@article {pmid41508907,
year = {2026},
author = {Liu, CF and Leon, S and Herrig, I and Wessely, O and Tang, WHW},
title = {Generation of Mice Harboring Bicc1 Conditional Null Alleles.},
journal = {Genesis (New York, N.Y. : 2000)},
volume = {64},
number = {1},
pages = {e70038},
pmid = {41508907},
issn = {1526-968X},
support = {MF-2203-02413//G. Harold and Leila Y. Mathers Charitable Foundation/ ; R01DK080745/DK/NIDDK NIH HHS/United States ; },
mesh = {Animals ; Mice ; Alleles ; Mice, Knockout ; CRISPR-Cas Systems ; *Polycystic Kidney Diseases/genetics ; *RNA-Binding Proteins/genetics ; Gene Editing ; Exons ; Female ; },
abstract = {Bicaudal C1 (Bicc1) encodes an RNA-binding protein critical for many organ development and epithelial tissue homeostasis. Bicc1 null mutations have been shown to lead to the development of polycystic kidney disease (PKD) and death at an early prenatal stage. To elucidate the tissue-specific functions of Bicc1, we engineered two independent conditional knockout (cKO) mouse lines targeting distinct exonic regions of the gene. The first line was generated using a traditional embryonic stem (ES) cell-based approach, wherein loxP sites were inserted flanking exon 4 (E4), enabling Cre-mediated excision of a functionally essential coding region. The second line was created using CRISPR/Cas9 genome editing, introducing loxP sites around both exon 4 and exon 5 (E4-5) in a double-step zygote injection strategy. Both alleles were validated by PCR genotyping, sequencing, and functional recombination was confirmed via a tissue-specific Cre driver. These independent cKO models provide a robust platform for dissecting the role of Bicc1 in specific tissues and developmental stages, and offer new avenues for studying the mechanistic basis of PKD and other Bicc1-related pathologies.},
}

Animals
Mice
Alleles
Mice, Knockout
CRISPR-Cas Systems
*Polycystic Kidney Diseases/genetics
*RNA-Binding Proteins/genetics
Gene Editing
Exons
Female

@article {pmid41508607,
year = {2026},
author = {Wang, F and Lu, S and Zhu, C and Yang, L},
title = {PCdb: A comprehensive plant genome editing database integrating sgRNA efficiency, off-target predictions, and epigenomic landscapes.},
journal = {Plant communications},
volume = {},
number = {},
pages = {101708},
doi = {10.1016/j.xplc.2026.101708},
pmid = {41508607},
issn = {2590-3462},
abstract = {CRISPR/Cas-based genome editing has revolutionized plant biotechnology, enabling precise genomic modifications for crop improvement and functional genomics. The success of these applications hinges on designing single guide RNAs (sgRNAs) that maximize on-target efficiency while minimizing off-target effects. Current resources for sgRNA design and performance evaluation in plants are fragmented and lack integration with genomic and epigenomic context, which influences both editing efficacy and specificity. Here, we present PCdb (Plant CRISPR Database; https://gmo.sjtu.edu.cn/pcdb), a comprehensive plant-focused database by integrating experimentally validated sgRNAs, their annotated genomic contexts, genome-wide off-target predictions, and multi-layered epigenomic annotations. PCdb encompasses 6,172 manually curated editing records from 2,132 publications, covering 4,320 unique sgRNAs and 6,117,424 predicted off-target sites across nine major plant species. Uniquely, PCdb contextualizes potential editing outcomes-both on-target and off-target-within the chromatin landscape by incorporating DNA methylation profiles, chromatin accessibility data, and histone modification patterns. The database features an intuitive web interface supporting flexible queries, interactive visualization tools, and comprehensive analytical modules for both sgRNA efficiency assessment and off-target analysis. A case study reanalysis of Oryza sativa yield-related genes demonstrates PCdb's capability to provide a comprehensive performance profile, evaluating both on-target characteristics and off-target risks within their native epigenomic context. Through systematic analysis of the database, we reveal critical sequence and chromatin features influencing editing outcomes, providing novel insights for improved gene editing efficacy and specificity.},
}

@article {pmid41506049,
year = {2026},
author = {Yaşar, S and Gehrke, F and Capdeville, N and Puchta, H},
title = {Recent progress in plant genome engineering: from large insertions to chromosome number changes.},
journal = {Current opinion in biotechnology},
volume = {97},
number = {},
pages = {103426},
doi = {10.1016/j.copbio.2025.103426},
pmid = {41506049},
issn = {1879-0429},
abstract = {The adaptation of the CRISPR/Cas system as a biotechnological tool has enabled a wide spectrum of targeted genome modifications. Whereas earlier approaches focused on small sequence changes, recent years have seen a shift toward larger-scale alterations. Advances in homology-directed gene targeting now enable efficient, scar-free kilobase insertions, while combining nuclease-deficient Cas effectors with recombinases or transposases allows the integration of much larger sequences. Prime editing further expands this scope, enabling inversions, replacements, and deletions spanning hundreds of kilobases to several megabases. More recently, genome engineering has reached a new stage with chromosome fission and fusion, demonstrating the feasibility of controlled karyotype restructuring. Together, these advances open new opportunities for crop improvement, from establishing reproductive barriers and mimicking evolutionary processes to trait stacking on Plant Artificial Chromosomes.},
}

@article {pmid41506042,
year = {2026},
author = {Jiang, H and Yang, J and Qian, C and Li, A and Liu, Y and Zhang, F and Deng, Y and Duan, J and Lv, X},
title = {Logic-gated CRISPR-Cas12a assay with engineered signal amplification for sensitive multiplexed detection of HCC miRNAs.},
journal = {Biosensors & bioelectronics},
volume = {297},
number = {},
pages = {118376},
doi = {10.1016/j.bios.2026.118376},
pmid = {41506042},
issn = {1873-4235},
abstract = {Rapid, sensitive, and accurate multi-target analysis is of great significance in biomedical detection. Traditional methods, however, often function as a mere "one-pot" collection of individual assays, ignoring the intrinsic relationships among biomarkers. To address this, we established a novel biosensing platform that integrates circle-to-circle AND logic gate circuit with an engineered CRISPR-Cas system for the early screening of hepatocellular carcinoma. In this design, the logic gate processes multiple miRNA inputs (e.g., miRNA 122 and miRNA 223), and only upon co-recognition, generates a unified DNA output. This output then directly activates a CRISPR-Cas12a system, which has been enhanced by a multi-legged crRNA assembled on a DNA tetrahedra and a cube-based framework probe to enhance the trans-cleavage activity and reaction kinetics. This integration converts complex multi-target recognition into a single, amplified detection signal, minimizing systemic interference. Under optimal conditions, the method achieved detection limits as low as 78.88 fM for miRNA 122 and 65.26 fM for miRNA 223, with serum recovery rates of 89.66 %-108.08 %. Clinical validation using 36 samples showed that excellent correlation with RT-qPCR (all R[2] > 0.98) and areas under the ROC curves of 0.8514 and 0.9244, effectively distinguishing liver cancer patients from healthy individuals. Combining high sensitivity, specificity, and clinical applicability, this strategy provides a universal platform for logic-operated multiple biomarkers analysis. Looking forward, integration with microfluidic systems could enable automated, high-throughput testing, further enhancing its utility in point-of-care diagnostics. This approach holds great promise not only for early hepatocellular carcinoma screening but also, with adaptation of the input logic, for the detection of a broad spectrum of cancers and other diseases.},
}

@article {pmid41505099,
year = {2026},
author = {Xu, B and Li, S and Li, Y and Zhao, S and Li, X and Han, J and Wu, D and Li, S and Chen, L and Xie, S and Han, X and Zhao, C},
title = {Enhanced SfaTnpB enables single-base-specific, one-pot nucleic acid detection for high-sensitivity diagnostics.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505099},
issn = {1362-4962},
support = {2023YFF1001000//National Key Research & Developmental Program of China/ ; SH23YCKY01//Basic Research Project of Yazhouwan National Laboratory/ ; 32202634//Natural Science Foundation of China/ ; //Yazhouwan National Laboratory/ ; },
mesh = {Humans ; Polymorphism, Single Nucleotide ; CRISPR-Cas Systems ; *Transposases/genetics/metabolism ; *Nucleic Acid Amplification Techniques/methods ; Papillomavirus Infections/diagnosis/virology ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {CRISPR/Cas12-based nucleic acid detection has revolutionized molecular diagnostics but shows limited single-nucleotide specificity, limited high-fidelity subtype discrimination, and limited compatibility with one-pot assays, restricting its broader clinical application. Here, we report a transposon-associated transposase B (TnpB) ortholog, SfaTnpB, with high trans-cleavage activity, robust single-base mismatch discrimination, and broad temperature tolerance. By stepwise engineering of its guide RNA (ωRNA), we developed an enhanced SfaTnpB (enSfaTnpB) system with markedly improved trans-cleavage efficiency. In combination with a TAM-independent split-activator strategy, this system enables precise detection of single-nucleotide polymorphisms. We further developed TOPIC (TnpB-based One-Pot nucleIC acid detection), a one-pot detection platform coupling enSfaTnpB with recombinase-aided amplification (RAA) or loop-mediated isothermal amplification that enables ultrasensitive detection of human papillomavirus (HPV) subtypes 16 and 18 (∼4 copies/μl) and African swine fever virus DNA (∼3 copies/μl). Finally, RAA-TOPIC accurately detected and genotyped 14 high-risk HPV subtypes with high-fidelity subtype discrimination, showing complete concordance with quantitative real-time PCR-based clinical diagnostics. These findings establish TOPIC as a compact, programmable, and scalable molecular detection tool with broad potential for precision diagnostics and point-of-care testing, particularly in resource-limited settings.},
}

Humans
Polymorphism, Single Nucleotide
CRISPR-Cas Systems
*Transposases/genetics/metabolism
*Nucleic Acid Amplification Techniques/methods
Papillomavirus Infections/diagnosis/virology
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41505097,
year = {2026},
author = {Wang, L and Xiang, X and Yin, G and Shu, H and Wu, Y and Chen, H and Na, R and Gijzen, M and Hou, Y and Dong, S},
title = {Effector gene silencing coordinated by histone methylation and small RNAs enhances host adaptation in a plant pathogen.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505097},
issn = {1362-4962},
support = {32400164//National Natural Science Foundation of China/ ; 32488302//National Natural Science Foundation of China/ ; 25ZR1401177//Natural Science Foundation of Shanghai/ ; //Shanghai Jiao Tong University/ ; },
mesh = {*Histones/metabolism/genetics ; *Gene Silencing ; *Phytophthora/genetics/pathogenicity ; Plant Diseases/microbiology/genetics/parasitology ; Methylation ; *Host-Pathogen Interactions/genetics ; Epigenesis, Genetic ; CRISPR-Cas Systems ; Virulence/genetics ; *RNA, Small Untranslated/genetics/metabolism ; },
abstract = {Pathogen adaptability driven by epigenetic processes remains poorly understood and poses a significant challenge to sustainable disease management. Histone 3 lysine 27 trimethylation (H3K27me3) and small RNA (sRNA)-mediated silencing of avirulence (Avr) genes are two major strategies that pathogens employ to evade recognition by host resistance (R) proteins. Here, we demonstrate that these two epigenetic mechanisms operate in a coordinated manner to silence Avr genes in the oomycete Phytophthora sojae. CRISPR/Cas9-mediated editing of PsSu(z)12, a core component of the Polycomb repressive complex 2 (PRC2), abolished H3K27me3 deposition at Avr1b and Avr3a, leading to transcriptional reactivation and loss of avirulence. Complementation with PsSu(z)12 restored H3K27me3 and silencing at Avr1b, but not at Avr3a. This prompted sRNA profiling at both loci, revealing differential co-enrichment of sRNA and H3K27me3. Integrated analysis of H3K27me3-enriched chromatin immunoprecipitation, RNA and sRNA sequencing data uncovered a strong locus-specific co-silencing pattern, with 11 out of 12 H3K27me3-regulated arginine-X-leucine-arginine effectors also targeted by sRNAs. Notably, epigenetic variation among field isolates indicated regulatory heterogeneity and plasticity in effector control. Together, our findings establish PsSu(z)12 as a central hub coordinating H3K27me3 and sRNA-mediated effector gene silencing, revealing a dual-layered epigenetic mechanism that enables immune evasion and promotes pathogen adaptation.},
}

*Histones/metabolism/genetics
*Gene Silencing
*Phytophthora/genetics/pathogenicity
Plant Diseases/microbiology/genetics/parasitology
Methylation
*Host-Pathogen Interactions/genetics
Epigenesis, Genetic
CRISPR-Cas Systems
Virulence/genetics
*RNA, Small Untranslated/genetics/metabolism

@article {pmid41505088,
year = {2026},
author = {Lee-Yow, YC and Valbuena, RC and Richter, CS and Chang, HY and Engreitz, JM},
title = {Junction-targeting designs limit the application of CRISPR-Cas13d in circular RNA perturbation studies.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
doi = {10.1093/nar/gkaf1447},
pmid = {41505088},
issn = {1362-4962},
support = {5T32GM007790/GF/NIH HHS/United States ; DGE-1656518//GRFP/ ; //Stanford Interdisciplinary Graduate Fellowship/ ; R35HG011324/HG/NHGRI NIH HHS/United States ; NNF21SA0072102//Novo Nordisk Foundation/ ; //Lucile Packard Children's Hospital at Stanford University/ ; /HHMI/Howard Hughes Medical Institute/United States ; },
mesh = {RNA, Circular/genetics ; Humans ; *CRISPR-Cas Systems ; RNA, Guide, CRISPR-Cas Systems/genetics/metabolism ; *RNA/genetics/metabolism ; K562 Cells ; Gene Knockdown Techniques/methods ; Exons ; },
abstract = {Circular RNAs (circRNAs) are RNA molecules formed through the backsplicing of linear exons. Several thousand have been identified, yet relatively few are functionally characterized due to challenges in distinguishing effects of circular from linear RNA targets. Recently, CRISPR-Cas13 systems have been utilized to directly target unique junctions formed through backsplicing, potentially allowing for selective degradation of circular isoforms. Applying this approach in pooled screens has indeed identified circRNAs proposed to affect viability in several cancer cell lines. However, the design limitations of applying Cas13d to study circRNAs are not fully characterized. Here, we assessed the limitations of Cas13d-mediated circRNA knockdowns by performing essentiality screens on 900 highly expressed circRNAs in K562, an ENCODE tier 1 cell line. We observed consistent off-target knockdown of linear isoforms by certain circRNA-targeting single-guide RNAs (sgRNAs). Re-analysis of existing Cas13d screens in other cell types revealed similar off-target effects. Using machine learning models that predict Cas13d sgRNA efficacy, we further found that most circRNA-targeting sgRNAs are unlikely to induce strong knockdown. After accounting for these design constraints, 0 of 346 circRNAs testable in our screens had detectable effects on proliferation. Our findings highlight key limitations of junction-targeting strategies, with implications for future circRNA perturbation studies.},
}

RNA, Circular/genetics
Humans
*CRISPR-Cas Systems
RNA, Guide, CRISPR-Cas Systems/genetics/metabolism
*RNA/genetics/metabolism
K562 Cells
Gene Knockdown Techniques/methods
Exons

@article {pmid41505087,
year = {2026},
author = {Di Pede, AC and Bagheri, N and Belforte, E and Palone, A and Rossetti, M and Porchetta, A},
title = {Triplex DNA clamp regulates Cas12a activation for ssDNA and RNA sensing.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41505087},
issn = {1362-4962},
support = {MFAG 2022-ID. 27151//AIRC/ ; //National Recovery and Resilience Plan/ ; //Italian Ministry of University and Research/ ; D53D23009090001//European Union/ ; 2022FPYZ2N//European Union/ ; PNRR M4C2-Investimento 1.4- CN00000041//Italian Ministry of Ministry of University and Research/ ; //Fondazione Umberto Veronesi/ ; },
mesh = {*DNA, Single-Stranded/metabolism/genetics/chemistry ; *CRISPR-Associated Proteins/metabolism/genetics ; *CRISPR-Cas Systems/genetics ; *RNA/metabolism/genetics/chemistry ; *DNA/chemistry/metabolism/genetics ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; },
abstract = {We present a molecular strategy that enables the programmable activation of the CRISPR-Cas12a system in response to triplex DNA formation triggered by single-stranded DNA (ssDNA) or RNA inputs. Our triplex-controlled Cas12a assay leverages the high specificity of clamp-like triplex structures to control a toehold-based strand displacement reaction within a rationally designed DNA hairpin (PAM-Switch). Upon displacement and protospacer adjacent motif (PAM) complementation, the Cas12a ribonucleoprotein (RNP) is activated, initiating trans-cleavage and producing a concentration-dependent fluorescent signal. By decoupling target recognition (via triplex formation) from direct hybridization with the Cas12a-crRNA complex, the assay eliminates the need for target-specific crRNAs. This design also allows multiple detection of distinct nucleic acid (NA) targets using a single Cas12a reaction mix. Through the use of triplex-based clamps, the proposed platform achieves enhanced specificity for single-nucleotide variants and supports the detection of both ssDNA and RNA targets across a broad range of lengths (10-20 nucleotides), addressing key limitations in current Cas12a-based diagnostics and opening new avenues for NA sensing.},
}

