@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 = {},
number = {},
pages = {},
pmid = {41720886},
issn = {2055-0278},
support = {IOS-2303522//National Science Foundation (NSF)/ ; DGE-2146752//National Science Foundation (NSF)/ ; DGE-2146752//National Science Foundation (NSF)/ ; },
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 {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.},
}

Animals
*Gene Editing/methods
Mice
*CRISPR-Cas Systems
*Deoxyribonuclease I/genetics/metabolism
Humans
Disease Models, Animal
*CRISPR-Associated Protein 9/genetics/metabolism
Point Mutation

@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)/ ; },
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.},
}

*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

@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 = {},
number = {},
pages = {106709},
doi = {10.1016/j.jinf.2026.106709},
pmid = {41720308},
issn = {1532-2742},
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 characterize the genomic feature and phylogenetic evolution of ST23-K1 in China.

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 characterized. Time-resolved phylogenetic analysis was performed using integrated locally generated and publicly available data.

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 β-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, were frequently co-located on IncFII-type plasmids.

CONCLUSION: ST23-K1 remains a hypervirulent lineage undergoing ongoing evolutionary expansion. The presence of acquired ARGs in ST23-K1 may be associated with AcrIE-harboring 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 {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 {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 {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.},
}

*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

@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},
doi = {10.1016/j.crmeth.2025.101297},
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.},
}

Animals
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Insect Proteins/genetics/metabolism
Genes, Reporter
*Insecta/genetics
*Blattellidae/genetics/metabolism

@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.

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.

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.

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.},
}

*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

@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.},
}

*Dependovirus/genetics
*Gene Editing/methods
*Genetic Vectors/genetics
*Genetic Therapy/methods
Humans
*CRISPR-Cas Systems
Animals
Gene Transfer Techniques

@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 & 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.},
}

Animals
Female
*Gryllidae/genetics/growth & development/physiology
CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
*Eye Color/genetics
Photoperiod
Gene Knockout Techniques
Gene Editing

@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},
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.

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.

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.

CONCLUSION: The one-pot RT-RAA/CRISPR-Cas13a assay provides a rapid and sensitive platform for NiV genotyping.},
}

@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},
doi = {10.1371/journal.pbio.3003658},
pmid = {41712626},
issn = {1545-7885},
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 {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 {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.},
}

Humans
*CRISPR-Cas Systems/genetics
*Systems Biology/methods
Animals
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Single-Cell Analysis
Immunoinformatics

@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 & 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.

METHODS: Murine β-TC-6 and human EndoC-βH1 β-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.

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.

DISCUSSION: These data indicate that single-gene deletions of RNLS or HIVEP2 are insufficient to protect β-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 β-cell graft survival.},
}

Animals
Mice
*Insulin-Secreting Cells/transplantation/immunology/metabolism
Humans
*Graft Rejection/genetics/immunology/prevention & control
Gene Knockout Techniques
*Spheroids, Cellular/immunology/transplantation/metabolism
CRISPR-Cas Systems
*Islets of Langerhans Transplantation
Cell Line
Gene Editing
Transplantation, Heterologous

@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 = {},
number = {},
pages = {},
doi = {10.1021/acssynbio.5c00868},
pmid = {41709870},
issn = {2161-5063},
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 {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.},
}

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

@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.},
}

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

@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.

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.

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.

CONCLUSION: These findings indicate that DRP1 inactivation supports NK cell function in hypoxia and metabolic engineering may enhance CAR-NK efficacy in solid tumors.},
}

*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

@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.},
}

*Gene Editing/methods
CRISPR-Cas Systems
*Dictyostelium/genetics
*Genome, Protozoan
Phylogeny

@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.},
}

*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

@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},
doi = {10.1002/advs.202517617},
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 & dosage ; *Nanoparticles/chemistry ; *Class I Phosphatidylinositol 3-Kinases/genetics/antagonists & 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.},
}

Humans
*RNA, Small Interfering/genetics/administration & dosage
*Nanoparticles/chemistry
*Class I Phosphatidylinositol 3-Kinases/genetics/antagonists & inhibitors/metabolism
Animals
Mice
Cell Line, Tumor
Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Lipids/chemistry
CRISPR-Cas Systems/genetics
Liposomes

@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 = {},
number = {},
pages = {e0204825},
doi = {10.1128/jvi.02048-25},
pmid = {41705810},
issn = {1098-5514},
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 {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 = {},
number = {},
pages = {},
doi = {10.1039/d6an00107f},
pmid = {41705504},
issn = {1364-5528},
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 {pmid41705235,
year = {2026},
author = {Li, Z and Yu, T and Ge, L 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.

