reveals the role of zasp52 gene in a non-model insect species for the first time. The results not only analyzed the role of Gmzasp52 gene in G. molesta, but also further characterized the function of zasp52 gene in non-model organisms and provided possible target gene for the control of this pest.},
}
@article {pmid40680952,
year = {2025},
author = {Gao, X and Dong, X and Song, H and Fu, Y and Li, J and Fan, G and Wang, T and Sun, Y and Wang, Y and Qiu, HJ and Luo, Y},
title = {A one-pot CRISPR-Cas12a-based assay for rapid, on-site detection of African swine fever virus.},
journal = {International journal of biological macromolecules},
volume = {321},
number = {Pt 2},
pages = {146109},
doi = {10.1016/j.ijbiomac.2025.146109},
pmid = {40680952},
issn = {1879-0003},
mesh = {*African Swine Fever Virus/genetics/isolation &amp; purification ; *CRISPR-Cas Systems/genetics ; Animals ; Swine ; *African Swine Fever/diagnosis/virology ; DNA, Viral/genetics ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases ; },
abstract = {African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and devastating disease threatening global swine production. The disease has caused substantial economic losses, driving the need for efficient diagnostic tools to enhance surveillance and control. Despite various available assays for ASF, field-deployable tools enabling rapid, accurate, and user-friendly detection remain urgently needed. Here, we developed and validated a novel one-pot recombinase polymerase amplification (RPA)-CRISPR-Cas12a assay for rapid and sensitive detection of ASFV by integrating all components into a single sealed tube, which requires only isothermal heating and ultraviolet visualization. The assay demonstrated a detection limit of 56 TCID50/mL and could be completed within 35 min, and without cross-reactivity with non-ASFV porcine viruses. In comparative testing of 150 clinical samples, the one-pot RPA-CRISPR-Cas12a assay exhibited 100 % agreement with gold standard quantitative PCR (qPCR). Notably, the assay identified ASFV genomic DNA in whole blood as early as 3 days post-infection with sensitivity comparable to the qPCR. This early detection capability, combined with a field-deployable format, provides a robust tool for implementing timely containment measures against ASF, especially in resource-limited setting.},
}
@article {pmid40681150,
year = {2025},
author = {Xi, C and Chiu, S and Voje, WE and Carothers, JM and Moon, TS},
title = {Conditional guide RNA deactivation by mRNA and small molecule triggers in Saccharomyces cerevisiae.},
journal = {New biotechnology},
volume = {89},
number = {},
pages = {105-118},
pmid = {40681150},
issn = {1876-4347},
support = {R01 AT009741/AT/NCCIH NIH HHS/United States ; },
mesh = {*Saccharomyces cerevisiae/genetics/metabolism ; *RNA, Messenger/genetics/metabolism ; *RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Gene Expression Regulation, Fungal ; },
abstract = {CRISPR interference (CRISPRi) technologies have revolutionized bioengineering by providing precise tools for gene expression modulation, enabling targeted gene perturbation and metabolic pathway optimization. Despite these advances, achieving dynamic control over gene expression by CRISPR-based regulation remains a challenge due to its inherently static nature. Utilizing toehold-mediated strand displacement and ligand-responsive ribozymes (aptazymes), this study introduces switchable guide RNAs (gRNAs) that facilitate tunable gene expression mediated by mRNA or small molecule signals. We demonstrate complete silencing of gRNA via strategically designed 5' or 3' extensions that impede the gRNA spacer or the dCas9 handle, with subsequent restoration of function through sequestration or cleavage of the obstructive sequence. The resulting toehold-embedded or aptazyme-embedded gRNAs can be deactivated by specific signals, including two full-length translatable mRNAs and two small molecule triggers, thereby lifting CRISPRi repression on targeted genes. This modular approach allows for gRNA-based biocomputing through multi-layer or multi-input genetic logic gates in Saccharomyces cerevisiae. Offering a versatile strategy for post-CRISPR regulation in response to environmental signals or cellular states, this methodology expands the toolkit in eukaryotic systems for reversible control of gene expression.},