*DNA, Single-Stranded/metabolism/genetics/chemistry
*CRISPR-Associated Proteins/metabolism/genetics
*CRISPR-Cas Systems/genetics
*RNA/metabolism/genetics/chemistry
*DNA/chemistry/metabolism/genetics
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics

@article {pmid41504765,
year = {2026},
author = {Osei, EK and O'Mahony, AK and O'Hea, R and Moriarty, J and O'Doherty, Á and Wilson, M and Garcia Manzanilla, E and Mahony, J and Kenny, JG},
title = {Genomics of Irish swine-derived Streptococcus suis: population structure, prophages and anti-viral defence mechanisms.},
journal = {Microbial genomics},
volume = {12},
number = {1},
pages = {},
doi = {10.1099/mgen.0.001590},
pmid = {41504765},
issn = {2057-5858},
mesh = {Animals ; *Streptococcus suis/genetics/virology/classification/isolation & purification ; Swine/microbiology ; *Prophages/genetics ; Ireland ; Phylogeny ; *Streptococcal Infections/microbiology/veterinary ; Whole Genome Sequencing ; Genomics ; *Swine Diseases/microbiology ; Multilocus Sequence Typing ; Genome, Bacterial ; Genetic Variation ; Serogroup ; },
abstract = {Streptococcus suis is a major pig pathogen with zoonotic potential, posing an occupational risk to farmers and meat handlers. We characterized 110 S. suis strains from diseased pigs in Ireland (2005-2022) using whole-genome sequencing to investigate population structure and phage-host dynamics. We identified 15 distinct serotypes, with serotypes 9 and 2 being the most dominant. In silico multi-locus sequence typing revealed high diversity within the collection, identifying several sequence types (STs), including 26 novel STs. Investigation of strain-level genomic clustering using PopPUNK against global S. suis genomes showed that the Irish isolates were phylogenetically dispersed across the broader global S. suis population rather than clustering in a single clonal group. The majority of Irish isolates fall within the ten established pathogenic lineages, including the highly virulent zoonotic lineage 1. A locally persistent clonal lineage was identified among Irish isolates, showing minimal genetic variation over a decade.Prophage analysis revealed novel viral taxa that were interspersed among known streptococcal phages, rather than clustering distinctly. Restriction-modification systems were the predominant anti-viral defence systems identified across genomes. CRISPR-Cas systems were present in limited strains but showed substantial targeting bias toward full-length prophages, indicating ongoing phage pressure. CRISPR spacers matched non-S. suis streptococcal phages, and phylogenomic analysis revealed that Vansinderenvirus phages clustered with S. suis rather than other Streptococcus thermophilus phages, suggesting evolutionary connections between phage lineages infecting different streptococci.This study presents the first comprehensive genomic characterization of S. suis in Ireland, revealing a diverse population with significant implications for animal and human health.},
}

Animals
*Streptococcus suis/genetics/virology/classification/isolation & purification
Swine/microbiology
*Prophages/genetics
Ireland
Phylogeny
*Streptococcal Infections/microbiology/veterinary
Whole Genome Sequencing
Genomics
*Swine Diseases/microbiology
Multilocus Sequence Typing
Genome, Bacterial
Genetic Variation
Serogroup

@article {pmid41503690,
year = {2026},
author = {Ma, N and Zhang, M and Ghonaim, AH and Zhou, P and Wang, C and Zhou, J and Guo, G and Lebbink, RJ and Bosch, BJ and Zhu, H and Li, W and He, Q},
title = {The essential role of heparan sulfate in the entry of PDCoV and other porcine coronaviruses.},
journal = {Virulence},
volume = {17},
number = {1},
pages = {2614154},
doi = {10.1080/21505594.2026.2614154},
pmid = {41503690},
issn = {2150-5608},
mesh = {Animals ; *Heparitin Sulfate/metabolism ; Swine ; *Deltacoronavirus/physiology ; *Virus Internalization/drug effects ; Swine Diseases/virology ; Humans ; *Porcine epidemic diarrhea virus/physiology ; *Coronavirus Infections/virology/veterinary ; Cell Line ; CRISPR-Cas Systems ; Receptors, Virus/metabolism ; Transmissible gastroenteritis virus/physiology ; Alphacoronavirus ; },
abstract = {Porcine enteric coronaviruses, including porcine deltacoronavirus (PDCoV), porcine epidemic diarrhea virus (PEDV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and transmissible gastroenteritis coronavirus (TGEV), can cause acute diarrhea, vomiting, dehydration, and high mortality in suckling piglets. Recent studies revealing human PDCoV infections and the potential of SADS-CoV to penetrate human cell lines have heightened apprehensions about the zoonotic transmission risks of these viruses. While heparan sulfate (HS) serves as a receptor in PDCoV binding, the key host genes involved in HS biogenesis and the specific molecular mechanisms underlying this process have not been fully examined. Enzymes involved in HS biosynthesis, including SLC35B2, EXT1, and NDST1, were identified as critical host factors via the use of CRISPR-Cas9 knockout cells. Moreover, inhibition assays using heparin sodium, a competitive HS mimic, demonstrated dose-dependent reductions in PDCoV infection in vitro. Additionally, mitoxantrone, an HS-binding drug, reduced PDCoV infection. Furthermore, HS was confirmed to facilitate the entry of other porcine enteric coronaviruses (SeCoVs), including PEDV, SADS-CoV, and TGEV, underscoring the conserved role of HS in CoV pathogenesis. These insights contribute to the understanding of porcine coronavirus-host interactions and support the development of innovative antiviral interventions.},
}

Animals
*Heparitin Sulfate/metabolism
Swine
*Deltacoronavirus/physiology
*Virus Internalization/drug effects
Swine Diseases/virology
Humans
*Porcine epidemic diarrhea virus/physiology
*Coronavirus Infections/virology/veterinary
Cell Line
CRISPR-Cas Systems
Receptors, Virus/metabolism
Transmissible gastroenteritis virus/physiology
Alphacoronavirus

@article {pmid41453683,
year = {2026},
author = {Akhter, R and Kitab, B and Kayesh, MEH and Shimizu, R and Onuma, H and Yamamoto, N and Ogawa, S and Sugiyama, M and Tanaka, Y and Sato, Y and Kohara, M and Tsukiyama-Kohara, K},
title = {Optimization of lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes for in vivo delivery of a CRISPR/Cas9 system targeting hepatitis B virus.},
journal = {Virus research},
volume = {363},
number = {},
pages = {199682},
doi = {10.1016/j.virusres.2025.199682},
pmid = {41453683},
issn = {1872-7492},
mesh = {*Hepatitis B virus/genetics/drug effects/physiology ; *CRISPR-Cas Systems ; Animals ; *Nanoparticles/chemistry/administration & dosage ; Mice ; Humans ; *Ribonucleoproteins/administration & dosage/genetics/chemistry ; Virus Replication/drug effects ; *Lipids/chemistry ; Genetic Therapy/methods ; *Hepatitis B, Chronic/therapy/virology ; RNA, Guide, CRISPR-Cas Systems/genetics/administration & dosage ; CRISPR-Associated Protein 9 ; Liposomes ; },
abstract = {Patients with chronic hepatitis B virus (HBV) infection may benefit from clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based gene therapy. We previously identified a guide RNA (WJ11) that suppressed HBV replication in vitro and in vivo; however, we were unable to achieve delivery at clinically feasible doses in vivo using an adeno-associated virus (AAV) vector. Lipid nanoparticle (LNP)-based WJ11/Cas9 ribonucleoprotein-oligonucleotide complex delivery suppressed HBV replication by 2-3-fold more than did AAV-based delivery. In the present study, we investigated the HBV replication-suppressive effects of LNP/WJ11/Cas9 complexes after intravenous administration to persistently HBV genotype C-infected humanized chimeric mice. CL4H6 (ionizable lipid) LNPs were selected as the first candidate for WJ11/Cas9 delivery based on their reported high encapsulation efficiency; however, no significant anti-HBV effect was noted in serum or hepatic tissue. The ionizable lipid candidate CL4F11_ε-3 improved absolute serum HBV values to a certain degree but had no significant effect on hepatic HBV DNA or covalently closed circular (ccc)DNA levels. CL4F11_ζ-2 LNP/WJ11/Cas9, a new complex prepared through structural optimization of the ionizable lipid and heat treatment of WJ11, showed suppressive effect for serum viral load along with a reduction of hepatic HBV DNA, HBV cccDNA, HBsAg, and HBcrAg levels when compared with controls. Therefore, LNP-based delivery of this CRISPR/Cas9 formula holds promise for the treatment of chronic HBV infection.},
}

*Hepatitis B virus/genetics/drug effects/physiology
*CRISPR-Cas Systems
Animals
*Nanoparticles/chemistry/administration & dosage
Mice
Humans
*Ribonucleoproteins/administration & dosage/genetics/chemistry
Virus Replication/drug effects
*Lipids/chemistry
Genetic Therapy/methods
*Hepatitis B, Chronic/therapy/virology
RNA, Guide, CRISPR-Cas Systems/genetics/administration & dosage
CRISPR-Associated Protein 9
Liposomes

@article {pmid41423170,
year = {2026},
author = {Sheri, V and Verma, PK and Lekkala, S and Janga, MR},
title = {Application of digital PCR and CRISPR/Cas13a-based fluorescent assay for accurate and on-site detection of cotton leafroll dwarf virus.},
journal = {Journal of virological methods},
volume = {341},
number = {},
pages = {115332},
doi = {10.1016/j.jviromet.2025.115332},
pmid = {41423170},
issn = {1879-0984},
mesh = {*Plant Diseases/virology ; *Gossypium/virology ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; Plant Leaves/virology ; Viral Load ; *Closteroviridae/isolation & purification/genetics ; RNA, Viral/genetics ; Texas ; *Polymerase Chain Reaction/methods ; },
abstract = {Cotton leafroll dwarf virus (CLRDV) is an emerging viral pathogen posing a significant threat to cotton production in the United States. Early and accurate detection is critical for effective disease surveillance and management. Although traditional reverse transcription PCR (RT-PCR) is commonly employed for CLRDV diagnosis, it suffers from limitations in sensitivity, quantification accuracy, and involves labor-intensive workflows. In this study, we evaluated two advanced molecular diagnostic approaches for detecting CLRDV, digital PCR (dPCR) and CRISPR/Cas13a-based fluorescent assay. Symptomatic cotton leaf samples from Lubbock and Brownfield, Texas, were screened and confirmed positive by RT-PCR. Digital PCR analysis enabled absolute quantification of viral load, revealing significantly higher titers in Brownfield (F2) samples and offered improved sensitivity over RT-PCR, particularly in samples with low viral loads. However, dPCR is resource-intensive and requires specialized instrumentation. To address the need for rapid, field-deployable diagnostics, we developed a CRISPR/Cas13a-based assay targeting the conserved ORF3, ORF2, and ORF3a regions of the CLRDV genome. Adapted from the SHERLOCK platform, this fluorescence-based assay uses collateral cleavage activity of Cas13a to enable highly specific visual detection. While the assay successfully enabled direct detection from crude leaf extracts without RNA purification, the sensitivity analysis was conducted using purified, in vitro transcribed RNA. Fluorescence signals were reliably observed with as few as 50 RNA copies, defining the assay's practical limit of detection. While dPCR is optimal for quantitative laboratory analysis, the CRISPR/Cas13a-based assay offers a rapid, sensitive, and cost-effective tool for field-level detection. Together, these complementary tools enhance CLRDV surveillance and management in cotton.},
}

*Plant Diseases/virology
*Gossypium/virology
Sensitivity and Specificity
*CRISPR-Cas Systems
Plant Leaves/virology
Viral Load
*Closteroviridae/isolation & purification/genetics
RNA, Viral/genetics
Texas
*Polymerase Chain Reaction/methods

@article {pmid40879055,
year = {2026},
author = {Wang, M and Liao, Q and Bai, S and Liu, X and Peng, Y and Liu, P and Lu, H and Zhu, JK and Zeng, C},
title = {Co-Delivery of Multiple RNAs via Lipid Nanoparticles Enables Precise Gene Editing of CAR-T Cells.},
journal = {Advanced healthcare materials},
volume = {15},
number = {1},
pages = {e01475},
doi = {10.1002/adhm.202501475},
pmid = {40879055},
issn = {2192-2659},
support = {KJZD20240903102703005//Shenzhen Science and Technology Program/ ; 2024B1111130001//Guangdong S&T Program/ ; 82402147//National Natural Science Foundation of China/ ; NCTIB2023XB01002//"Open Competition to Select the Best Candidates" Technology Breakthrough Project for Cell Therapy of NCTIB/ ; },
mesh = {*Gene Editing/methods ; Humans ; *Nanoparticles/chemistry ; Animals ; Mice ; CRISPR-Cas Systems ; Cell Line, Tumor ; *Lipids/chemistry ; *Receptors, Chimeric Antigen/genetics/metabolism ; *T-Lymphocytes/metabolism ; *Immunotherapy, Adoptive/methods ; Programmed Cell Death 1 Receptor/genetics/metabolism ; *RNA ; Gene Transfer Techniques ; Liposomes ; },
abstract = {Over the past decade, CAR-T cell therapy has achieved remarkable success in treating hematological malignancies. However, traditional CAR-T cell engineering employs viral vectors, which has several limitations. Additionally, the immunosuppressive tumor microenvironment, particularly mediated by the PD-1/PD-L1 pathway, significantly restricts CAR-T cell efficacy. CRISPR/Cas9-mediated PD-1 knockout can enhance CAR-T cell anti-tumor activity, but traditional electroporation (EP) method often damages T cells. Herein, a novel lipid nanoparticles (LNPs)-mediated delivery technology are introduced to engineer CAR-T cells. The LNPs platform enables the simultaneous expression of CAR cassette and CRISPR/Cas9 gene editor in T cells via co-delivery of multiplex RNAs (CD19 CAR mRNA+Cas9 mRNA+sgRNA targeting PD-1). Importantly, LNPs exhibit higher transfection efficiency and superior cell viability compared to traditional electroporation method. The engineered CAR-T cells with PD-1 knockout, which express anti-CD19 CAR, can specifically kill CD19+ Nalm-6 tumor cells in vitro and display enhanced anti-tumor activity in vivo. Furthermore, LNPs-mediated co-delivery of Cas9 mRNA and sgRNAs targeting PD-1, TRAC, and B2M enables triple-knockout of T cells with high editing efficiencies (76% for PD-1, 86% for TRAC, and 80% for B2M), highlighting the ability for multiplex gene editing. This LNP-mediated delivery strategy has great potentials for the development of safer and more efficacious CAR-T cells.},
}

*Gene Editing/methods
Humans
*Nanoparticles/chemistry
Animals
Mice
CRISPR-Cas Systems
Cell Line, Tumor
*Lipids/chemistry
*Receptors, Chimeric Antigen/genetics/metabolism
*T-Lymphocytes/metabolism
*Immunotherapy, Adoptive/methods
Programmed Cell Death 1 Receptor/genetics/metabolism
*RNA
Gene Transfer Techniques
Liposomes