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.

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.

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.},
}

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

@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 & purification/genetics ; *Nucleic Acid Amplification Techniques/methods ; Enterotoxigenic Escherichia coli/isolation & 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.},
}

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 & purification/genetics
*Nucleic Acid Amplification Techniques/methods
Enterotoxigenic Escherichia coli/isolation & purification/genetics

@article {pmid41704216,
year = {2026},
author = {Khweis, S and Blackburn, M and Perdigao, C and Pierce, M and Lewis, C and Dunkle, J},
title = {Cas10 residues lining the target RNA binding channel regulate interference by distinguishing cognate target RNA from mismatched targets.},
journal = {RNA biology},
volume = {},
number = {},
pages = {},
doi = {10.1080/15476286.2026.2633385},
pmid = {41704216},
issn = {1555-8584},
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 {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.},
}

*Saccharomyces cerevisiae/metabolism/genetics
Metabolic Engineering/methods
*CRISPR-Cas Systems/genetics
Optogenetics/methods
Light

@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 & 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.},
}

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

@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.},
}

*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

@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.},
}

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

@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 & 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.

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.

RESULTS: Our custom library knockout screens revealed single-guide RNAs targeting Pten, Fbxw7, and genes encoding several components of the transforming growth factor-ß 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.

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.},
}

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

@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//Fundação de Amparo à Pesquisa do Estado de São 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.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Meristem/genetics
*Ribonucleoproteins/genetics/metabolism
*Populus/genetics
*Citrus/genetics
*Biolistics/methods
Plants, Genetically Modified
Transgenes/genetics

@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.

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.

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.},
}

*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

@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 & 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.

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.

RESULTS: LAC12b-2M is sensitive to detect SFTSV with a LOD of 1 copy/μL at 61 °C within 30 min. Compared to clinical RT-qPCR, LAC12b-2M demonstrated a sensitivity of 98.8 % (82/83), a specificity of 100.0 % (96/96), and an accuracy of 99.4 % (178/179) in cohort 1 (n=179), and an accuracy of 100.0 % in cohort 2 (n=36).

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.},
}

Humans
*Phlebovirus/genetics/isolation & 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

@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 {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 = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.01.024},
pmid = {41702404},
issn = {1097-4164},
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 {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 = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2026.01.022},
pmid = {41702403},
issn = {1097-4164},
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 {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},
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 {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.},
}

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

@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 & 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.},
}

Animals
Swine
*CRISPR-Cas Systems/genetics
*Deltacoronavirus/genetics/isolation & purification
RNA, Viral/genetics/analysis
Sensitivity and Specificity
Nucleic Acid Amplification Techniques/methods
*Swine Diseases/diagnosis/virology

@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.},
}

Humans
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
Dependovirus/genetics
*CRISPR-Associated Proteins/genetics/metabolism

@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.},
}

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

@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 ; *Germ Cells/cytology/metabolism ; *Gene Editing/methods ; Male ; CRISPR-Cas Systems ; Female ; Cell Proliferation ; Cell Line ; },
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.},
}

Animals
*Chickens/genetics
*Germ Cells/cytology/metabolism
*Gene Editing/methods
Male
CRISPR-Cas Systems
Female
Cell Proliferation
Cell Line

@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)/ ; },
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.},
}

*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

@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.},
}

*Bacillus subtilis/virology/genetics
*Genome, Viral/genetics
CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Bacillus Phages/genetics
*Genetic Engineering/methods

@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 & 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.},
}

*Kluyveromyces/genetics/metabolism/enzymology/growth & development
Mannitol/metabolism
Sorbitol/metabolism
CRISPR-Cas Systems
*Fungal Proteins/genetics/metabolism
L-Iditol 2-Dehydrogenase/genetics/metabolism

@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 & 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.

RESULTS: The assay achieved a detection limit of 2 copies/µL and demonstrated high specificity against seven other common avian pathogens. Detection was visualized within 1 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.