}
@article {pmid40681400,
year = {2026},
author = {Huang, HC and Wu, LF and Liu, K and Ma, BG},
title = {Opto-CRISPR: new prospects for gene editing and regulation.},
journal = {Trends in biotechnology},
volume = {44},
number = {1},
pages = {22-39},
doi = {10.1016/j.tibtech.2025.06.018},
pmid = {40681400},
issn = {1879-3096},
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Optogenetics/methods ; Humans ; Animals ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Clustered regularly interspaced short palindromic repeats (CRISPR) technology represents a landmark advance in the field of gene editing. However, conventional CRISPR/Cas systems are limited by inadequate temporal and spatial control. In recent years, the development of optically controlled CRISPR (Opto-CRISPR) technology has offered a novel solution to this issue. As a combination of optogenetics and the CRISPR technology, the Opto-CRISPR technology enables dynamic space-time-specific gene editing and regulation in cells and organisms. In this review, we concisely introduce the basic principles of Opto-CRISPR, summarize its operational mechanisms, and discuss its applications and recent advances across various research fields. In addition, this review analyzes the limitations of Opto-CRISPR, aiming to provide a reference for the development of this emerging field.},
}
@article {pmid40681749,
year = {2025},
author = {de Lima Balico, L and Gaucher, EA},
title = {Genomic insertion of ancestral uricase into human liver cells to determine metabolic consequences of pseudogenization.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26093},
pmid = {40681749},
issn = {2045-2322},
mesh = {Humans ; *Urate Oxidase/genetics/metabolism ; Uric Acid/metabolism ; *Hepatocytes/metabolism ; Animals ; Triglycerides/metabolism ; Fructose/metabolism ; *Liver/metabolism/cytology ; CRISPR-Cas Systems ; },
abstract = {The biological role of urate (uric acid) during primate evolution has been unclear ever since it was discovered over 100 years ago that humans have unusually high levels of the small molecule compared to most other mammals. Humans (including all apes) are uncharacteristically susceptible to the build-up of urate because we no longer have a functional uricase enzyme capable of oxidizing this highly insoluble molecule. We have now utilized CRISPR technology to insert functional ancestral uricase into the genome of human liver cells to address recent metabolic hypotheses that our ancestral primates inactivated uricase as a mechanism to increase triglyceride production in response to fructose and/or starvation. Uricase expression is confirmed in both hepatocyte monolayer and spheroid tissue cultures, and its expression reduces intracellular urate levels. The presence of uricase is also shown to prevent an increase in triglyceride production upon cellular uptake of fructose in both culture conditions. Our results make progress that further describes a potential advantageous biological role of urate during primate evolution.},
}
@article {pmid40681864,
year = {2025},
author = {Ronda, C and Perdue, T and Schwanz, L and Rivera Gelsinger, D and Brockmann, L and Kaufman, A and Huang, Y and Sternberg, SH and Wang, HH},
title = {Precise virulence inactivation using a CRISPR-associated transposase for combating Enterobacteriaceae gut pathogens.},
journal = {Nature biomedical engineering},
volume = {9},
number = {12},
pages = {2017-2027},
pmid = {40681864},
issn = {2157-846X},
support = {R21 AI168976/AI/NIAID NIH HHS/United States ; R01EB031935//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; 527896//Simons Foundation/ ; DP2 HG011650/HG/NHGRI NIH HHS/United States ; RM1 HG009490/HG/NHGRI NIH HHS/United States ; R21 AI126305/AI/NIAID NIH HHS/United States ; R21AI68976//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; P30 DK132710/DK/NIDDK NIH HHS/United States ; 1016691//Burroughs Wellcome Fund (BWF)/ ; R21 AI146817/AI/NIAID NIH HHS/United States ; 2R01AI132403//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; DP2HG011650//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 DK118044/DK/NIDDK NIH HHS/United States ; R01 AI132403/AI/NIAID NIH HHS/United States ; R01 EB031935/EB/NIBIB NIH HHS/United States ; 1R21AI146817//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; R01 EB027793/EB/NIBIB NIH HHS/United States ; 1R01EB031935//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; S-168-4X5-001//U.S. Department of Defense (United States Department of Defense)/ ; 1R01DK118044//U.S. Department of Health &amp; Human Services | National Institutes of Health (NIH)/ ; },