@article {pmid41503845,
year = {2026},
author = {Li, MF and Zubair, A and Wdidi, S and He, S},
title = {Disrupting Viral Persistence: CRISPR/Cas9-Based Strategies for Hepatitis B and C Treatment, and Challenges.},
journal = {Journal of cellular and molecular medicine},
volume = {30},
number = {1},
pages = {e70986},
pmid = {41503845},
issn = {1582-4934},
mesh = {*CRISPR-Cas Systems/genetics ; Humans ; *Hepatitis B virus/genetics ; *Hepatitis B/therapy/virology/genetics ; Gene Editing/methods ; *Hepatitis C/therapy/virology/genetics ; *Hepacivirus/genetics ; Animals ; Antiviral Agents/therapeutic use/pharmacology ; Genome, Viral ; DNA, Circular/genetics ; DNA, Viral/genetics ; Genetic Therapy ; Virus Replication/genetics ; },
abstract = {Hepatitis B and C viruses (HBV and HCV) remain among the leading causes of liver disease worldwide. Current antiviral drugs, such as nucleotide analogues (NAs), can reduce the replication of new HBV and HCV infections but cannot completely eliminate chronic infections. This is primarily because a stable form of viral DNA, known as covalently closed circular DNA (cccDNA), persists in liver cells and continues to sustain the infection. In recent years, the CRISPR/Cas9 gene-editing system has emerged as a powerful tool for precisely cutting and inactivating specific DNA sequences. Due to its efficiency and ease of use, researchers have applied CRISPR/Cas9 in numerous studies to directly target and disrupt the HBV genome, demonstrating promising antiviral effects in both cell cultures and animal models. Targeting multiple sites within the HBV genome has been shown to further enhance its effectiveness, paving the way for potential combination therapies aimed at disabling both cccDNA and HBV and HCV DNA integrated into the host genome. Despite its potential, CRISPR/Cas9 still faces significant challenges before clinical application, most notably the risk of off-target effects-unintended cleavage of non-target DNA sequences-and the difficulty of delivering the system efficiently into liver cells in vivo. Future progress will depend on improving the tool's precision, efficiency, flexibility and delivery methods. In this review, we explore recent advances in designing guide RNAs (gRNAs) for targeting HBV and HCV, as well as the delivery systems used to transport CRISPR/Cas9 into cells. We also discuss the remaining challenges and potential strategies for advancing CRISPR/Cas9 from the laboratory toward a viable clinical cure for HBV and HCV.},
}

*CRISPR-Cas Systems/genetics
Humans
*Hepatitis B virus/genetics
*Hepatitis B/therapy/virology/genetics
Gene Editing/methods
*Hepatitis C/therapy/virology/genetics
*Hepacivirus/genetics
Animals
Antiviral Agents/therapeutic use/pharmacology
Genome, Viral
DNA, Circular/genetics
DNA, Viral/genetics
Genetic Therapy
Virus Replication/genetics

@article {pmid41502163,
year = {2026},
author = {Manojkumar, C and Limbola, M and Paul, S and Thangadurai, K and Rajendran, KV and Roy, A and Mandal, B and Jeena, K and Bedekar, MK},
title = {CRISPR-Cas12a-based lateral flow detection of white spot syndrome virus: a dual-target approach for detection of early and latent infection.},
journal = {Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc},
volume = {},
number = {},
pages = {10406387251399147},
doi = {10.1177/10406387251399147},
pmid = {41502163},
issn = {1943-4936},
abstract = {White spot syndrome virus (WSSV; family Nimaviridae; taxon species White spot syndrome virus) is a major viral pathogen that poses a significant threat to the global shrimp industry, with early detection being the most effective strategy for disease control. We developed a CRISPR-Cas12a-based dual-target detection assay for WSSV, specifically targeting the VP28 gene (gene product is a major envelope protein) and WSSV366 (a latency-associated gene), optimized using Indian WSSV isolates. Our CRISPR RNAs for both targets had high efficiency, and we evaluated the assay using fluorescence-based and lateral flow strip (LFS) endpoint detection. In fluorescence assays, the Cr-WSSV assay (without recombinase polymerase amplification, RPA) detected WSSV at 3 × 10[5] copies/μL; RPA integration significantly enhanced sensitivity, allowing detection at as low as 20 and 200 copies for VP28 and WSSV366, respectively, with 100% specificity. We developed a CRISPR-based LFS assay with optimized FAM-biotin reporter concentrations of 100 nM and 250 nM, yielding robust and reproducible results for improved field applicability. Performance evaluation confirmed lack of cross-reactivity to other WOAH-listed shrimp pathogens, while maintaining detection limits of 20 and 200 copies of VP28 and WSSV366. Clinical validation further demonstrated that the RPA-Cr-WSSV-LFS assay successfully detected WSSV366 even in VP28-negative samples, underscoring the importance of detecting WSSV366 in latent infections. Our rapid, cost-effective, and highly sensitive CRISPR-Cas-based assay enhances WSSV surveillance and biosecurity in shrimp aquaculture by incorporating structural and latency-associated gene markers, making it a promising alternative to conventional molecular testing.},
}

@article {pmid41501880,
year = {2026},
author = {Zhu, M and Wu, Y and Ou, H and Liu, X and Wang, Y and Liu, X and Zou, C and Yang, G and Du, M and Yu, D and Zheng, D and He, L and Zhang, K and Zhang, W and Wang, S and Qin, H and Hao, Q and He, Y and Lin, W and Zhang, Y and Gu, J and Li, M and Qin, W and Cao, Z},
title = {Targeting TRIM25 as a therapeutic strategy to enhance ferroptosis in glioblastoma cells.},
journal = {Journal of nanobiotechnology},
volume = {24},
number = {1},
pages = {20},
pmid = {41501880},
issn = {1477-3155},
support = {01-SWKJYCJJ17//the National Defense Biotechnology Fund for Outstanding Young Talents/ ; 2025GTKP008//the National Key Laboratory of Oncology/ ; },
mesh = {*Ferroptosis/drug effects ; Humans ; *Glioblastoma/metabolism/pathology/drug therapy ; Animals ; Cell Line, Tumor ; *Tripartite Motif Proteins/metabolism/genetics ; *Ubiquitin-Protein Ligases/metabolism/genetics ; Mice ; *Brain Neoplasms/metabolism/pathology ; CRISPR-Cas Systems ; Gene Editing ; Mitochondria/metabolism ; Nanoparticles/chemistry ; Mice, Nude ; Ubiquitination ; },
abstract = {BACKGROUND: Glioblastoma (GBM) is the most common malignant brain tumor with a dismal prognosis (< 7% 5-year survival) under current first-line treatment. While inducing programmed cell death (PCD) is a promising antitumor strategy, its effectiveness in GBM remains controversial. Ferroptosis emerged as the most enriched PCD process and was highly correlated with GBM malignant progression.

METHODS: We performed a CRISPR-Cas9 loss-of-function screen to identify critical ferroptosis contributors. Mechanistic studies involved assessing mitochondrial function and morphology. Protein interaction and degradation pathways were investigated using immunoprecipitation and ubiquitination assays. We developed a blood-brain-barrier-penetrating genome editing delivery system, Angiopep-2-modified nanoparticles with disulfide bonds (ANPSS), loaded with Cas9/sgRNA complexes.

RESULTS: Voltage-dependent anion channel 2 (VDAC2) was identified as a critical contributor to ferroptosis. VDAC2 overexpression induced mitochondrial dysfunction and characteristic ferroptotic mitochondrial morphology. The E3 ubiquitin ligase TRIM25 was identified as a key suppressor of VDAC2, directly interacting with it and inducing its K48-linked polyubiquitination and subsequent proteasomal degradation. In vivo, the ANPSS(sgTRIM25) system effectively targeted GBM cells, significantly promoted ferroptosis, and inhibited GBM progression.

CONCLUSIONS: Our findings demonstrate that TRIM25 is a critical negative regulator of VDAC2-dependent ferroptosis in GBM. Targeting TRIM25 using the ANPSS(sgTRIM25) genome editing system effectively overcomes ferroptosis resistance and suppresses tumor growth, representing a viable therapeutic approach for GBM.},
}

*Ferroptosis/drug effects
Humans
*Glioblastoma/metabolism/pathology/drug therapy
Animals
Cell Line, Tumor
*Tripartite Motif Proteins/metabolism/genetics
*Ubiquitin-Protein Ligases/metabolism/genetics
Mice
*Brain Neoplasms/metabolism/pathology
CRISPR-Cas Systems
Gene Editing
Mitochondria/metabolism
Nanoparticles/chemistry
Mice, Nude
Ubiquitination

@article {pmid41501459,
year = {2026},
author = {Dmytrenko, O and Yuan, B and Crosby, KT and Krebel, M and Chen, X and Nowak, JS and Chramiec-Głąbik, A and Filani, B and Gribling-Burrer, AS and van der Toorn, W and von Kleist, M and Achmedov, T and Smyth, RP and Glatt, S and Bravo, JPK and Heinz, DW and Jackson, RN and Beisel, CL},
title = {RNA-triggered Cas12a3 cleaves tRNA tails to execute bacterial immunity.},
journal = {Nature},
volume = {},
number = {},
pages = {},
pmid = {41501459},
issn = {1476-4687},
abstract = {In all domains of life, tRNAs mediate the transfer of genetic information from mRNAs to proteins. As their depletion suppresses translation and, consequently, viral replication, tRNAs represent long-standing and increasingly recognized targets of innate immunity[1-5]. Here we report Cas12a3 effector nucleases from type V CRISPR-Cas adaptive immune systems in bacteria that preferentially cleave tRNAs after recognition of target RNA. Cas12a3 orthologues belong to one of two previously unreported nuclease clades that exhibit RNA-mediated cleavage of non-target RNA, and are distinct from all other known type V systems. Through cell-based and biochemical assays and direct RNA sequencing, we demonstrate that recognition of a complementary target RNA by the CRISPR RNA triggers Cas12a3 to cleave the conserved 5'-CCA-3' tail of diverse tRNAs to drive growth arrest and anti-phage defence. Cryogenic electron microscopy structures further revealed a distinct tRNA-loading domain that positions the tRNA tail in the RuvC active site of the nuclease. By designing synthetic reporters that mimic the tRNA acceptor stem and tail, we expanded the capacity of current CRISPR-based diagnostics for multiplexed RNA detection. Overall, these findings reveal widespread tRNA inactivation as a previously unrecognized CRISPR-based immune strategy that broadens the application space of the existing CRISPR toolbox.},
}

@article {pmid41500141,
year = {2026},
author = {Zheng, S and Tie, H and Chai, S and Wang, M and Wang, S and Zeng, YY and Wu, G and Zhang, TG},
title = {Molecular mechanisms and biotechnological advances in herbicide resistance: Insights into the development of herbicide-tolerant crops.},
journal = {Journal of plant physiology},
volume = {317},
number = {},
pages = {154690},
doi = {10.1016/j.jplph.2026.154690},
pmid = {41500141},
issn = {1618-1328},
abstract = {Herbicides play a pivotal role in modern agriculture by controlling weed populations and safeguarding crop yields. However, the long-term and extensive use of herbicides has accelerated the evolution of herbicide-resistant weeds, thereby diminishing their efficacy and posing a serious threat to global food security. Recent advances in molecular biology and plant biotechnology have greatly expanded our understanding of herbicide resistance mechanisms and enabled the development of crops with enhanced herbicide resistance. Herbicide resistance genes function primarily by encoding detoxifying enzymes, modifying herbicide target sites, or activating specific metabolic pathways that mitigate herbicidal toxicity. Emerging genetic tools, including transgenic approaches and CRISPR/Cas-mediated genome editing, have further facilitated the precise introduction of resistance traits into major crops. It is noteworthy that this review offers novel insights into the latest CRISPR/Cas applications, including base editing and prime editing for developing novel, non-transgenic herbicide-resistant crops. Furthermore, it provides a systematic overview of advanced strategies for engineering multi-gene stacking traits to combat complex or evolving weed resistance. This review integrates recent progress in elucidating the molecular targets of herbicides and the underlying resistance mechanisms, and highlights the potential of modern biotechnological strategies for engineering herbicide-resistant crops to promote sustainable and environmentally responsible weed management.},
}

@article {pmid41499404,
year = {2026},
author = {Wan, X and Cohen, SM and Yu, Y and Hoan Le, H and Park, H and Groaz, A and Moreno, R and Tan, M and Schneider, J and Gronquist, MR and Shinya, R and Schroeder, FC and Sternberg, PW},
title = {O-acyltransferase genes involved in the production of volatile sex pheromones in Caenorhabditis elegans.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
volume = {123},
number = {2},
pages = {e2524778123},
doi = {10.1073/pnas.2524778123},
pmid = {41499404},
issn = {1091-6490},
support = {R24OD023041//HHS | NIH (NIH)/ ; DGE 1745301//NSF | NSF Graduate Research Fellowship Program (GRFP)/ ; 00000//Tianqiao and Chrissy Chen Institute for Neuroscience/ ; 00000//Chuck Lorre Research Scholars Program/ ; },
mesh = {Animals ; *Sex Attractants/biosynthesis/metabolism/genetics ; *Caenorhabditis elegans/genetics/metabolism/enzymology ; Male ; *Acyltransferases/genetics/metabolism ; Female ; *Caenorhabditis elegans Proteins/genetics/metabolism ; Gas Chromatography-Mass Spectrometry ; CRISPR-Cas Systems ; Sexual Behavior, Animal ; },
abstract = {Gene family expansions are critical for functional diversification, yet the contributions of paralogs to metabolic pathways are often unclear. In Caenorhabditis, the expanded O-acyltransferase (OAC) family-enzymes that transfer acyl groups to hydroxylated substrates-remains poorly characterized despite having been implicated in lipid metabolism. Using CRISPR-Cas9 mutagenesis, behavioral assays, gas chromatographic-mass spectral (GC-MS) analyses, and metabolomics, we systematically analyzed 59 OAC-family protein-coding genes to define their roles in regulating signaling molecules. We found that four adjacent paralogs (oac-13, oac-16, oac-25, and oac-28) on chromosome I are required for synthesizing volatile sex pheromones-airborne signals critical for male mate-searching. Specifically, oac-13 and oac-16 are necessary for producing both major pheromone components, while the identical tandem paralogs oac-25 and oac-28 regulate the production of the later-eluting component in gas chromatography. Disruption of these genes reduced production of key pheromone components and impaired male attraction. Metabolomics revealed that oac-16 and other OACs also modulate the synthesis and secretion of nonvolatile ascaroside pheromones, indicating dual roles in chemical signaling. This work uncovers functional specialization within an expanded gene family, illustrating how redundancy and divergence enable adaptive evolution of communication systems.},
}

Animals
*Sex Attractants/biosynthesis/metabolism/genetics
*Caenorhabditis elegans/genetics/metabolism/enzymology
Male
*Acyltransferases/genetics/metabolism
Female
*Caenorhabditis elegans Proteins/genetics/metabolism
Gas Chromatography-Mass Spectrometry
CRISPR-Cas Systems
Sexual Behavior, Animal

@article {pmid41498982,
year = {2026},
author = {Mangal, H and Mathur, S and Kumar, S and Chaurasia, A and Ranjan, R},
title = {Role of CRISPR in bioremediation of heavy metal(loid): a breakthrough in environmental biotechnology.},
journal = {World journal of microbiology & biotechnology},
volume = {42},
number = {1},
pages = {34},
pmid = {41498982},
issn = {1573-0972},
mesh = {*Metals, Heavy/metabolism ; *Biodegradation, Environmental ; *CRISPR-Cas Systems ; *Biotechnology/methods ; Gene Editing/methods ; Bacteria/genetics/metabolism ; Biosensing Techniques ; Plants/genetics/metabolism ; *Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {A number of technological advancements have made bioremediation an emerging and innovative technology, including its economic viability, increased competence, and natural environment friendliness. The efficiency, scalability, and specificity of conventional physical, chemical, and biological remediation techniques are still limited, despite their partial success. Recent developments in CRISPR-based genome engineering have made it possible to precisely manipulate metal transporters, detoxification enzymes, and stress-response pathways in microorganisms and plants, opening up new possibilities to improve bioremediation. This review offers a thorough and integrated examination of enzyme engineering, biosensing systems, microbial bioremediation, and CRISPR-enabled phytoremediation. This work is novel because it presents a unified roadmap for next-generation bioremediation technologies by integrating CRISPR editing with multi-omics, synthetic biology, and emerging CRISPR-based biosensors. We also go over ecological risks, current difficulties, legal issues, and potential field deployment scenarios in the future. These revelations collectively demonstrate the revolutionary potential of CRISPR in creating highly effective, sustainable, and scalable remedies for heavy metal pollution.},
}