CONCLUSIONS: This simple, rapid, field-deployable method is valuable for timely MG surveillance and effective disease management in poultry production.},
}

Animals
*Mycoplasma gallisepticum/isolation & 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

@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.},
}

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

@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.},
}

*Gene Editing/methods
Humans
*Artificial Intelligence
*CRISPR-Cas Systems/genetics
Animals
Machine Learning
Deep Learning

@article {pmid40738974,
year = {2026},
author = {Qu, Y and Huang, K and Yin, M and Zhan, K and Liu, D and Yin, D and Cousins, HC and Johnson, WA and Wang, X and Shah, M and Altman, RB and Zhou, D and Wang, M and Cong, L},
title = {CRISPR-GPT for agentic automation of gene-editing experiments.},
journal = {Nature biomedical engineering},
volume = {10},
number = {2},
pages = {245-258},
pmid = {40738974},
issn = {2157-846X},
support = {R35HG011316, 1R01GM141627//U.S. Department of Health & Human Services | NIH | National Human Genome Research Institute (NHGRI)/ ; 1653435//National Science Foundation (NSF)/ ; },
mesh = {Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; Artificial Intelligence ; Automation ; Cell Line, Tumor ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Performing effective gene-editing experiments requires a deep understanding of both the CRISPR technology and the biological system involved. Meanwhile, despite their versatility and promise, large language models (LLMs) often lack domain-specific knowledge and struggle to accurately solve biological design problems. We present CRISPR-GPT, an LLM agent system to automate and enhance CRISPR-based gene-editing design and data analysis. CRISPR-GPT leverages the reasoning capabilities of LLMs for complex task decomposition, decision-making and interactive human-artificial intelligence (AI) collaboration. This system incorporates domain expertise, retrieval techniques, external tools and a specialized LLM fine tuned with open-forum discussions among scientists. CRISPR-GPT assists users in selecting CRISPR systems, experiment planning, designing guide RNAs, choosing delivery methods, drafting protocols, designing assays and analysing data. We showcase the potential of CRISPR-GPT by knocking out four genes with CRISPR-Cas12a in a human lung adenocarcinoma cell line and epigenetically activating two genes using CRISPR-dCas9 in a human melanoma cell line. CRISPR-GPT enables fully AI-guided gene-editing experiment design and analysis across different modalities, validating its effectiveness as an AI co-pilot in genome engineering.},
}

Humans
*Gene Editing/methods
*CRISPR-Cas Systems/genetics
Artificial Intelligence
Automation
Cell Line, Tumor
RNA, Guide, CRISPR-Cas Systems/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41699287,
year = {2026},
author = {Pindi, C and Palermo, G},
title = {Computation and deep-learning-driven advances in CRISPR genome editing.},
journal = {Nature structural & 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.},
}

*Deep Learning
*Gene Editing/methods
*CRISPR-Cas Systems
Humans
Neural Networks, Computer
Algorithms

@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.

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.

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.

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 {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.},
}

*Deep Learning
*RNA, Guide, CRISPR-Cas Systems/genetics
*CRISPR-Cas Systems
Escherichia coli/genetics
*CRISPR-Associated Protein 9/genetics/metabolism
*Gene Editing/methods

@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 = {FP106237//G. Harold and Leila Y. Mathers Charitable Foundation/ ; //Dr. Ralph and Marian Falk Medical Research Trust/ ; //Bank of America/ ; /EB/NIBIB NIH HHS/United States ; R01-EB035016/NH/NIH HHS/United States ; DGE-2022294368//National Science Foundation/ ; },
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.},
}

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

@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.},
}

*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

@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.},
}

*Vaccines, Attenuated/genetics/immunology
Humans
*Vaccine Development/methods
Gene Editing
*Molecular Biology/methods
Animals
CRISPR-Cas Systems
Biotechnology

@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.},
}

*Food Safety/methods
*Food Contamination/analysis
Biosensing Techniques/methods
*Food Analysis/methods
Humans
Food Microbiology

@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 & 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 & purification/genetics ; Fluorescent Dyes/chemistry ; *Escherichia coli/genetics/isolation & 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.},
}

*CRISPR-Cas Systems/genetics
*Copper/chemistry
*Biosensing Techniques/methods
*DNA, Bacterial/isolation & purification/genetics
Fluorescent Dyes/chemistry
*Escherichia coli/genetics/isolation & purification
*Metal Nanoparticles/chemistry
Humans
Limit of Detection
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@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.},
}

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

@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.},
}

*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

@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.},
}

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

@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.},
}

Humans
Animals
*Ischemic Stroke/therapy/genetics
*CRISPR-Cas Systems/genetics
*Gene Editing/methods
*Genetic Therapy/methods

@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 & 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.},
}

*African Swine Fever Virus/genetics/isolation & 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

@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 & bioelectronics},
volume = {300},
number = {},
pages = {118482},
doi = {10.1016/j.bios.2026.118482},
pmid = {41643424},
issn = {1873-4235},
mesh = {*MicroRNAs/genetics/isolation & 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.},
}

*MicroRNAs/genetics/isolation & 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