mesh = {Animals ; *Transposases/genetics/metabolism ; Mice ; *Enterobacteriaceae/pathogenicity/genetics ; Virulence/genetics ; *CRISPR-Cas Systems/genetics ; *Gastrointestinal Microbiome/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Gene Editing/methods ; Shiga Toxin/genetics ; *Enterobacteriaceae Infections/microbiology ; Humans ; },
abstract = {Targeted gene manipulation in a complex microbial community is an enabling technology for precise microbiome editing. Here we introduce BACTRINS, an in situ microbiome engineering platform designed for efficient and precise genomic insertion of a desired payload and simultaneous knockout of target genes. This system leverages conjugation-mediated delivery of CRISPR-associated transposases to achieve RNA-guided genomic integration, allowing precise insertion of a therapeutic payload while neutralizing pathogen virulence without causing cell death. When applied against an Enterobacteriaceae Shiga toxin-producing pathogen in the gut, this system delivers a CRISPR-associated transposase by bacterial conjugation for site-specific inactivation of the Shiga toxin gene and integration of a nanobody therapeutic payload to disrupt pathogen attachment. A single dose of this therapy results in high-efficiency Shiga gene inactivation and improved survival in a murine infection model of Shiga-producing pathogen. This work establishes a new type of live bacterial therapeutic capable of reducing gut infections by transforming toxigenic pathogens into commensal protectors.},
}
@article {pmid40682144,
year = {2025},
author = {Motosugi, N and Hasegawa, K and Kurosaki, N and Kawaguchi, E and Izumi, K and Iida, Y and Higashiseto, M and Yokoyama, K and Sasaki, A and Nakabayashi, K and Fukuda, A},
title = {Highly efficient XIST reactivation in female hPSC by transient dual inhibition of TP53 and DNA methylation during Cas9 mediated genome editing.},
journal = {Stem cell research &amp; therapy},
volume = {16},
number = {1},
pages = {389},
pmid = {40682144},
issn = {1757-6512},
support = {22bm0804030h0002//Japan Agency for Medical Research and Development/ ; },
mesh = {Humans ; *RNA, Long Noncoding/genetics/metabolism ; Female ; *Gene Editing/methods ; *DNA Methylation/genetics ; *Tumor Suppressor Protein p53/metabolism/genetics/antagonists &amp; inhibitors ; *CRISPR-Cas Systems/genetics ; X Chromosome Inactivation/genetics ; *Pluripotent Stem Cells/metabolism/cytology ; },
abstract = {The irreversible erosion of X-chromosome inactivation (XCI) due to repression of the long non-coding RNA XIST presents a major challenge for disease modeling and raises safety concerns for the clinical application of female human pluripotent stem cells (hPSCs) due to the aberrant overexpression of X-linked genes. While Cas9-mediated non-homologous end joining (NHEJ) targeting the XIST promoter can induce DNA demethylation and restore XCI by reactivating XIST, its efficiency remains low. Here, we introduce a highly efficient strategy for XIST reactivation by combining TP53 inhibition with suppression of DNA methylation maintenance during Cas9-mediated NHEJ. This dual-inhibition approach increased the proportion of XIST-positive hPSCs from ~ 5 to ~ 43.7%, providing a robust method for stabilizing XCI in female hPSCs for diverse applications.},
}
@article {pmid40682401,
year = {2025},
author = {Moso, MA and Roche, M and Cevaal, PM and Lewin, SR},
title = {CRISPR/Cas9 for achieving postintervention HIV control.},
journal = {Current opinion in HIV and AIDS},
volume = {20},
number = {5},
pages = {432-440},
pmid = {40682401},
issn = {1746-6318},
mesh = {Humans ; *HIV Infections/therapy/prevention &amp; control/virology ; *CRISPR-Cas Systems ; *Genetic Therapy/methods ; Gene Editing/methods ; *HIV-1/genetics ; },