*Metals, Heavy/metabolism
*Biodegradation, Environmental
*CRISPR-Cas Systems
*Biotechnology/methods
Gene Editing/methods
Bacteria/genetics/metabolism
Biosensing Techniques
Plants/genetics/metabolism
*Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41498549,
year = {2026},
author = {Gopalaswamy, R and Subbian, S},
title = {The power of resistance: mechanisms of antimicrobial resistance in Mycobacterium tuberculosis and its impact on tuberculosis management.},
journal = {Clinical microbiology reviews},
volume = {},
number = {},
pages = {e0019425},
doi = {10.1128/cmr.00194-25},
pmid = {41498549},
issn = {1098-6618},
abstract = {SUMMARYThe global resurgence of drug-resistant tuberculosis (DR-TB) presents a formidable challenge to public health, driven by a complex interplay of mycobacterial evolution, dynamics and outcomes of host-pathogen interactions and systemic gaps in diagnosis and treatment strategies. This comprehensive review delineates the multifactorial basis of antimicrobial resistance (AMR) in Mycobacterium tuberculosis (Mtb), integrating molecular, immunological, and pharmacological perspectives to inform next-generation strategies for effective TB control. We reconceptualize TB as a dynamic clinical spectrum-ranging from asymptomatic infection to overt disease-shaped by granuloma biology and bacterial adaptation. This spectrum underpins both diagnostic ambiguity and therapeutic failure, particularly in the context of phenotypic drug tolerance/resistance to current anti-TB drugs. We discuss Mtb's intrinsic and extrinsic resistance mechanisms, including the lipid-rich cell envelope, efflux systems, and enzymatic drug modification, which are compounded by acquired mutations that disrupt drug activation, alter targets, and confer cross-resistance. These adaptations are further potentiated by granuloma-induced pharmacokinetic heterogeneity and host-induced metabolic quiescence. We highlight the emerging role of therapeutic drug monitoring and pharmacokinetic/pharmacodynamic modeling in optimizing individualized therapy, particularly for novel regimens incorporating bedaquiline, pretomanid, and linezolid. Moreover, we underscore the diagnostic limitations in detecting heteroresistance and early-stage disease, advocating for expanded deployment of advanced and targeted molecular diagnostic modalities. Finally, we propose a paradigm shift toward integrated, precision-based TB management, leveraging host-directed therapies, biofilm-disrupting agents, and real-time pharmacokinetics-guided dosing to preempt resistance emergence and improve clinical outcomes. This review provides a translational framework for addressing the biological and operational complexities of DR-TB in the era of AMR.},
}

@article {pmid41475346,
year = {2026},
author = {Behera, AK and Kim, JJ and Kordale, S and Pekovic, F and Damodaran, AP and Kumari, B and Vidak, S and Dickson, E and Xiao, MS and Duncan, G and Andresson, T and Misteli, T and Valkov, E and Gonatopoulos-Pournatzis, T},
title = {RNA-coupled CRISPR screens reveal ZNF207 as a regulator of LMNA aberrant splicing in progeria.},
journal = {Molecular cell},
volume = {86},
number = {1},
pages = {41-59.e15},
doi = {10.1016/j.molcel.2025.12.003},
pmid = {41475346},
issn = {1097-4164},
mesh = {Humans ; *Progeria/genetics/metabolism/pathology ; *Lamin Type A/genetics/metabolism ; *Alternative Splicing ; *CRISPR-Cas Systems ; Ribonucleoprotein, U1 Small Nuclear/metabolism/genetics ; HEK293 Cells ; *Clustered Regularly Interspaced Short Palindromic Repeats ; RNA Precursors/genetics/metabolism ; Zinc Fingers ; },
abstract = {Despite progress in understanding pre-mRNA splicing, the regulatory mechanisms controlling most alternative splicing events remain unclear. We developed CRASP-seq (CRISPR-based identification of regulators of alternative splicing with phenotypic sequencing), a method that integrates pooled CRISPR-based genetic perturbations with deep sequencing of splicing reporters, to quantitatively assess the impact of all human genes on alternative splicing from a single RNA sample. CRASP-seq identified both known and untested regulators, enriched for proteins involved in RNA splicing and metabolism. As a proof-of-concept, CRASP-seq analysis of the LMNA cryptic splicing event linked to progeria uncovered ZNF207, primarily known for mitotic spindle assembly, as a regulator of progerin splicing. ZNF207 depletion enhances canonical LMNA splicing and decreases progerin protein levels in patient-derived cells. We further show that ZNF207's zinc-finger domain broadly impacts alternative splicing through direct interactions with U1 small nuclear ribonucleoprotein (snRNP) components. These findings position ZNF207 as a U1 snRNP auxiliary factor and demonstrate the power of CRASP-seq to uncover key regulators and domains of alternative splicing.},
}

Humans
*Progeria/genetics/metabolism/pathology
*Lamin Type A/genetics/metabolism
*Alternative Splicing
*CRISPR-Cas Systems
Ribonucleoprotein, U1 Small Nuclear/metabolism/genetics
HEK293 Cells
*Clustered Regularly Interspaced Short Palindromic Repeats
RNA Precursors/genetics/metabolism
Zinc Fingers

@article {pmid41421338,
year = {2026},
author = {Du, R and Flynn, MJ and Mahe, K and Honsa, M and Gu, B and Li, D and McGeary, SE and Gradinaru, V and Jungmann, R and Elowitz, MB},
title = {miRNA modules for precise, tunable control of gene expression.},
journal = {Molecular cell},
volume = {86},
number = {1},
pages = {194-212.e7},
doi = {10.1016/j.molcel.2025.11.028},
pmid = {41421338},
issn = {1097-4164},
mesh = {Animals ; *MicroRNAs/genetics/metabolism ; Mice ; Humans ; Dependovirus/genetics ; Transgenes ; *Gene Expression Regulation ; Gene Editing/methods ; Neurons/metabolism ; CRISPR-Cas Systems ; Gene Dosage ; HEK293 Cells ; },
abstract = {Accurate control of transgene expression is important for research and therapy but is challenging to achieve in most settings. MicroRNA (miRNA)-based regulatory circuits can be incorporated within transgenes for improved control. However, the design principles, performance limits, and applications of these circuits in research and biotechnology have not been systematically determined. Here, combining modeling and experiments, we introduce miRNA-based circuit modules, termed "dosage invariant miRNA-mediated expression regulators" (DIMMERs), that establish precise, tunable control of transgene expression across diverse cell types to facilitate imaging, editing, and gene therapy. The circuits use multivalent miRNA regulatory interactions to achieve nearly uniform, tunable protein expression over two orders of magnitude variation in gene dosage. They function across diverse cell types and can be multiplexed for the independent regulation of multiple genes. DIMMERs reduce off-target CRISPR base editing, improve single-molecule imaging, and allow live tracking of adeno-associated virus (AAV)-delivered transgene expression in mouse cortical neurons. DIMMERs thus enable accurate regulation for research and biotechnology applications.},
}

Animals
*MicroRNAs/genetics/metabolism
Mice
Humans
Dependovirus/genetics
Transgenes
*Gene Expression Regulation
Gene Editing/methods
Neurons/metabolism
CRISPR-Cas Systems
Gene Dosage
HEK293 Cells

@article {pmid41369550,
year = {2026},
author = {Smith, DJ},
title = {Complementary human gene interaction maps from radiation hybrids and CRISPRi.},
journal = {Physiological genomics},
volume = {58},
number = {1},
pages = {42-57},
doi = {10.1152/physiolgenomics.00075.2025},
pmid = {41369550},
issn = {1531-2267},
support = {C25CR8562//University of California Cancer Research Coordinating Committee/ ; //Norton Simon Research Foundation/ ; },
mesh = {Humans ; Genome-Wide Association Study ; *Gene Regulatory Networks/genetics ; *CRISPR-Cas Systems/genetics ; *Radiation Hybrid Mapping/methods ; *Protein Interaction Maps/genetics ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Alleles ; },
abstract = {The only comprehensive human genetic interaction map was constructed using increased gene copy numbers in radiation hybrid (RH) cells. Recently, a second map restricted to essential genes was created using CRISPR interference (CRISPRi)-induced loss-of-function alleles. Here, the two maps are compared to understand their similarities and differences. Both maps showed significant overlap with protein-protein interaction databases and identified a shared set of interacting genes, although the specific gene pairs differed between approaches. Notably, the RH map exhibited strong overlap with genome-wide association study (GWAS) networks, whereas the CRISPRi map did not. These findings demonstrate how gain- and loss-of-function alleles reveal distinct yet complementary genetic interaction landscapes.NEW & NOTEWORTHY This study compared two mammalian genetic interaction networks for cell growth: the radiation hybrid (RH) network used extra gene copies and the CRISPRi network used partial gene suppression. Both networks overlapped with protein-protein interaction data and identified common interacting genes, yet specific gene pair interactions differed dramatically. Only the RH network predicted genome-wide association study (GWAS) networks. As the first comparison of large-scale mammalian genetic interaction networks, this work reveals how gain- and loss-of-function variants capture diverse biological perspectives.},
}

Humans
Genome-Wide Association Study
*Gene Regulatory Networks/genetics
*CRISPR-Cas Systems/genetics
*Radiation Hybrid Mapping/methods
*Protein Interaction Maps/genetics
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics
Alleles

@article {pmid41355773,
year = {2026},
author = {Tian, S and Yao, L and Gong, F and Li, Y and Zhao, Y and Yang, Y},
title = {Rapid and sensitive detection of circulating tumor DNA via a CRISPR/Cas12a-based catalytic hairpin assembly.},
journal = {Analytical methods : advancing methods and applications},
volume = {18},
number = {1},
pages = {115-123},
doi = {10.1039/d5ay01624j},
pmid = {41355773},
issn = {1759-9679},
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Circulating Tumor DNA/blood/genetics ; Limit of Detection ; *Endodeoxyribonucleases/metabolism/genetics ; Biomarkers, Tumor/blood/genetics ; Biosensing Techniques/methods ; Neoplasms/blood/diagnosis/genetics ; Bacterial Proteins ; CRISPR-Associated Proteins ; },
abstract = {Cancer is one of the major diseases that endanger the human health. Circulating tumor DNA (ctDNA) is an ideal biomarker for the real-time monitoring of cancer. In the present work, a rapid and sensitive assay coupled with CRISPR/Cas12a and CHA (Cas12a-CHA) was constructed for the detection of ctDNA. We designed and prepared a trigger, which was the substrate of Cas12a. On the addition of ctDNA, crRNA-guided ctDNA activated the trans-endonuclease activity of Cas12a. After being activated, Cas12a exhibited a high trans-cleavage activity on the trigger, which resulted in a decrease in fluorescence. Owing to this design, the Cas12a-CHA assay enabled the sensitive detection of ctDNA with a linear range of 10 fM to 50 pM. Furthermore, a limit-of-detection of 5.8 fM was achieved within 40 min. Besides, the proposed assay had an excellent base mismatch recognition ability and worked well in human serum samples. Conclusively, this detection platform holds significant potential for application in early cancer diagnosis.},
}

Humans
*CRISPR-Cas Systems/genetics
*Circulating Tumor DNA/blood/genetics
Limit of Detection
*Endodeoxyribonucleases/metabolism/genetics
Biomarkers, Tumor/blood/genetics
Biosensing Techniques/methods
Neoplasms/blood/diagnosis/genetics
Bacterial Proteins
CRISPR-Associated Proteins

@article {pmid41345100,
year = {2025},
author = {Bai, M and Zhang, J and Lin, W and Zhou, Y and Jiang, M and Wu, H and Peng, C and Lin, J and He, F and Kuang, H and Guan, Y},
title = {A flanking-nicks prime editor (FLICK-PE) system to boost prime editing in dicots.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {337},
pmid = {41345100},
issn = {2041-1723},
mesh = {*Gene Editing/methods ; *Glycine max/genetics/drug effects ; *Nicotiana/genetics ; Plants, Genetically Modified/genetics ; Glyphosate ; Glycine/analogs & derivatives/pharmacology ; CRISPR-Cas Systems/genetics ; Herbicide Resistance/genetics ; 3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics ; RNA, Guide, CRISPR-Cas Systems/genetics ; Genome, Plant ; },
abstract = {Prime editing (PE) enables precise genome modifications to mammalian cells and monocot staple crops, but remains relatively challenging in dicot plants. Here, we develop a Flanking-Nicks Prime Editor (FLICK-PE) system that boosts editing efficiency in soybean and tobacco. We show that optimization for PE by adding a nicking sgRNA could dramatically enhance intended-editing efficiency in soybean. Inspired by this observation, we design a FLICK-PE strategy to confer a pair of nicks flanking the target site. In soybean, FLICK-PE achieves on average a 15.7-fold increase in intended-editing efficiency compared to PE2, and a 2.2-fold increase compared to PE3. Using FLICK-PE, we efficiently engineer glyphosate resistance in soybean by introducing TAP-IVS mutations in EPSPS1a, achieving three amino-acid substitutions and an intended editing efficiency of 21.1%. This approach yields stable edited soybean varieties with vigorous glyphosate tolerance and minimal growth penalties in a field trial. FLICK-PE also demonstrates efficacy in tobacco, underscoring its broad applicability and versatility for rapid, precision breeding in agriculturally vital crops.},
}

*Gene Editing/methods
*Glycine max/genetics/drug effects
*Nicotiana/genetics
Plants, Genetically Modified/genetics
Glyphosate
Glycine/analogs & derivatives/pharmacology
CRISPR-Cas Systems/genetics
Herbicide Resistance/genetics
3-Phosphoshikimate 1-Carboxyvinyltransferase/genetics
RNA, Guide, CRISPR-Cas Systems/genetics
Genome, Plant

@article {pmid41344285,
year = {2026},
author = {Dasanayaka, BP and Pathirana, SL and Jayawardana, A and Handunnetti, SM and Fernando, N and Galhena, BP and Weerasena, SJ and Nitsche, A and Iddamaldeniya, SS and Dietzsch, AK},
title = {A roadmap in detecting frequently reported bovine babesiosis: From blood smear to CRISPR.},
journal = {Veterinary parasitology},
volume = {342},
number = {},
pages = {110662},
doi = {10.1016/j.vetpar.2025.110662},
pmid = {41344285},
issn = {1873-2550},
mesh = {Animals ; Cattle ; *Babesiosis/diagnosis/parasitology/blood ; *Cattle Diseases/diagnosis/parasitology/blood ; *CRISPR-Cas Systems ; *Babesia/isolation & purification/genetics ; Babesia bovis/isolation & purification/genetics ; Real-Time Polymerase Chain Reaction/veterinary ; Sensitivity and Specificity ; },
abstract = {Current diagnosis of Babesia bovis and B. bigemina relies on direct microscopy, nucleic acid detection, and serology. Light-microscopic analysis of Giemsa-stained smears still serves as the primary diagnostic modality at the point of care. However, carrier cattle, particularly those harbouring B. bovis, often carry parasites at levels far below the detection threshold, and such levels are sensitive only to DNA-based detection approaches. Early probe-hybridization techniques have been largely replaced by conventional Polymerase Chain Reaction (PCR), nested formats, and real-time quantitative PCR (qPCR)-which enable species-specific discrimination within closed-tube systems, thereby minimizing contamination risk. Species‑level identification is essential for clinical management, surveillance, and experimental studies. Duplex TaqMan qPCRs simultaneously distinguish B. bovis from B. bigemina, surpassing the analytical sensitivity offered by nested PCR (nPCR) methodologies. Reverse-line-blot (RLB) hybridisation broadens the diagnostic scope by concurrently detecting co-infections of B. bovis and B. bigemina and mixed haemoparasitic species in one workflow. However, the analytical sensitivity of RLB remains inferior to that of qPCR in detecting low-density and carrier-state infections of B. bigemina. RLB remains useful for retrospective genotyping when amplification is impractical or fails. Field-ready isothermal approaches have expanded the scope of molecular diagnostics beyond laboratory settings, facilitating field-level application and rapid on-site detection. Coupling Loop-mediated isothermal amplification (LAMP) with a lateral-flow dipstick (LFD) (LAMP-LFD) enables pen-side direct visual detection. Antibody tests are essential tools for herd-level surveillance. A recent chimeric Enzyme-Linked Immunosorbent Assay (ELISA) that combines three immunodominant B. bovis antigens broaden strain coverage and boosts diagnostic reliability. Recombinase polymerase amplification coupled to CRISPR-Cas12a cleavage has achieved single-target detection of B. bigemina from tick salivary-gland DNA, paving the way for innovative pen-side platforms, once cost and technical hurdles are overcome. Importantly, vector-based detection using appropriate tissues bearing an optimum level of ticks is species-dependent. B. bigemina sporozoites concentrate in nymph/adult salivary glands, whereas B. bovis sporozoites are produced mainly in larval salivary glands, so monitoring programs should stratify sampling accordingly.},
}

Animals
Cattle
*Babesiosis/diagnosis/parasitology/blood
*Cattle Diseases/diagnosis/parasitology/blood
*CRISPR-Cas Systems
*Babesia/isolation & purification/genetics
Babesia bovis/isolation & purification/genetics
Real-Time Polymerase Chain Reaction/veterinary
Sensitivity and Specificity