@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.},
}

*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

@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 & bioelectronics},
volume = {300},
number = {},
pages = {118449},
doi = {10.1016/j.bios.2026.118449},
pmid = {41638184},
issn = {1873-4235},
mesh = {*Hydrogen Peroxide/isolation & 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.},
}

*Hydrogen Peroxide/isolation & 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

@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.},
}

*Escherichia coli/genetics
*Gene Expression Regulation, Bacterial
*Transposases/genetics/metabolism
*CRISPR-Cas Systems
*Transcription, Genetic
Plasmids/genetics

@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},
doi = {10.1111/nph.70951},
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.},
}

*Crops, Agricultural/genetics
*Gene Transfer, Horizontal/genetics
Gene Editing
*CRISPR-Cas Systems/genetics
*Genetic Engineering/methods
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@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 & 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.},
}

Animals
*Distemper Virus, Canine/isolation & purification/genetics
Sensitivity and Specificity
Dogs
*CRISPR-Cas Systems
*Distemper/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
*Molecular Diagnostic Techniques/methods
RNA, Viral/genetics

@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.

RESULTS: In this study, we screen and identify high-activity IscB variants for rice. A version of pIscB-v3, combining enOgeuIscB and ωRNA-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.

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.},
}

*Oryza/genetics
*Gene Editing/methods
CRISPR-Cas Systems
Genome, Plant
*Endonucleases/genetics/metabolism

@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},
doi = {10.1016/j.scr.2026.103905},
pmid = {41579593},
issn = {1876-7753},
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.},
}

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

@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.},
}

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

@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.},
}

Humans
*CRISPR-Cas Systems/genetics
*Human Embryonic Stem Cells/metabolism/cytology
Cell Line
*Glycerolphosphate Dehydrogenase/genetics/metabolism/deficiency
*Gene Knockout Techniques
Cell Differentiation

@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},
doi = {10.1128/jb.00376-25},
pmid = {41532790},
issn = {1098-5530},
support = {R03AI185593//National Institute of Allergy and Infectious Diseases/ ; R15NS131921/NS/NINDS NIH HHS/United States ; R01AI177991//National Institute of Allergy and Infectious Diseases/ ; R01AI182835//National Institute of Allergy and Infectious Diseases/ ; R21AI178067//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.

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.},
}

*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

@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},
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 {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.},
}

*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

@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 = {},
number = {},
pages = {},
doi = {10.1186/s12951-026-04122-w},
pmid = {41689002},
issn = {1477-3155},
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 {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 & development ; *CRISPR-Cas Systems/genetics ; *Plants, Genetically Modified/genetics/growth & 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.},
}

*Gene Editing/methods
*Crops, Agricultural/genetics/growth & development
*CRISPR-Cas Systems/genetics
*Plants, Genetically Modified/genetics/growth & development
Genome, Plant

@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.},
}

*CRISPR-Cas Systems/genetics
Humans
Flow Cytometry/methods
*Melanoma/genetics/immunology/pathology
*Immune Checkpoint Proteins/genetics
Animals
Cell Line, Tumor

@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.},
}

Cell-Free System
Humans
Animals
*Nanostructures/chemistry
*Biocompatible Materials/chemistry

@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 = {},
number = {},
pages = {150873},
doi = {10.1016/j.ijbiomac.2026.150873},
pmid = {41687992},
issn = {1879-0003},
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 {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 = {},
doi = {10.1242/bio.062472},
pmid = {41630603},
issn = {2046-6390},
support = {R35GM137878/NH/NIH HHS/United States ; GR000042507/NH/NIH HHS/United States ; P30CA014520/NH/NIH HHS/United States ; R21OD037634/NH/NIH HHS/United States ; R01HL151522/NH/NIH HHS/United States ; 7000320//National Institute of Food and Agriculture/ ; 2019-67017-29179//National Institute of Food and Agriculture/ ; 2137434//National Science Foundation/ ; //NIH/ ; },
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.},
}

Animals
*Zebrafish/genetics
*Gene Editing/methods
*Gene Knock-In Techniques/methods
Genome
CRISPR-Cas Systems
Genetic Vectors
Exons