abstract = {PURPOSE OF REVIEW: Recent advances in gene therapy have led to the first clinically approved CRISPR/Cas9 therapy for β-thalassaemia and sickle cell disease. Gene therapy could play an important role in targeting HIV persistence and achieving postintervention HIV control. Here, we review recent updates in CRISPR/Cas9-based HIV gene therapy approaches, including CCR5-editing (protect), proviral targeting (excise or modify), and immune cell engineering (attack).<br><br>RECENT FINDINGS: Recent studies provide additional safety data for use of CRISPR/Cas9-based gene therapies, however low in vivo editing efficiency highlights the need for improved delivery methods. This is particularly relevant for strategies requiring transfection of all HIV-infected cells containing intact proviruses, such as proviral excision. For ex vivo editing approaches, poor engraftment and durability of edited cells present additional challenges. Newer methods such as lipid nanoparticle delivery could provide a mechanism to overcome current limitations with ex vivo and in vivo delivery. Several studies have demonstrated proof-of-concept of combination gene therapy approaches, including gene editing strategies to generate HIV-resistant cells with immune effector functions, providing novel approaches to control and durably suppress viral replication.<br><br>SUMMARY: Several studies have demonstrated feasibility of gene therapy approaches in achieving postintervention HIV control. Improvements in both ex vivo and in vivo delivery methods are required to progress current gene therapy approaches to the clinic.},
}
@article {pmid40682549,
year = {2025},
author = {Yang, L and Liu, R and Meng, Y and Deng, Z and Bu, S and Liu, J and Huang, A and Wu, S and Kan, X},
title = {Fruit Phenotype Analysis of SlSAHH2-CRISPR Tomato and Methylation Mechanism of SlSAHH2 Promoting Fruit Ripening.},
journal = {Journal of agricultural and food chemistry},
volume = {73},
number = {30},
pages = {18691-18705},
doi = {10.1021/acs.jafc.5c05207},
pmid = {40682549},
issn = {1520-5118},
mesh = {*Solanum lycopersicum/genetics/growth &amp; development/metabolism/enzymology ; *Fruit/genetics/growth &amp; development/metabolism/enzymology ; *Plant Proteins/genetics/metabolism ; Gene Expression Regulation, Plant ; Ethylenes/biosynthesis/metabolism ; *Adenosylhomocysteinase/genetics/metabolism ; *Plants, Genetically Modified/genetics/metabolism/growth &amp; development/enzymology ; DNA Methylation ; Methylation ; Phenotype ; CRISPR-Cas Systems ; },
abstract = {S-adenosyl-l-homocysteine hydrolase (SAHH EC 3.3.1.1) is a key enzyme that maintains methylation homeostasis. In this study, the knockout of SlSAHH2 delayed tomato fruit ripening. Compared with the wild type (WT), most ripening-related biochemical characteristics were significantly reduced in SlSAHH2-CRISPR fruits. Additionally, the expression of genes related to ethylene synthesis and ripening was generally down-regulated. In transcriptome data, a total of 3701 up-regulated and 3134 down-regulated differentially expressed genes (DEGs) were identified and analyzed. Bisulfite sequencing PCR (BSP) results showed that the methylation levels of the promoters of ethylene-related genes in SlSAHH2-OE fruits were higher. Furthermore, in the presence of a methylation inhibitor, the expression of E4, E8, ACO1, ACO3, and ACS2 was generally up- or down-regulated in SlSAHH2-OE or SlSAHH2-CRISPR fruits, respectively. In summary, it can be inferred that in the balance between methylation and ethylene synthesis, SlSAHH2 was more inclined to promote the latter process during tomato fruit ripening.},
}
@article {pmid40682826,
year = {2025},
author = {Zheng, H and Mao, C and Chen, S and Hou, S and Sun, D},
title = {A quorum sensing-controlled type I CRISPRi toolkit for dynamically regulating metabolic flux.},
journal = {Nucleic acids research},
volume = {53},
number = {14},
pages = {},
pmid = {40682826},
issn = {1362-4962},
support = {32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; 2020C02031//Research and Development Program of Zhejiang Province/ ; 32170083//National Natural Science Foundation of China/ ; 31670084//National Natural Science Foundation of China/ ; },