@article {pmid41338106,
year = {2026},
author = {Paenkaew, S and Euppayo, T and Tungtrakanpoung, R and Teapunvong, W and Nganvongpanit, K and Buddhachat, K},
title = {Rapid and specific detection of Babesia vogeli using RPA/CRISPR-Cas12a: A feasible field-friendly diagnostic for canine babesiosis.},
journal = {Veterinary parasitology},
volume = {342},
number = {},
pages = {110660},
doi = {10.1016/j.vetpar.2025.110660},
pmid = {41338106},
issn = {1873-2550},
mesh = {Animals ; Dogs ; *Babesiosis/diagnosis/parasitology ; *Dog Diseases/diagnosis/parasitology ; *Babesia/isolation & purification/genetics ; Sensitivity and Specificity ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/veterinary/methods ; Recombinases/metabolism ; },
abstract = {Babesia vogeli is a protozoan parasite causing canine babesiosis, a tick-borne disease prevalent in tropical and subtropical regions. Its microscopic identification is challenging due to morphological similarity with other Babesia spp., and serological assays often yield inaccurate results. To address this issue, we developed a rapid, equipment-minimal diagnostic method combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a (RPA/CRISPR-cas12a) for B. vogeli-specific detection. The RPA assay enables DNA amplification for both B. vogeli and Hepatozoon canis, while CRISPR/Cas12a using gRNA_Bab ensures specificity for B. vogeli, even in co-infections and other pathogens. This approach detects as few as 10[5] copies within two hours for both readout platforms such as fluorescence and lateral flow dipstick (LFD). Forty canine blood samples were detected by RPA/CRISPR-cas12a to examine its performance. Results showed high concordance with qPCR-high resolution melting (HRM) (Cohen's kappa: 0.93 for fluorescence, 0.81 for LFD), outperforming conventional PCR. The clinical sensitivity and specificity of RPA/CRISPR-cas12a were 100 % and 96.8 %, respectively and the concordance with qPCR-HRM was 97.5 %. RPA/CRISPR-cas12a for Babesia spp. detection provided a simple, rapid, and accurate method, demonstrating promise for point-of-care diagnosis of canine babesiosis in resource-limited settings. This method showed high potential as a practical diagnostic tool in veterinary clinics, with accelerated surveillance to control outbreaks of Babesia-associated canine babesiosis.},
}

Animals
Dogs
*Babesiosis/diagnosis/parasitology
*Dog Diseases/diagnosis/parasitology
*Babesia/isolation & purification/genetics
Sensitivity and Specificity
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/veterinary/methods
Recombinases/metabolism

@article {pmid41145673,
year = {2026},
author = {Benz, T and Larghero, P and Meyer, C and Hanewald, T and Brüggmann, D and Hentrich, AE and Louwen, F and Marschalek, R},
title = {CRISPR/Cas9-mediated t(4;11) translocation in human hematopoietic stem/precursor cells demonstrates plasticity to differentiate into either the myeloid or lymphoid lineage.},
journal = {Leukemia},
volume = {40},
number = {1},
pages = {72-86},
pmid = {41145673},
issn = {1476-5551},
support = {2022.070.1//Wilhelm Sander-Stiftung (Wilhelm Sander Foundation)/ ; },
mesh = {Humans ; *CRISPR-Cas Systems/genetics ; *Translocation, Genetic ; *Hematopoietic Stem Cells/metabolism/cytology ; *Chromosomes, Human, Pair 11/genetics ; *Cell Differentiation/genetics ; *Chromosomes, Human, Pair 4/genetics ; Myeloid-Lymphoid Leukemia Protein/genetics ; Histone-Lysine N-Methyltransferase/genetics ; *Myeloid Cells/cytology/metabolism ; *Lymphocytes/cytology/metabolism ; Cell Lineage/genetics ; Oncogene Proteins, Fusion/genetics ; DNA-Binding Proteins/genetics ; Transcriptional Elongation Factors ; },
abstract = {The chromosomal translocation t(4;11)(q21;q23) is frequently diagnosed in KMT2A-r Acute Leukemia patients. Although we understand much about the function of both wildtype KMT2A and AFF1 multiprotein complexes, little is known about the molecular actions the two fusion proteins KMT2A::AFF1 and AFF1::KMT2A during the very early steps of disease onset and progression. Most published data have been generated in t(4;11) cell lines or transplanted mouse models, where exactly this process remains a black box. Here, we present the results of our efforts to establish a t(4;11) chromosomal translocation in human hematopoietic stem/precursor cells by CRISPR/Cas9. These genetically modified cells can be expanded over 5-6 months in vitro and their potential to differentiate was examined with IL-7 supplementation. The benefit of this model system is that (1) both reciprocal fusion proteins are concomitantly present, and (2) a molecular surveillance is possible at any timepoint through analysis of RNA, DNA or protein. Thus, the CRISPR/Cas9 technique allowed us to create a bona fide model system to study the very early steps of leukemia onset at the molecular level. In conclusion, this approach is the fastest way to investigate and characterize KMT2A-r fusions in primary human cells.},
}

Humans
*CRISPR-Cas Systems/genetics
*Translocation, Genetic
*Hematopoietic Stem Cells/metabolism/cytology
*Chromosomes, Human, Pair 11/genetics
*Cell Differentiation/genetics
*Chromosomes, Human, Pair 4/genetics
Myeloid-Lymphoid Leukemia Protein/genetics
Histone-Lysine N-Methyltransferase/genetics
*Myeloid Cells/cytology/metabolism
*Lymphocytes/cytology/metabolism
Cell Lineage/genetics
Oncogene Proteins, Fusion/genetics
DNA-Binding Proteins/genetics
Transcriptional Elongation Factors

@article {pmid40759332,
year = {2025},
author = {Yang, J and Huang, C and Feng, Y and He, J and Liu, Y and Zhang, P and Liu, C},
title = {Adaptor protein complex 1 gamma 1 subunit is an important host factor involved in both Zika virus and dengue virus infections.},
journal = {Virologica Sinica},
volume = {40},
number = {6},
pages = {874-883},
doi = {10.1016/j.virs.2025.07.012},
pmid = {40759332},
issn = {1995-820X},
mesh = {Humans ; *Dengue Virus/physiology/genetics ; *Zika Virus/physiology/genetics ; Virus Replication ; *Zika Virus Infection/virology/metabolism ; *Dengue/virology/metabolism ; Cell Line ; Virus Internalization ; *Host-Pathogen Interactions ; Viral Envelope Proteins/metabolism ; CRISPR-Cas Systems ; Animals ; RNA Interference ; },
abstract = {Mosquito-borne flaviviruses, such as Zika virus (ZIKV) and dengue virus (DENV), cause diverse severe clinical manifestations including fever, rash, hepatitis, arthralgia, and congenital anomalies. Here, we identified a host factor, the adaptor protein complex 1 gamma 1 subunit (AP1G1), which plays an important role in both ZIKV and dengue virus 2 (DENV2) infections. We explored the role of AP1G1 in ZIKV and DENV2 infections using CRISPR/Cas9 gene editing technology and RNA interference (RNAi) techniques. Knockout or silencing of AP1G1 decreases the replication of ZIKV and DENV2 in multiple human cell lines. Intriguingly, depletion of AP1G1 results in a significant reduction in ZIKV at an early stage, but decreases DENV2 replication levels during the late stage, suggesting that AP1G1 plays distinct roles in the infection by ZIKV and DENV2. Furthermore, we determined that AP1G1 mediates ZIKV-endosomal membrane fusion through inhibitor experiments and fluorescence labeling assays. Mechanistically, we found that AP1G1 exerts its pro-viral effect through binding to the ZIKV envelope glycoprotein (E protein). This interaction promotes the fusion of viral and endosomal membranes, during which the ZIKV genomic RNAs are released from the endosome into the cytoplasm, a process that facilitates viral replication. However, for DENV2 infection, AP1G1 primarily affects its viral RNA replication stage, rather than the fusion of virus-endosomal membrane. Taken together, our work demonstrates that AP1G1 plays a pro-viral role in both ZIKV and DENV2 infections via distinct mechanisms, highlighting its potential as a therapeutic target for antiviral strategies.},
}

Humans
*Dengue Virus/physiology/genetics
*Zika Virus/physiology/genetics
Virus Replication
*Zika Virus Infection/virology/metabolism
*Dengue/virology/metabolism
Cell Line
Virus Internalization
*Host-Pathogen Interactions
Viral Envelope Proteins/metabolism
CRISPR-Cas Systems
Animals
RNA Interference

@article {pmid41497669,
year = {2025},
author = {Kumbara, A and Tognon, M and Carone, G and Fontanesi, A and Bombieri, N and Giugno, R and Pinello, L},
title = {CRISPR-HAWK: Haplotype- and Variant-aware guide design toolkit for CRISPR-Cas.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41497669},
issn = {2692-8205},
abstract = {MOTIVATION: Current CRISPR guide RNA design tools rely on reference genomes, overlooking how genetic variation impacts editing outcomes. As genome editing advances toward clinical applications, incorporating population diversity becomes essential for ensuring therapeutic efficacy across diverse populations.

RESULTS: We present CRISPR-HAWK, a framework integrating individual- and population-scale variants and haplotypes into gRNA design. Analyzing therapeutic targets across 79,648 genomes reveals that genetic variants substantially alter guide performance. For the clinically approved sickle cell disease therapeutic guide targeting BCL11A, we identify haplotypes that completely abolish predicted cutting activity. Across seven therapeutic loci, 82.5% of guides contain variants modifying on-target activity. Variants also create novel protospacer adjacent motif sites generating individual-specific guides invisible to reference-based design. These findings demonstrate that variant-aware selection is critical for equitable genome editing.

AVAILABILITY: CRISPR-HAWK is available at https://github.com/pinellolab/CRISPR-HAWK and https://github.com/InfOmics/CRISPR-HAWK.},
}

@article {pmid41497644,
year = {2025},
author = {Stringer, AM and Wade, JT},
title = {Identification of PAM Requirements for the Vibrio cholerae type I-E CRISPR-Cas System.},
journal = {bioRxiv : the preprint server for biology},
volume = {},
number = {},
pages = {},
pmid = {41497644},
issn = {2692-8205},
abstract = {CRISPR-Cas systems are prokaryotic adaptive immune systems that use RNA-guided protein complexes to target invading nucleic acid. A surveillance complex consisting of protein and a CRISPR-RNA (crRNA) binds target nucleic acid via base-pairing interactions, typically leading to processing of the target nucleic acid by a nuclease. CRISPR-Cas systems are classified based on their mechanism of action, with type I systems being the most prevalent in nature. Type I CRISPR-Cas systems target DNA, and require extensive complementarity between the crRNA and the target DNA. Moreover, type I systems require the presence of a "Protospacer Adjacent Motif" (PAM) sequence in the target DNA immediately adjacent to the expected region of base-pairing with the crRNA. Classical biotypes of the bacterial pathogen Vibrio cholerae have active type I-E CRISPR-Cas systems. While the optimal PAM sequence for this CRISPR-Cas system is known to be AAY, the activity of other sequences as possible PAMs has not been determined. Here, we quantify the effectiveness of all possible trinucleotide sequences in the PAM position for the V. cholerae type I-E CRISPR-Cas system. Our data indicate a hierarchy of PAM efficacy, with 15 of the 64 trinucleotide sequences functioning as a PAM.},
}

@article {pmid41496934,
year = {2026},
author = {Khizar, M and Aminpoor, H and Zaib, M and Ali, Q and Karimi, H},
title = {Nanoparticle-enhanced CRISPR delivery: paving the path for in vivo tumor gene editing.},
journal = {Annals of medicine and surgery (2012)},
volume = {88},
number = {1},
pages = {1054-1055},
pmid = {41496934},
issn = {2049-0801},
abstract = {Nanoparticle-based delivery systems are redefining how CRISPR/Cas technology can be used in cancer treatment. By encapsulating CRISPR components within lipid, polymeric, or inorganic nanoparticles, researchers have improved their stability, circulation time, and tumor-targeting precision. The NTLA-2001 trial demonstrated the first successful use of lipid nanoparticles for in vivo CRISPR delivery in humans, paving the way for potential applications in oncology. Preclinical studies have shown promising results, with efficient gene knockout and tumor suppression across multiple models. Despite these advances, barriers remain, including limited delivery to solid tumors, potential off-target effects, and inconsistent nanoparticle formulations. Global research efforts spanning the United States, China, Europe, and India are now focused on refining delivery platforms and standardizing protocols. This letter highlights current progress, ongoing challenges, and the need for transparent, globally coordinated development. Nanoparticle-enhanced CRISPR delivery has the potential to bring genetic precision therapy from the laboratory to the clinic, offering a new avenue for durable and accessible cancer care.},
}

@article {pmid41496894,
year = {2026},
author = {Kantor, B and Duke, L and Bhide, PG},
title = {CRISPR-Cas editing technologies for viral-mediated gene therapies of human diseases: Mechanisms, progress, and challenges.},
journal = {Molecular therapy. Nucleic acids},
volume = {37},
number = {1},
pages = {102786},
pmid = {41496894},
issn = {2162-2531},
abstract = {The gene therapy landscape has evolved substantially in recent years, beginning with the approval of the first adeno-associated virus-based gene therapy, Luxterna, in 2017. Since then, the US FDA has approved nearly 30 new viral gene therapy programs, with notable examples including Zolgensma, Spinraza, Hemgenix, Zynteglo, Lyfgenia, Kymriah, Skysona, and Tecelra. Remarkably, all these products rely on delivery via adeno-associated vectors (AAVs) and lentiviral vectors (LVs). Improvements in viral-mediated gene transfer efficiency and clinical-scale manufacturing, together with immense commercial interest, have greatly propelled the clinical adoption of gene therapy products. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR) and its related Cas proteins (CRISPR-Cas) have made significant advances in gene therapy, offering next-generation approaches for curative gene editing to treat genetic diseases and disorders. In this review, we examine the range of these therapeutics and their viral carriers, focusing primarily on LVs and AAVs. We provide a snapshot of the current status of the field and highlight some of the current challenges in the clinical application of gene therapy, with particular emphasis on viral CRISPR-Cas-based technologies and their future potential.},
}

@article {pmid41496629,
year = {2026},
author = {Li, J and Wang, L and Yang, S and Zhou, X and Gou, Q and Cai, J and Yang, H and Wang, Q and Li, S},
title = {Chicken Shank Color Determined by Inhibition of Dermal Melanin (ID) is Mediated by a Structural Variation Regulating CDKN2A Expression.},
journal = {Pigment cell & melanoma research},
volume = {39},
number = {1},
pages = {e70072},
doi = {10.1111/pcmr.70072},
pmid = {41496629},
issn = {1755-148X},
support = {2022hszd006//Major Project of Hubei Hongshan Laboratory/ ; 31772585//National Natural Science Foundation of China/ ; },
mesh = {Animals ; *Chickens/genetics ; *Melanins/metabolism ; *Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism ; *Skin Pigmentation/genetics ; Melanocytes/metabolism ; *Gene Expression Regulation ; Polymorphism, Single Nucleotide/genetics ; Female ; Male ; CRISPR-Cas Systems/genetics ; Genome-Wide Association Study ; *Skin/metabolism ; },
abstract = {Shank color in chickens is a classic quantitative trait governed by four genetic loci. Among these, the Inhibition of dermal melanin (ID) locus, which suppresses dermal melanogenesis in the shank, is the sole sex-linked mutation and its molecular mechanisms remain elusive. To identify the causal mutation, we established a resource population segregating for shank colors. A genome-wide association study utilizing FarmCPU software identified a top-associated SNP on the Z chromosome. Linkage mapping subsequently narrowed the candidate region, within which we discovered a candidate structural variant associated with the yellow shank phenotype. This variant is characterized by a 143 bp deletion coupled with a 2 bp insertion. CDKN2A was the only gene within the same topologically associating domain to exhibit differential expression. Functional validation via CRISPR/Cas9-edited cells demonstrated that this mutation regulates CDKN2A transcription and is responsible for the ID shank color in chickens. We propose that the resulting absence of melanocytes is likely due to apoptosis. This work resolves the molecular basis of the ID locus, thereby completing the genetic puzzle of chicken shank color. This discovery enables the development of molecular markers for auto-sexing of day-old chicks, a tool with significant potential for the poultry industry.},
}

Animals
*Chickens/genetics
*Melanins/metabolism
*Cyclin-Dependent Kinase Inhibitor p16/genetics/metabolism
*Skin Pigmentation/genetics
Melanocytes/metabolism
*Gene Expression Regulation
Polymorphism, Single Nucleotide/genetics
Female
Male
CRISPR-Cas Systems/genetics
Genome-Wide Association Study
*Skin/metabolism