@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.},
}

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

@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.},
}

*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

@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.},
}

*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

@article {pmid40905941,
year = {2026},
author = {Chaiyadet, S and Ittiprasert, W and Smout, MJ and Khowawisetsut, L and Ruangsuwast, A and Brindley, PJ and Loukas, A and Laha, T},
title = {Gene Editing of a Carcinogenic Liver Fluke Tetraspanin Impairs Parasite Surface Biogenesis and Extracellular Vesicle Uptake by Human Host Cells.},
journal = {The Journal of infectious diseases},
volume = {233},
number = {2},
pages = {e510-e520},
doi = {10.1093/infdis/jiaf466},
pmid = {40905941},
issn = {1537-6613},
support = {/CA/NCI NIH HHS/United States ; /NH/NIH HHS/United States ; //National Research Council of Thailand/ ; //Fundamental Fund of Khon Kaen University/ ; //the National Science, Research and Innovation Fund/ ; //National Health and Medical Research Council/ ; },
mesh = {*Extracellular Vesicles/metabolism ; Animals ; *Opisthorchis/genetics/metabolism ; Humans ; *Tetraspanins/genetics/metabolism ; *Gene Editing ; *Opisthorchiasis/parasitology ; *Helminth Proteins/genetics/metabolism ; CRISPR-Cas Systems ; Host-Parasite Interactions ; },
abstract = {Opisthorchiasis remains a significant public health concern throughout Southeast Asia. The liver fluke Opisthorchis viverrini resides within the biliary tract, and chronic infection leads to bile duct cancer, or cholangiocarcinoma. Here, we examined the functions of liver fluke tetraspanins, 4-transmembrane domain proteins expressed on the surface of the fluke tegument, and extracellular vesicles (EVs) derived from this syncytial surface. We undertook CRISPR-Cas9 gene knockout (KO) of the O viverrini tetraspanin 2 (Ov-tsp-2) gene and found that Ov-tsp-2-KO flukes had abnormal tegument biogenesis. The tegument of Ov-tsp-2-KO flukes was increasingly vacuolated, and fewer EVs were secreted. EVs that were secreted were deficient in Ov-TSP-2, and their uptake by cholangiocytes was diminished. The findings indicate a critical role for Ov-TSP-2 in maintenance of the tegument, EV production, and uptake by host target cells; they also support the development of this parasite antigen as an anti-infection and anticancer vaccine for opisthorchiasis and opisthorchiasis-associated cholangiocarcinoma.},
}

*Extracellular Vesicles/metabolism
Animals
*Opisthorchis/genetics/metabolism
Humans
*Tetraspanins/genetics/metabolism
*Gene Editing
*Opisthorchiasis/parasitology
*Helminth Proteins/genetics/metabolism
CRISPR-Cas Systems
Host-Parasite Interactions

@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.},
}

*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

@article {pmid41686849,
year = {2026},
author = {Dueñas, E and Tirado, I and Huaihua, P and Del Riego, AP 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},
doi = {10.1371/journal.pntd.0013456},
pmid = {41686849},
issn = {1935-2735},
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).

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.

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 {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.},
}

*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

@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 {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 & 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.},
}

Humans
Female
*Proto-Oncogene Proteins p21(ras)/genetics/metabolism/antagonists & 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

@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.},
}

*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

@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//Consejería de Universidad, Investigación e Innovación/ ; },
mesh = {Humans ; *Gene Editing/ethics/legislation & 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.},
}

Humans
*Gene Editing/ethics/legislation & jurisprudence/methods
*Genetic Therapy/ethics/methods
Animals
CRISPR-Cas Systems
*Translational Research, Biomedical/ethics

@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},
doi = {10.1021/acsnano.5c17709},
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.},
}

*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

@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.},
}

*Biosensing Techniques/methods
Humans
*Colorectal Neoplasms/genetics/diagnosis
*CRISPR-Cas Systems
*Fluorescent Dyes/chemistry
*RNA/analysis/genetics
Aptamers, Nucleotide/genetics/chemistry

@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 & 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.},
}

*SARS-CoV-2/genetics/isolation & purification
Humans
*COVID-19/diagnosis/virology
*Nucleic Acid Amplification Techniques/methods
*CRISPR-Cas Systems
*Clustered Regularly Interspaced Short Palindromic Repeats

@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 & purification ; Humans ; *Nucleic Acid Amplification Techniques/methods ; *RNA, Viral/analysis/genetics ; *COVID-19/diagnosis ; Influenza A virus/genetics/isolation & 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.},
}

*SARS-CoV-2/genetics/isolation & purification
Humans
*Nucleic Acid Amplification Techniques/methods
*RNA, Viral/analysis/genetics
*COVID-19/diagnosis
Influenza A virus/genetics/isolation & purification
Limit of Detection
*CRISPR-Cas Systems
Influenza, Human/diagnosis
*CRISPR-Associated Proteins/genetics

@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.},
}

*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