mesh = {*Quorum Sensing/genetics ; *Bacillus subtilis/genetics/metabolism ; *CRISPR-Cas Systems ; *Metabolic Engineering/methods ; Gene Expression Regulation, Bacterial ; Riboflavin/biosynthesis ; Synthetic Biology/methods ; Bacterial Proteins/genetics/metabolism ; },
abstract = {Synthetic biology advances have enabled dynamic metabolic regulation via quorum sensing (QS) and CRISPR systems. However, the integration of QS with CRISPR-based systems for dynamic control remains largely underexplored. Here, we developed a QS-controlled type I CRISPR interference (QICi) toolkit that modulates target gene expression in response to cell density. By streamlining CRISPR RNA (crRNA) vector construction and optimizing key QS components PhrQ and RapQ, we achieved a twofold enhancement in QICi efficacy. We subsequently implemented the optimized QICi to reprogram Bacillus subtilis for d-pantothenic acid (DPA) and riboflavin (RF) biosynthesis. Dynamic regulation of the citrate synthase gene citZ by QICi, coupled with pantoate pathway engineering, cofactor supply enhancement, and suppression of sporulation, elevated DPA titers to 14.97 g/l in 5-l fed-batch fermentations without precursor supplementation. QICi-mediated metabolic rewiring of key nodes boosted RF production by 2.49-fold. Together, our work provides a robust tool for reprogramming microbial metabolism and advancing sustainable biomanufacturing.},
}
@article {pmid40683408,
year = {2025},
author = {Zhou, J and Zhou, C and Jiang, G and Wang, Y and Pan, L and Jia, X and Tu, X and Li, W and Wang, C},
title = {Engineering an Escherichia coli with performance-enhanced switch utilizing CRISPR-Cas9 system as living quorum quencher for biofilm formation inhibition.},
journal = {Environmental research},
volume = {285},
number = {Pt 2},
pages = {122383},
doi = {10.1016/j.envres.2025.122383},
pmid = {40683408},
issn = {1096-0953},
mesh = {*Biofilms/growth &amp; development ; *Escherichia coli/genetics/physiology ; *Quorum Sensing/genetics ; *CRISPR-Cas Systems ; Genetic Engineering ; },
abstract = {Quorum quenching (QQ) of signal molecules plays a critical role in disrupting bacterial communication, thereby suppressing biofilm formation. However, the wild-type QQ bacteria lacks the regulatory capacity to modulate gene expression levels. In this study, the QQ gene aiiO and reporter gene GFP were chromosomally integrated into Escherichia coli BW25113 using the clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9) system. The performance-enhanced switch in the engineering bacteria (EB) allowed it to express aiiO weakly without the inducer isopropyl-beta-D-thiogalactopyranoside (IPTG) and express aiiO strongly with IPTG, and 1.00 mM IPTG induction enhanced EB's QQ activity by 2.34-fold. In activated sludge systems, the inoculation of EB reduced biofilm formation by 18.56 % versus controls after 168 h, with the performance-enhanced switch enhancing inhibition to 24.72 %. EB reduced biofilm thickness by 22.96 %, total microbial biomass by 57.68 %, and significantly decreased extracellular polymeric substances secretion and adhesion strength of the biofilm (maximum reductions: 29.88 % and 34.31 %, respectively) across all sampling points versus controls. 1.00 mM IPTG addition sustainedly intensified these biofilm-inhibitory effects by EB, demonstrating the genetic switch's persistent functionality under environmentally relevant conditions. Furthermore, the genetically modified strain exhibited minimal environmental impact according to standardized assessments. Therefore, this study successfully constructed an implementable strategy for engineering bacteria-mediated biofilm control, with demonstrated applicability in complex environmental systems.},
}
@article {pmid40683724,
year = {2025},
author = {Chen, J and Lu, Y and Meng, X and Chen, D and Wu, C and Song, D},
title = {Visual and self-contained diagnosis of Salmonella using RPA-CRISPR on a centrifugal force driven microfluidic system.},
journal = {Food microbiology},
volume = {132},
number = {},
pages = {104844},
doi = {10.1016/j.fm.2025.104844},
pmid = {40683724},
issn = {1095-9998},