@article {pmid41495909,
year = {2026},
author = {Derollez, E and Roson-Calero, N and Rouzé, P and Dedieu-Berne, A and Ballesté-Delpierre, C and Fraikin, N and Iorga, BI and Huang, TD and Bigot, S and Vila, J and Bogaerts, P and Lesterlin, C},
title = {Specific killing and resensitization of pathogenic Escherichia coli strains carrying blaCTX-M-15 β-lactamase using targeted-antibacterial-plasmids (TAPs).},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495909},
issn = {1362-4962},
support = {//Joint Programming Initiative on Antimicrobial Resistance/ ; JPIAMR2021-194//JPIAMR/ ; FRM-EQU202103012587//Foundation for Medical Research/ ; ANR-20-PAMR-0010//French Priority Research Program/ ; //Agence Nationale de la Recherches/ ; },
mesh = {*beta-Lactamases/genetics/metabolism ; *Escherichia coli/genetics/drug effects/enzymology ; *Plasmids/genetics ; CRISPR-Cas Systems ; Humans ; Anti-Bacterial Agents/pharmacology ; Conjugation, Genetic ; Drug Resistance, Multiple, Bacterial/genetics ; Escherichia coli Infections/microbiology ; },
abstract = {Targeted-Antibacterial-Plasmids (TAPs) offer a precise approach to combat multidrug-resistant bacteria by selectively removing resistant strains while preserving commensals. Here, we assess TAPs that deliver CRISPR/Cas systems via conjugation to kill or resensitize extended-spectrum β-lactamase (ESBL)-producing Escherichia coli carrying the blaCTX-M-15 gene. We systematically tested multiple variables in the TAP approach, including two commensal E. coli donors, three distinct helper plasmids encoding the transfer machineries, and six recipient strains harbouring the resistance gene either chromosomally or on a plasmid. Cas9-based TAPs induced double-stranded breaks in chromosomal blaCTX-M-15 genes, resulting in immediate bacterial death. When the target gene was plasmid-borne, Cas9 cleavage triggered plasmid loss and partial toxin-antitoxin-mediated killing. In contrast, dCas9-based TAPs inhibited blaCTX-M-15 expression without affecting cell viability, thereby restoring third-generation cephalosporin susceptibility. In mixed-culture experiments, TAPs specifically eliminated only blaCTX-M-15-carrying E. coli while sparing other non-targeted bacterial species. Conjugation assays in human faeces demonstrated substantial suppression of cefotaxime-resistant (CtxR) E. coli by both Cas9- and dCas9-based TAPs, underscoring their efficacy in complex microbial environments. These findings highlight TAPs' decolonization promise, paving the way for future microbiome-editing interventions against multidrug-resistant carriage or infection.},
}

*beta-Lactamases/genetics/metabolism
*Escherichia coli/genetics/drug effects/enzymology
*Plasmids/genetics
CRISPR-Cas Systems
Humans
Anti-Bacterial Agents/pharmacology
Conjugation, Genetic
Drug Resistance, Multiple, Bacterial/genetics
Escherichia coli Infections/microbiology

@article {pmid41495894,
year = {2026},
author = {Ishihara, K and Matsumoto, S and Gerle, C and Gopalasingam, CC and Shigematsu, H and Shirai, T and Numata, T},
title = {Sequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495894},
issn = {1362-4962},
support = {20H02916//Japan Society for the Promotion of Science/ ; 24H00505//Japan Society for the Promotion of Science/ ; //Japan Foundation for Applied Enzymology/ ; //Naito Foundation/ ; //Institute for Fermentation, Osaka/ ; //Noda Institute for Scientific Research/ ; 23KJ1734//JSPS/ ; },
mesh = {*CRISPR-Cas Systems/genetics ; *CRISPR-Associated Proteins/chemistry/genetics/metabolism ; *DNA Transposable Elements/genetics ; *RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism ; *Bacterial Proteins/chemistry/genetics/metabolism ; Vibrio parahaemolyticus/genetics ; DNA/chemistry/metabolism/genetics ; Cryoelectron Microscopy ; Models, Molecular ; },
abstract = {Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.},
}

*CRISPR-Cas Systems/genetics
*CRISPR-Associated Proteins/chemistry/genetics/metabolism
*DNA Transposable Elements/genetics
*RNA, Guide, CRISPR-Cas Systems/genetics/chemistry/metabolism
*Bacterial Proteins/chemistry/genetics/metabolism
Vibrio parahaemolyticus/genetics
DNA/chemistry/metabolism/genetics
Cryoelectron Microscopy
Models, Molecular

@article {pmid41495892,
year = {2026},
author = {Zhang, Z and Wang, J and Guo, T and Yu, X and Wang, F and Zhang, H and Liu, Y and Li, W and Cheng, Y and Peng, Y and Yan, G and Cui, J and Ma, L},
title = {Argonaute-mediated RNA editing selectively repairs point mutations.},
journal = {Nucleic acids research},
volume = {54},
number = {1},
pages = {},
pmid = {41495892},
issn = {1362-4962},
support = {2022YFA0911800//National Key Research and Development Program of China/ ; 2024BCA001//Technological Innovation Plan in Hubei Province/ ; 2024040701010046//Natural Science Foundation of Wuhan City/ ; 2025CSA051//Science and Technology Program Project of Hubei Province/ ; 32300517//National Natural Science Foundation of China/ ; 32400513//National Natural Science Foundation of China/ ; NYWSWZX2025-2027-10//Hubei Provincial Major Special Project for the Development of the Agricultural Microbiology Industry/ ; 2021CSA066//Hubei Province Central Government Guided Special Fund for Local Science and Technology Development/ ; },
mesh = {*Argonaute Proteins/metabolism/genetics ; Humans ; *RNA Editing ; *Point Mutation ; HEK293 Cells ; RNA, Guide, CRISPR-Cas Systems/genetics ; Animals ; Ribonucleoproteins/metabolism/genetics ; Adenosine Deaminase/genetics/metabolism ; },
abstract = {RNA editing enzymatically modifies RNA molecules post-transcriptionally, enabling precise sequence alterations. Advantages include reversibility and temporal control without genomic DNA changes, allowing dynamic regulation of gene expression while preserving original genetic information. In this study, we characterized McAgo derived from Monosporascus cannonballus, which functions as a programmable nuclease guided by 14-30 nt gRNAs, demonstrating robust RNA cleavage activity at physiological temperature. Furthermore, we delivered McAgo RNP (ribonucleoprotein) complexes into mammalian cells, achieving >90% RNA knockdown efficiency with minimal innate immune responses. A catalytically inactive mutant (dMcAgo) using a gRNA as short as 20 nt, conjugated to the hADAR2 deaminase domain (hADAR2dd E488Q), achieved up to 90% RNA editing efficiency in vitro. This study establishes, for the first time, the effective targeting of endogenous RNA by a heterologous Argonaute in mammalian cells, alongside its demonstrated utility for RNA editing-thereby expanding the functional repertoire of Argonaute proteins.},
}

*Argonaute Proteins/metabolism/genetics
Humans
*RNA Editing
*Point Mutation
HEK293 Cells
RNA, Guide, CRISPR-Cas Systems/genetics
Animals
Ribonucleoproteins/metabolism/genetics
Adenosine Deaminase/genetics/metabolism

@article {pmid41461227,
year = {2026},
author = {Su, S and Zhang, X and Wang, X and Qiu, C and Xu, Z and Piñero, JC and Peng, X and Li, F and Zuo, Y and Chen, M},
title = {CRISPR/Cas9-based evidence that overexpression of Gm-mGST1 mediates abamectin resistance in the oriental fruit moth, Grapholita molesta.},
journal = {Insect biochemistry and molecular biology},
volume = {187},
number = {},
pages = {104472},
doi = {10.1016/j.ibmb.2025.104472},
pmid = {41461227},
issn = {1879-0240},
mesh = {Animals ; *Ivermectin/analogs & derivatives/pharmacology ; *Moths/genetics/drug effects/metabolism ; *Insecticide Resistance/genetics ; CRISPR-Cas Systems ; *Insecticides/pharmacology ; *Glutathione Transferase/genetics/metabolism ; *Insect Proteins/genetics/metabolism ; Chloride Channels/genetics/metabolism ; Larva/genetics/growth & development/drug effects ; },
abstract = {Abamectin-based insecticides are widely used in integrated pest management and are particularly effective against fruit borers such as the oriental fruit moth, Grapholita molesta. However, rapid resistance evolution threatens their long-term efficacy. This study elucidates the role of the glutathione S-transferase gene Gm-mGST1 in abamectin resistance in G. molesta. A laboratory-selected resistant strain (AB-R) exhibited an 85.5-fold increase in resistance compared with a susceptible strain (AB-S). Sequencing of glutamate-gated chloride channel (GmGluCl) gene revealed no target-site mutations, implicating a metabolic resistance mechanism. In AB-R, GST enzymatic activity was significantly elevated. GST synergist diethyl maleate (DEM) increased the toxicity of abamectin more strongly in the abamectin-resistant G. molesta strain than in the susceptible strain, indicating that GSTs contribute to abamectin resistance. Gm-mGST1 showed strong and stage-specific overexpression under abamectin exposure. Functional analysis using CRISPR/Cas9 knockout of Gm-mGST1 in the AB-R strain reduced resistance 16.3-fold, providing the definitive evidence that a GST gene directly mediates abamectin resistance in G. molesta. The catalytic activity of recombinant Gm-mGST1 was verified in vitro using CDNB as the substrate. Additionally, abamectin exhibited a certain degree of inhibitory effect on the activity of Gm-mGST1. HPLC analysis further revealed that the peak area of abamectin significantly decreased in the presence of recombinant Gm-mGST1, while ectopic expression in Drosophila melanogaster increased abamectin tolerance by 1.97-fold. There is a significant positive correlation between the abamectin resistance levels and the expression levels of Gm-mGST1 in field populations of G. molesta. These findings identify Gm-mGST1 as a critical gene involved in abamectin resistance and establish it as a potential molecular marker for monitoring resistance in field populations. More broadly, this study sets a precedent for integrating CRISPR/Cas9 gene editing into insecticide resistance research, bridging the gap between correlative evidence and functional validation, and providing a framework for developing GST-targeted resistance management strategies in orchard pests. This study provides evidence using CRISPR/Cas9 to confirm the contribution of GST to abamectin resistance in insects.},
}

Animals
*Ivermectin/analogs & derivatives/pharmacology
*Moths/genetics/drug effects/metabolism
*Insecticide Resistance/genetics
CRISPR-Cas Systems
*Insecticides/pharmacology
*Glutathione Transferase/genetics/metabolism
*Insect Proteins/genetics/metabolism
Chloride Channels/genetics/metabolism
Larva/genetics/growth & development/drug effects

@article {pmid41423037,
year = {2026},
author = {Ma, L and Wu, B and Li, S and Zhang, X and Zhao, X and Zhang, J and Zhang, M and Zhang, M and Ma, L and Guo, C and Zhang, T},
title = {CRISPR/Cas9-mediated LmSerpin5 knockout causes midgut dysplasia and leads to embryonic lethality in Locusta migratoria.},
journal = {Journal of insect physiology},
volume = {168},
number = {},
pages = {104925},
doi = {10.1016/j.jinsphys.2025.104925},
pmid = {41423037},
issn = {1879-1611},
mesh = {Animals ; *Locusta migratoria/genetics/growth & development/embryology/immunology ; CRISPR-Cas Systems ; *Insect Proteins/genetics/metabolism ; Nymph/growth & development/genetics ; Immunity, Innate/genetics ; Gene Knockout Techniques ; },
abstract = {Serpins play a crucial role in in various physiological processes of insects. Previous studies have suggested that Serpins regulated processes like egg diapause, melanization, and antimicrobial peptide synthesis in Locusta migratoria, but their overall functional characterization remains insufficient. In this study, the functions of LmSerpin5 in regulating developmental processes and innate immunity were investigated via CRISPR/Cas9-mediated knockout. Homozygous LmSerpin5 mutation caused complete embryonic lethality. By contrast, chimeric mutants showed elevated mortality during embryonic-to-first-instar nymph transition, though chitinous tissue development remained unaffected. Additionally, adult mutants exhibited no external malformations but displayed pathological changes in immune organs, including fat body cells with enlarged lipid droplets and nuclei, and midgut absorptive cells lacking brush borders. Furthermore, pro-nymphal midguts exhibited reduced microvilli density, structural defects, and inflammatory intestinal folds. Molecular analysis confirmed upregulation of Toll pathway downstream genes (LmMyd88, LmPelle and LmTube) in mutant tissues, with midgut-specific activation of LmTube and LmPelle linking structural damage to immune dysregulation. These results demonstrated LmSerpin5 maintains homeostasis through dual mechanisms: ensuring embryonic survival and suppressing excessive Toll activation.},
}

Animals
*Locusta migratoria/genetics/growth & development/embryology/immunology
CRISPR-Cas Systems
*Insect Proteins/genetics/metabolism
Nymph/growth & development/genetics
Immunity, Innate/genetics
Gene Knockout Techniques

@article {pmid41353342,
year = {2025},
author = {He, L and Yao, Y and You, Y and Wei, X and Ma, Y and Yuan, W and Lang, Z and Zhu, JK},
title = {Versatile molecular tools enabling customizable DNA methylation editing in Arabidopsis.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {251},
pmid = {41353342},
issn = {2041-1723},
support = {32188102//National Natural Science Foundation of China (National Science Foundation of China)/ ; 32100458//National Natural Science Foundation of China (National Science Foundation of China)/ ; },
mesh = {*Arabidopsis/genetics ; *DNA Methylation/genetics ; *Gene Editing/methods ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Genome, Plant ; },
abstract = {Tools to edit DNA methylation in a targeted manner are vital for establishing causal relationships between DNA methylation and its function, as well as for plant breeding and gene therapy. Here, by constructing dCas9 fusions to a panel of effectors and cofactors, we develop a range of highly effective tools for editing DNA methylation in Arabidopsis, including five tools for DNA methylation and six tools for DNA demethylation. Our tools show a diversity of performance features in terms of specificity and efficiency, offering either the capacity to edit DNA methylation in a target-specific manner or the ability to edit DNA methylation genome-wide due to potent off-target effect. Importantly, DNA methylation edited by these tools is inherited in the absence of transgene. These versatile tools pave the way for diverse applications of DNA methylation editing in not only research but also epigenetic breeding of crops.},
}

*Arabidopsis/genetics
*DNA Methylation/genetics
*Gene Editing/methods
CRISPR-Cas Systems/genetics
Plants, Genetically Modified
Genome, Plant

@article {pmid41213129,
year = {2026},
author = {Ajdanian, L and Villot, S and Karikari, B and Torkamaneh, D},
title = {Technological advances in trait development: from conventional breeding and untargeted mutagenesis to precision genome editing.},
journal = {Genome},
volume = {69},
number = {},
pages = {1-13},
doi = {10.1139/gen-2025-0020},
pmid = {41213129},
issn = {1480-3321},
mesh = {*Gene Editing/methods ; *Plant Breeding/methods ; *Crops, Agricultural/genetics ; *Mutagenesis ; Plants, Genetically Modified/genetics ; *Genome, Plant ; CRISPR-Cas Systems ; },
abstract = {Plant biotechnology has revolutionized modern agriculture by enabling precise and efficient crop improvement strategies. This review explores the evolution of selective breeding, mutation breeding, and precision breeding, highlighting their applications in Canada's agricultural sector. Conventional selective breeding has been instrumental in developing high-yielding and disease-resistant cultivars, while mutation breeding, through physical and chemical mutagenesis, has introduced valuable genetic diversity. The advent of transgenic breeding allowed for the direct insertion of foreign genes, leading to the development of crops with herbicide tolerance, pest resistance, and improved nutritional content. However, concerns over regulatory restrictions and public acceptance have driven the rapid adoption of genome editing tools, which enable precise modifications without introducing foreign DNA. Canada has played a key role in applying these biotechnological innovations, successfully developing genetically modified canola, CRISPR-edited wheat, stress-resistant soybean, and barley and oat cultivars improved for stress resistance and yield. While each breeding approach presents distinct advantages and limitations, integrating conventional and molecular techniques is essential for maximizing genetic potential, ensuring agriculture, and effectively food security challenges.},
}

*Gene Editing/methods
*Plant Breeding/methods
*Crops, Agricultural/genetics
*Mutagenesis
Plants, Genetically Modified/genetics
*Genome, Plant
CRISPR-Cas Systems

@article {pmid41047629,
year = {2026},
author = {Zhang, C and Chen, Z and Cao, J and Zhang, Z and Li, WK and Zhang, X and Chen, J and Liu, J and Yuan, Z and Gao, F and Shi, Z and Zhao, XM and Chen, J and Zhan, C and Cheng, TL},
title = {RNA inosine sensor-guided TadA mutational scanning for toxicity minimization of adenine base editors.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {380-396},
doi = {10.1016/j.ymthe.2025.10.011},
pmid = {41047629},
issn = {1525-0024},
mesh = {*Inosine/genetics/metabolism ; Humans ; *RNA Editing ; Animals ; *Adenine/metabolism ; *Mutation ; *Gene Editing/methods ; Mice ; HEK293 Cells ; CRISPR-Cas Systems ; Biosensing Techniques/methods ; },
abstract = {The TadA component of adenine base editors (ABEs) induces widespread RNA off-target edits and raises safety concerns for their applications. However, the extent of RNA editing-related toxicity remains elusive, and high-throughput engineering of ABEs focusing on RNA editing activities remains challenging. Here, we demonstrate that RNA off-target editing of classical ABEs leads to substantial toxicity in vitro and in vivo. We then design a rapid, cost-effective, and sensitive fluorescent RNA inosine sensor to accelerate RNA off-target editing evaluation and high-throughput screening in mammalian cells. Deep mutation scanning with the RNA sensor identifies various TadA8e mutants displaying minimized RNA editing activity, with the representative H52L/D53R mutant compatible with both SpCas9 and the compact IscB nickase. We show that the engineered ABEs could efficiently target clinically relevant sites in vitro and in vivo with enhanced precision, thereby providing promising tools for applications in which RNA editing-related toxicity should be carefully evaluated and minimized.},
}

*Inosine/genetics/metabolism
Humans
*RNA Editing
Animals
*Adenine/metabolism
*Mutation
*Gene Editing/methods
Mice
HEK293 Cells
CRISPR-Cas Systems
Biosensing Techniques/methods

@article {pmid41017153,
year = {2026},
author = {Ye, C and Ma, Y and Shrestha, R and Cai, J and Liu, Y and Peng, L and Yu, J and Cai, H},
title = {Extracellular vesicle-mediated delivery of CRISPR machinery silences androgen receptor in castration-resistant prostate cancer cells.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {281-299},
doi = {10.1016/j.ymthe.2025.09.045},
pmid = {41017153},
issn = {1525-0024},
mesh = {Humans ; Male ; *Receptors, Androgen/genetics ; *Extracellular Vesicles/metabolism/genetics ; *Prostatic Neoplasms, Castration-Resistant/genetics/therapy/pathology/metabolism ; Cell Line, Tumor ; *CRISPR-Cas Systems ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Gene Silencing ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cell Proliferation ; CRISPR-Associated Protein 9 ; Ribonucleoproteins/genetics ; },
abstract = {CRISPR-mediated gene editing is a promising technology for treatment of diseases by silencing a driver gene at the genomic DNA level. However, delivery of CRISPR machinery remains challenging for potential therapeutic application. Here, we developed a platform using extracellular vesicles (EVs) as a vehicle to deliver Cas9/single-guide RNA (sgRNA) ribonucleoprotein (RNP) complex to silence androgen receptor (AR) gene in prostate cancer (PCa) cells. A genetic modification conferred the N-myristoylation to the Cas9 protein, which enhanced the encapsulation of Cas9/sgRNA RNP into EVs and silenced both ectopic and endogenous AR gene. Interestingly, gene editing efficiency varied across PCa cell lines, associated with different chromatin accessibility at the target site. Functional analyses demonstrated that Cas9/sgRNA RNP (targeting the N-terminal domain of the AR gene) did not change gene-edited AR mRNA levels, but significantly inhibited expression levels of AR downstream genes, thereby attenuating PCa cell proliferation. Importantly, EV-mediated delivery of the Cas9/sgRNA RNP introduced indels into the AR gene and inhibited proliferation of enzalutamide-resistant PCa cells. This study highlights a therapeutic strategy for treatment of castration-resistant PCa using a programmable EV-mediated delivery platform.},
}

Humans
Male
*Receptors, Androgen/genetics
*Extracellular Vesicles/metabolism/genetics
*Prostatic Neoplasms, Castration-Resistant/genetics/therapy/pathology/metabolism
Cell Line, Tumor
*CRISPR-Cas Systems
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Gene Silencing
Clustered Regularly Interspaced Short Palindromic Repeats
Cell Proliferation
CRISPR-Associated Protein 9
Ribonucleoproteins/genetics

@article {pmid40994008,
year = {2026},
author = {Marco, E and Sousa, P and Janoudi, T and de Dreuzy, E and Heath, JM and Viswanathan, R and Zuris, JA and Gotta, GM and Giannoukos, G and Hansen, S and Wood, DK and Walters, MC and Tisdale, JF and Wilson, CJ and Chang, KH},
title = {Nonclinical evaluation of renizgamglogene autogedtemcel for SCD and TDT.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {249-265},
doi = {10.1016/j.ymthe.2025.09.031},
pmid = {40994008},
issn = {1525-0024},
mesh = {*Anemia, Sickle Cell/genetics/therapy ; Humans ; *Gene Editing/methods ; Animals ; Mice ; *beta-Thalassemia/genetics/therapy ; Fetal Hemoglobin/genetics/metabolism ; CRISPR-Cas Systems ; Promoter Regions, Genetic ; Repressor Proteins ; Erythroid Cells/metabolism ; Carrier Proteins/genetics/metabolism ; },
abstract = {Sickle cell disease and transfusion-dependent β-thalassemia can be treated by fetal hemoglobin upregulation. Disruption of the distal BCL11A binding site at the HBG1/2 promoters to induce fetal hemoglobin using either SpCas9 or AsCas12a mimics multiple hereditary persistence of fetal hemoglobin mutations. AsCas12a showed higher editing efficiency, higher specificity, and increased fetal hemoglobin induction potential compared with SpCas9. AsCas12a-edited healthy donor CD34[+] cells exhibited long-term, multi-lineage, and polyclonal engraftment in immunocompromised mice. High-level fetal hemoglobin induction was observed in erythroid progeny derived in vivo from edited healthy donor CD34[+] cells and sickle cell disease or transfusion-dependent β-thalassemia donor CD34[+] cells in vitro. In erythroid cells from patients with sickle cell disease, gene editing reduced sickling and improved rheological behaviors under deoxygenated conditions. In erythroid cells from patients with β-thalassemia, gene editing ameliorated ineffective erythropoiesis and significantly increased hemoglobin content per cell. A comprehensive off-target editing evaluation in edited CD34[+] cells showed AsCas12a to be highly specific, with no off-target editing detected. In summary, editing CD34[+] cells at the HBG1/2 promoter distal BCL11A binding site using AsCas12a phenocopied hereditary persistence of fetal hemoglobin mutations, demonstrating its potential as a gene editing approach for the treatment of β-hemoglobinopathies.},
}

*Anemia, Sickle Cell/genetics/therapy
Humans
*Gene Editing/methods
Animals
Mice
*beta-Thalassemia/genetics/therapy
Fetal Hemoglobin/genetics/metabolism
CRISPR-Cas Systems
Promoter Regions, Genetic
Repressor Proteins
Erythroid Cells/metabolism
Carrier Proteins/genetics/metabolism

@article {pmid40914806,
year = {2026},
author = {Liu, H and Singh, S and Mullen, TJ and Bullock, C and Keegan, S and Patterson, T and Thakur, S and Lundberg, A and Shenker, S and Couto, R and Yadav, C and Dastagir, S and Li, L and Bainter, W and Liberzon, E and Malloy, CR and Lazzarotto, CR and Ohsumi, TK and Chilakala, S and Chen, HM and Kshirsagar, R and Hohmann, AF and Arlauckas, SP and Lazorchak, A and Scull, C and Morgan, RA},
title = {A precision gene-engineered B cell medicine producing sustained levels of active factor IX for hemophilia B therapy.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {266-280},
doi = {10.1016/j.ymthe.2025.09.001},
pmid = {40914806},
issn = {1525-0024},
mesh = {*Hemophilia B/therapy/genetics ; *Factor IX/genetics/metabolism ; Animals ; *Genetic Therapy/methods ; Mice ; Humans ; *B-Lymphocytes/metabolism ; CRISPR-Cas Systems ; Gene Editing ; Dependovirus/genetics ; Receptors, CCR5/genetics ; Genetic Vectors/genetics ; Genetic Engineering ; Transgenes ; Disease Models, Animal ; },
abstract = {Hemophilia B gene therapy treatments have not addressed the need for predictable, durable, active, and redosable factor IX (FIX). Unlike conventional gene therapy, engineered B cell medicines (BCMs) are durable, redosable, and titratable and thus have the potential to address significant unmet needs in the hemophilia B treatment paradigm. BE-101 is an autologous BCM comprising expanded and differentiated B lymphocyte lineage cells genetically engineered ex vivo to secrete factor IX (FIX)-Padua. CRISPR-Cas9-mediated gene editing at the C-C chemokine receptor type 5 (CCR5) locus was used to facilitate transgene insertion of an adeno-associated virus 6-encoded DNA template via homology-directed repair. Transgene insertion did not alter B cell biology, viability, or differentiation into plasma cells. Appreciable levels of BE-101-derived FIX-Padua were detected within 1 day after IV administration in mice, and steady state was reached within 2 weeks and persisted for over 184 days. Redosing produced an increase in FIX-Padua production close to linear dose proportionality. Comprehensive genotoxicity analysis found no off-target issues of concern. No safety signals were observed in animal tolerability and Good Laboratory Practice toxicology studies. In conclusion, BE-101 produces sustained levels of active FIX-Padua with the ability to engraft without host preconditioning and with the potential for redosing and titratability.},
}

*Hemophilia B/therapy/genetics
*Factor IX/genetics/metabolism
Animals
*Genetic Therapy/methods
Mice
Humans
*B-Lymphocytes/metabolism
CRISPR-Cas Systems
Gene Editing
Dependovirus/genetics
Receptors, CCR5/genetics
Genetic Vectors/genetics
Genetic Engineering
Transgenes
Disease Models, Animal

@article {pmid40911442,
year = {2026},
author = {Tang, J and Yang, S and Li, S and Yue, X and Jin, T and Yang, X and Zhang, K and Yang, Q and Liu, T and Zhao, S and Gai, J and Li, Y},
title = {Editing a gibberellin receptor gene improves yield and nitrogen fixation in soybean.},
journal = {Journal of integrative plant biology},
volume = {68},
number = {1},
pages = {75-95},
doi = {10.1111/jipb.70026},
pmid = {40911442},
issn = {1744-7909},
support = {32372192//National Natural Science Foundation of China/ ; JBGS-2021-014//Core Technology Development for Breeding Program of Jiangsu Province/ ; BM2024005//Jiangsu Key Laboratory of Soybean Biotechnology and Intelligent Breeding/ ; },
mesh = {*Glycine max/genetics/metabolism/growth & development ; *Nitrogen Fixation/genetics ; *Gene Editing ; *Gibberellins/metabolism ; *Plant Proteins/genetics/metabolism ; CRISPR-Cas Systems/genetics ; Plants, Genetically Modified ; Gene Expression Regulation, Plant ; Seeds ; },
abstract = {Soybean is an important source of oil, protein, and feed. However, its yield is far below that of major cereal crops. The green revolution increased the yield of cereal crops partially through high-density planting of lodging-resistant semi-dwarf varieties, but required more nitrogen fertilizers, posing an environmental threat. Genes that can improve nitrogen use efficiency need to be integrated into semi-dwarf varieties to avoid the overuse of fertilizers without the loss of dwarfism. Unlike cereal crops, soybean can assimilate atmospheric nitrogen through symbiotic bacteria. Here, we created new alleles of GmGID1-2 (Glycine max GIBBERELLIN INSENSITIVE DWARF 1-2) using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) editing, which improved soybean architecture, yield, seed oil content, and nitrogen fixation, by regulation of important pathways and known genes related to branching, lipid metabolism, and nodule symbiosis. GmGID1-2 knockout reduced plant height, and increased stem diameter and strength, number of branches, nodes on the primary stem, pods, and seeds per plant, leading to an increase in seed weight per plant and yield in soybean. The nodule number, nodule weight, nitrogenase activity, and nitrogen content were also improved in GmGID1-2 knockout soybean lines, which is novel compared with the semi-dwarf genes in cereal crops. No loss-of-function allele for GmGID1-2 was identified in soybean germplasm and the edited GmGID1-2s are superior to the natural alleles, suggesting the GmGID1-2 knockout mutants generated in this study are valuable genetic resources to further improve soybean yield and seed oil content in future breeding programs. This study illustrates the pleiotropic functions of the GID1 knockout alleles with positive effects on plant architecture, yield, and nitrogen fixation in soybean, which provides a promising strategy toward sustainable agriculture.},
}

*Glycine max/genetics/metabolism/growth & development
*Nitrogen Fixation/genetics
*Gene Editing
*Gibberellins/metabolism
*Plant Proteins/genetics/metabolism
CRISPR-Cas Systems/genetics
Plants, Genetically Modified
Gene Expression Regulation, Plant
Seeds

@article {pmid40898613,
year = {2026},
author = {Yuan, L and Xiong, Y and Zhang, Y and Gu, S and Lei, Y},
title = {Epigenome editing based treatment: Progresses and challenges.},
journal = {Molecular therapy : the journal of the American Society of Gene Therapy},
volume = {34},
number = {1},
pages = {46-67},
doi = {10.1016/j.ymthe.2025.08.047},
pmid = {40898613},
issn = {1525-0024},
mesh = {Humans ; *Gene Editing/methods ; DNA Methylation ; *Epigenome ; *Epigenesis, Genetic ; *Genetic Therapy/methods ; CRISPR-Cas Systems ; Animals ; *Epigenomics/methods ; Gene Expression Regulation ; Epigenome Editing ; },
abstract = {Epigenome editing is emerging as a transformative approach in clinical treatment, enabling precise modifications to gene expression without altering the underlying DNA sequence. The ongoing transition of epigenome editing techniques from foundational research to clinical applications highlights several key strategies. These include targeted DNA methylation/demethylation, histone modification, and transcriptional regulation. These approaches offer the potential for durable and reversible gene expression modulation, paving the way for precisely tailored therapies for genetic and complex diseases. Here, we review pioneering research, technological advancements, granted patents, and clinical trials that have been reported during the past decade. By synthesizing current research and development efforts, this review aims to provide insights into the promising landscape of epigenome editing and its potential to promote therapeutic interventions.},
}

Humans
*Gene Editing/methods
DNA Methylation
*Epigenome
*Epigenesis, Genetic
*Genetic Therapy/methods
CRISPR-Cas Systems
Animals
*Epigenomics/methods
Gene Expression Regulation
Epigenome Editing

@article {pmid41494892,
year = {2026},
author = {Wang, MR and Mu, W and Zhen, A and Kitchen, SG},
title = {CRISPR/Cas strategies to enhance CAR T-cell function and persistence via metabolic reprogramming.},
journal = {Trends in biotechnology},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.tibtech.2025.12.001},
pmid = {41494892},
issn = {1879-3096},
abstract = {While chimeric antigen receptor (CAR) T-cell therapy has become a standard of care in various blood cancers, its full curative potential for other diseases has yet to be maximized. One key limiting factor is progressive T-cell exhaustion and differentiation over time, leading to the loss of the CAR-expressing cells. CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) gene manipulation to enhance CAR T-cell therapy has revolutionized the field in recent years. In this review, we will examine the application of CRISPR/Cas aimed at improving CAR T-cell function and persistence to combat the issues of exhaustion and dysfunction, with a focus on metabolic reprogramming. Understanding current preclinical CRISPR/Cas strategies for modulating CAR T-cell metabolism is critical in advancing CAR-T therapies to clinical applications.},
}

@article {pmid41494604,
year = {2026},
author = {Zeng, Y and Zhao, G and Wu, S and Hu, B and Forn-Cuní, G and Knol, R and El Ghalbzouri, A and Snaar-Jagalska, E and Kros, A},
title = {CD44-targeted lipid nanoparticles for enhanced CRISPR/Cas9 delivery in cancer gene editing.},
journal = {Journal of controlled release : official journal of the Controlled Release Society},
volume = {391},
number = {},
pages = {114598},
doi = {10.1016/j.jconrel.2025.114598},
pmid = {41494604},
issn = {1873-4995},
abstract = {Skin cancer is the third most common malignancy, with melanoma being the most challenging due to its resistance to current therapies. Gene editing technologies like CRISPR/Cas9 offer a promising strategy for targeting cancer-specific genes, but the efficient delivery of these tools to tumor sites remains a significant challenge. Lipid nanoparticles (LNPs) have emerged as the leading platform for gene editing tools due to their ability to protect and transport large payloads. To enhance the precision of gene editing in melanoma, we developed CD44-specific peptide-modified LNPs for targeted delivery of CRISPR/Cas9 mRNA and guide RNA against polo-like kinase 1 (sgPLK1). Our approach led to enhanced targeting and gene editing efficacy by specifically delivering CRISPR/Cas9 and sgPLK1 to melanoma tumor cells, resulting in significant inhibition of tumor growth in both in vitro and in vivo skin melanoma models. Moreover, this platform showed the capacity to reach metastatic melanoma in the brain and resulting in substantial suppression of tumor growth in brain metastasis models. We envision that this peptide-modification strategy could be further employed to improve the targeting capabilities and therapeutic outcomes of LNPs for CRISPR/Cas9-based gene editing, paving the way for more precise and effective cancer treatments.},
}