mesh = {*Salmonella/genetics/isolation &amp; purification ; *Nucleic Acid Amplification Techniques/methods/instrumentation ; *CRISPR-Cas Systems ; Food Microbiology/methods ; Recombinases/genetics/metabolism ; Food Contamination/analysis ; Lab-On-A-Chip Devices ; Bacterial Proteins/genetics ; DNA, Bacterial/genetics ; },
abstract = {Rapid and sensitive nucleic acid detection is very important for the detection of Salmonella in food. Convenient detection methods show great potential in food safety. CRISPR-Cas12a system has been widely used for nucleic acid detection. In one-pot reaction, Cas12a can perform cis- and trans-cleavage of amplicons and primers, which reduces the sensitivity of the reaction and has certain limitations. In this study, we integrated the Cas12a cutting system with recombinase polymerase amplification (RPA) into one microfluidic chip to avoid cap opening pollution. A portable observation device was integrated to enable visual detection of signals with the naked eye. The detection technology could specifically detect 1 × 10° CFU/mL Salmonella in 50 min at 37 °C. In the field test, there was no need for professional equipment or high-tech means.},
}
@article {pmid40683956,
year = {2025},
author = {Zhang, C and Li, H and Poluektova, LY and Gendelman, HE and Dash, PK},
title = {Unique molecular signatures in rebound viruses from antiretroviral drug and CRISPR-treated HIV-1-infected humanized mice.},
journal = {Communications biology},
volume = {8},
number = {1},
pages = {1077},
pmid = {40683956},
issn = {2399-3642},
support = {R01 MH115860/MH/NIMH NIH HHS/United States ; R01MH115860//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R56 AG087827/AG/NIA NIH HHS/United States ; R01MH121402//U.S. Department of Health &amp; Human Services | NIH | National Institute of Mental Health (NIMH)/ ; R24OD018546//U.S. Department of Health &amp; Human Services | NIH | NIH Office of the Director (OD)/ ; R01 MH121402/MH/NIMH NIH HHS/United States ; R01 NS034239/NS/NINDS NIH HHS/United States ; R24 OD018546/OD/NIH HHS/United States ; },
mesh = {Animals ; *HIV-1/genetics/drug effects ; *HIV Infections/virology/drug therapy/genetics ; Mice ; Humans ; *CRISPR-Cas Systems ; *Anti-HIV Agents/pharmacology/therapeutic use ; Mutation ; *Anti-Retroviral Agents/pharmacology/therapeutic use ; Pyridones ; Heterocyclic Compounds, 3-Ring ; Oxazines ; Piperazines ; Dolutegravir ; },
abstract = {HIV-1 elimination from a subset of virus-infected humanized mice (hu-mice) is reported following sequential dual treatment with long-acting (LA) antiretroviral (ART) and CRISPR-Cas9 therapies. However, viral rebound is observed in > 50% of the dual-treated animals. The molecular signatures of the rebound virus, recovered from plasma, have now been determined. The LA-ART treatment contains nanoformulated dolutegravir, lamivudine, abacavir, and rilpivirine combinations, and the HIV-1 excision treatment is CRISPR-Cas9 targeting the HIV-1-LTR-gag. One-step reverse transcriptase polymerase chain reaction, which avoids spontaneous preparatory mutations, is performed on plasma-derived RNA. Sanger and Next-Generation Sequencing are employed targeting the HIV-1-gag, pol, and env genes. HIV-1 env shows the most divergence. LA-ART, with or without CRISPR, is responsible for the new mutations. The primary and accessory mutations are detected by deep sequencing. Viral evolution reflects changes in the virus as reported by ART-treated and HIV-1-infected patients. No major CRISPR-specific mutations are observed. The molecular viral signatures demonstrate an accelerated HIV-1 drug resistance escape from ART rather than from the generation of CRISPR mutants. These define viral rebound in the dual-treated hu-mice. The data underscores the limited role of CRISPR excision in generating these rebound HIV-1 mutants from dual-treated hu-mice.},
}
@article {pmid40683967,
year = {2025},
author = {Chang, W and Zhang, B and Yang, S and Zhang, X and Zhang, L and Zhang, S and Lu, J and Wang, W and Shang, P and Yue, Z},
title = {Genome-wide CRISPR/Cas9 screening identifies PTGR2 as a potential therapeutic target for sunitinib resistance in clear cell renal cell carcinoma.},
journal = {Scientific reports},
volume = {15},
number = {1},
pages = {26263},
pmid = {40683967},
issn = {2045-2322},
support = {PR5124025//the Special Fund Project for Doctoral Training Program of Lanzhou Univers