@article {pmid41492065,
year = {2026},
author = {Wang, Z and Wang, Y and Gao, H and Dai, J and Tang, N and Wang, Y and Ji, Q},
title = {Phage-associated Cas12p nucleases require binding to bacterial thioredoxin for activation and cleavage of target DNA.},
journal = {Nature microbiology},
volume = {11},
number = {1},
pages = {81-93},
pmid = {41492065},
issn = {2058-5276},
mesh = {*Thioredoxins/metabolism/genetics ; *CRISPR-Cas Systems ; *Bacteriophages/genetics/enzymology/metabolism ; *CRISPR-Associated Proteins/metabolism/genetics/chemistry ; Cryoelectron Microscopy ; Protein Binding ; *Endodeoxyribonucleases/metabolism/genetics ; *Bacterial Proteins/metabolism/genetics ; DNA Cleavage ; DNA/metabolism ; Escherichia coli/genetics/virology/metabolism ; },
abstract = {The evolutionary competition within phage-host systems led to the emergence of CRISPR-Cas defence mechanisms in bacteria and anti-CRISPR elements in bacteriophages. Although anti-CRISPR elements are well characterized, the role of bacterial factors that influence CRISPR-Cas efficacy has been comparatively overlooked. Type V CRISPR-Cas12 systems display striking functional and mechanistic diversity for nucleic acid targeting. Here we use a bioinformatic approach to identify Cas12p, a phage-associated nuclease that forms complexes with the bacterial thioredoxin protein TrxA to enable target DNA degradation. This represents an unexpected phage-bacteria interaction, in which the bacteriophage co-opts a bacterial factor to augment its own genome degradation machinery, potentially against competing phages. Biochemical characterization, cryo-EM-based structural analysis of the Cas12p-TrxA-sgRNA-dsDNA complex at 2.67 Å and bacterial defence assays reveal that TrxA directly binds and activates Cas12p, enabling its nuclease activity and subsequent CRISPR immunity. These findings expand our understanding of the multilayered intricacies of phage-bacteria molecular interactions.},
}

*Thioredoxins/metabolism/genetics
*CRISPR-Cas Systems
*Bacteriophages/genetics/enzymology/metabolism
*CRISPR-Associated Proteins/metabolism/genetics/chemistry
Cryoelectron Microscopy
Protein Binding
*Endodeoxyribonucleases/metabolism/genetics
*Bacterial Proteins/metabolism/genetics
DNA Cleavage
DNA/metabolism
Escherichia coli/genetics/virology/metabolism

@article {pmid41489362,
year = {2026},
author = {Shin, J and Barrangou, R},
title = {Occurrence and applications of CRISPR-Cas systems in bifidobacteria.},
journal = {Applied and environmental microbiology},
volume = {},
number = {},
pages = {e0170325},
doi = {10.1128/aem.01703-25},
pmid = {41489362},
issn = {1098-5336},
abstract = {Bifidobacterium is a key member of the human gut microbiota, and many strains are widely used as probiotics due to their health-promoting properties. Despite growing interest, genetic studies in Bifidobacterium have been relatively limited, primarily due to the lack of available genome editing tools. Recent advances in genomics and CRISPR-Cas systems provide opportunities for targeted genome modification in this genus. In this review, we provide an overview of the occurrence, diversity, and distribution of CRISPR-Cas systems across Bifidobacterium species and examine the editing tools developed and implemented to date. We also highlight practical challenges such as strain variability and low transformation efficiency and introduce future avenues of research such as large-payload insertion and in situ editing. Expanding the genetic toolbox for Bifidobacterium will broaden our understanding of this important genus and enable the development of next-generation probiotics.},
}

@article {pmid41488985,
year = {2026},
author = {Kim, K and Lee, J and Lee, N and Cho, BK},
title = {CRISPR-Based Approaches to Engineer Nonmodel Bacteria for Bioproduction and Biotherapeutics.},
journal = {Biochemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.biochem.5c00613},
pmid = {41488985},
issn = {1520-4995},
abstract = {Microbial diversity encompasses vast genetic and functional capacities, with immense potential for biotechnological applications. Yet, most biotechnological advances have been confined to a narrow set of model organisms, leaving the broader repertoire of nonmodel microbes largely untapped due to species-specific barriers that hinder genetic manipulation. Over the past decade, the advent of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) systems has transformed microbial engineering by enabling precise, programmable, and scalable control of genomes and gene expression. Importantly, the relative independence of many CRISPR effectors from host cofactors has facilitated their use in microbes previously challenging to engineer, thus expanding opportunities to exploit their unique metabolic and biosynthetic traits. In this review, we summarize the major CRISPR-Cas toolkits and highlight recent innovations, with particular emphasis on translational applications in nonmodel organisms such as C1-gas-fixing acetogens, antibiotic-producing Streptomyces, and gut commensal Bacteroides. We emphasize three areas of emerging impact: engineering microbial cell factories for sustainable biomanufacturing, accelerating natural product discovery, and development of next-generation live biotherapeutics. Finally, we discuss current limitations and future opportunities, underscoring how the integration of genome editing, synthetic biology, and systems-level approaches is reshaping the landscape of microbial biotechnology.},
}

@article {pmid41488303,
year = {2025},
author = {Tsolakidou, PJ},
title = {CRISPR-Cas systems against carbapenem resistance: from proof-of-concept to clinical translation.},
journal = {Frontiers in microbiology},
volume = {16},
number = {},
pages = {1725247},
pmid = {41488303},
issn = {1664-302X},
abstract = {Carbapenem-resistant Enterobacterales (CRE) pose a major global threat, driven by plasmid-borne carbapenemase genes such as bla KPC, bla NDM and bla OXA-48. CRISPR-Cas systems offer programmable strategies to selectively eliminate these resistance determinants. This mini-review summarizes recent advances in Cas9-based plasmid curing, RNA-targeting approaches such as Cas13a and Cas13d, and DNA-targeting Cas3-enhanced bacteriophage therapeutics that have entered early clinical evaluation. Particular attention is given to conjugative CRISPR-Cas9 plasmid systems, which enable targeted plasmid eradication without laboratory transformation and broaden the delivery toolbox beyond phage vectors. We further discuss major translational challenges, including delivery efficiency, phage host-range constraints, ecological risks of horizontal CRISPR dissemination, and off-target effects. Finally, we highlight emerging delivery platforms-outer membrane vesicles, lipid and polymeric nanoparticles, conjugative plasmids with containment circuits, and engineered live biotherapeutics-that may complement or overcome current limitations. Collectively, these developments illustrate the potential of CRISPR-based antimicrobials to augment traditional therapies through precise gene-level suppression of carbapenem resistance.},
}

@article {pmid41487293,
year = {2025},
author = {},
title = {Erratum: Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing [Corrigendum].},
journal = {Biologics : targets & therapy},
volume = {19},
number = {},
pages = {745-746},
doi = {10.2147/BTT.S585961},
pmid = {41487293},
issn = {1177-5475},
abstract = {[This corrects the article DOI: 10.2147/BTT.S326422.].},
}

@article {pmid41486484,
year = {2025},
author = {Boogari, M and Mohebbi, M and Hadidi, N},
title = {Genetically Engineered Probiotics: Design, Therapeutics, and Clinical Translation.},
journal = {Iranian biomedical journal},
volume = {29},
number = {6},
pages = {374-383},
doi = {10.61882/ibj.5197},
pmid = {41486484},
issn = {2008-823X},
abstract = {Genetically engineered probiotics aim to address transient colonization and the intra- and inter-subject variability that limit conventional probiotics. These strains utilizes CRISPR/Cas editing, programmable gene circuits, and biosensors in chassis such as E. coli Nissle 1917 and L. lactis. This narrative review summarizes the current engineering toolkits and standards (e.g., SEVA), chassis selection criteria, biocontainment strategies, and translational requirements under CMC/GMP frameworks and discusses regulatory considerations for clinical translation. Representative examples include IL-10-secreting L. lactis and phenylalanine-metabolizing strains for PKU (SYNB1618/SYNB1934), which illustrate pharmacodynamic target engagement and short-term preclinical safety. We outline clinical advancements in predefined pharmacodynamics, durability of function, monitoring shedding and HGT, and genomic-microbiome-informed patient stratification. Systems modeling approaches (GEM/ABM) are discussed as tools to guide rational design. GEPs offer programmable "sense-and-respond" therapeutics, with successful clinical adoption depending on durable efficacy, long-term safety, and clearly defined regulatory pathways.},
}

@article {pmid41484149,
year = {2026},
author = {Kremer, N and Mueller, F and Nguyen, H and Schulz, L and Popp, T and Artes, E and Wolters, J and Renner, M and Vetter, I and Maffini, S and Robles, MS and Musacchio, A and Bange, T},
title = {CUL4A-DDB1-DCAF10 is an N-recognin for N-terminally acetylated Src kinases.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {132},
pmid = {41484149},
issn = {2041-1723},
support = {5041 140321//Deutsche Forschungsgemeinschaft (German Research Foundation)/ ; },
mesh = {*Cullin Proteins/metabolism/genetics ; Humans ; Acetylation ; *src-Family Kinases/metabolism/genetics ; HEK293 Cells ; *DNA-Binding Proteins/metabolism/genetics ; Proteolysis ; Protein Processing, Post-Translational ; Ubiquitination ; Ubiquitin-Protein Ligases/metabolism ; CRISPR-Cas Systems ; },
abstract = {Co-translational N-terminal modifications such as methionine excision, acetylation, and myristoylation govern protein stability, localization, and folding. Disruption can expose N-terminal degrons that trigger ubiquitin-mediated degradation, safeguarding the proteome. N-terminal acetylation usually protects proteins from degradation, but can also promote it through the Ac/N-degron pathway. Src-family kinases (SFKs), signaling enzymes implicated in tumorigenesis, require N-terminal myristoylation for function. Using peptide pull-downs, mass spectrometry, and AlphaFold 3 predictions, we identify DCAF10 as the E3 ligase substrate receptor for alternatively N-terminally acetylated SFKs. Combining siRNA-mediated knockdown and CRISPR/Cas9-mediated knockout of endogenous Lyn with inducible Lyn-GFP variants confirms that DCAF10 regulates SFK levels by recognizing an N-terminal acetylated glycine residue. In vitro, a CUL4A-DDB1-DCAF10 complex ubiquitinates N-terminally acetylated SFKs. Thus, we define a novel N-degron pathway that monitors replacement of myristoylation by acetylation and activates degradation of SFKs upon acetylation. This mechanism may extend to other N-terminally myristoylated proteins beyond SFKs.},
}

*Cullin Proteins/metabolism/genetics
Humans
Acetylation
*src-Family Kinases/metabolism/genetics
HEK293 Cells
*DNA-Binding Proteins/metabolism/genetics
Proteolysis
Protein Processing, Post-Translational
Ubiquitination
Ubiquitin-Protein Ligases/metabolism
CRISPR-Cas Systems

@article {pmid41481841,
year = {2026},
author = {Jin, YM and Li, XD and Zhu, JK and Shao, CY and Huang, BB and Huang, HL and Wang, XW and Jiang, HB and Chen, W},
title = {Programmable adenine base editing in cyanobacteria using an engineered TadA-Cas9 fusion.},
journal = {The Plant journal : for cell and molecular biology},
volume = {125},
number = {1},
pages = {e70655},
doi = {10.1111/tpj.70655},
pmid = {41481841},
issn = {1365-313X},
support = {32470092//National Natural Science Foundation of China/ ; 32170108//National Natural Science Foundation of China/ ; 2024QL060//Ningbo Youth Leading Talent Project/ ; //Ningbo University Startup Funding/ ; },
mesh = {*Gene Editing/methods ; *CRISPR-Cas Systems/genetics ; *Adenine/metabolism ; *Synechocystis/genetics ; *Cyanobacteria/genetics ; Bacterial Proteins/genetics/metabolism ; *CRISPR-Associated Protein 9/genetics/metabolism ; *Anabaena/genetics ; },
abstract = {Cyanobacteria are photosynthetic prokaryotes with great potential in green biomanufacturing and basic research. Despite decades of pioneering achievements, the application of advanced genome editing tools, particularly CRISPR-based systems, has remained limited in cyanobacteria. In this study, we developed pCyABE, a new adenine base editor for efficient and precise A·T to G·C editing in cyanobacteria. This system utilizes a TadA-Cas9 nickase fusion and functions without double-strand breaks or donor templates. We demonstrated its high editing efficiency in Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120, highlighting its broad usability. pCyABE supports multiplex editing and enables start codon disruption for gene functional studies. Furthermore, this tool exhibits low off-target activity and can be effectively removed via sucrose counterselection. In conclusion, pCyABE provides a versatile and efficient genome editing platform that significantly expands the genetic toolbox for cyanobacterial research and biotechnology applications.},
}

*Gene Editing/methods
*CRISPR-Cas Systems/genetics
*Adenine/metabolism
*Synechocystis/genetics
*Cyanobacteria/genetics
Bacterial Proteins/genetics/metabolism
*CRISPR-Associated Protein 9/genetics/metabolism
*Anabaena/genetics

@article {pmid41481737,
year = {2026},
author = {Qi, S and Wei, L and Ding, Z and Zhong, F and Yang, S and Wu, L and Yang, X and Kang, B and Dan, M and Gan, J and Li, C and Su, X},
title = {Targeting transthyretin by one Cas9 variant with superfidelity and broad compatibility.},
journal = {Science advances},
volume = {12},
number = {1},
pages = {eadu6505},
pmid = {41481737},
issn = {2375-2548},
mesh = {*Prealbumin/genetics/metabolism ; Humans ; *Gene Editing/methods ; *CRISPR-Cas Systems ; *CRISPR-Associated Protein 9/genetics/metabolism/chemistry ; Amyloid Neuropathies, Familial/genetics/therapy ; HEK293 Cells ; },
abstract = {Amyloid transthyretin (ATTR) amyloidosis is a fatal disease caused by the accumulation of misfolded transthyretin proteins. Although knocking down the TTR gene by CRISPR-Cas9 represents a promising strategy for treating ATTR amyloidosis, its efficiency and safety remain to be further investigated. Here, we report a systematic investigation of SpCas9-based TTR editing. Besides the target site, wild-type SpCas9 and the reported variants induced extensive off-target edits. To improve the fidelity, we performed structural analysis and designed a series of SpCas9 variants. Studies demonstrated that SpCas9-Mut5 is an ultrahigh-fidelity variant, which induces extremely low levels of off-target edits and translocations without substantial impairment of on-target editing activity. SpCas9-Mut5 is compatible with the adenine base editor (ABE) system, markedly reducing off-target edits and narrowing the editing window. In conclusion, our study suggests that SpCas9-Mut5 is an excellent candidate for TTR gene editing. Besides ATTR amyloidosis, SpCas9-Mut5 and its derivative ABE could be widely used in the treatment of other diseases.},
}

*Prealbumin/genetics/metabolism
Humans
*Gene Editing/methods
*CRISPR-Cas Systems
*CRISPR-Associated Protein 9/genetics/metabolism/chemistry
Amyloid Neuropathies, Familial/genetics/therapy
HEK293 Cells

@article {pmid41481232,
year = {2026},
author = {Li, Y and Zhang, W and Wei, Z and Li, H and Liu, X and Zheng, T and Aziz, T and Xing, C and Meng, A and Wu, X},
title = {Stage- and tissue-specific gene editing using 4-OHT-inducible Cas9 in whole organism.},
journal = {The Journal of cell biology},
volume = {225},
number = {4},
pages = {},
pmid = {41481232},
issn = {1540-8140},
support = {#3258820001//National Natural Science Foundation of China/ ; #2023YFA1800300//National Key Research and Development Program of China/ ; 2018YFC1003304//National Key Research and Development Program of China/ ; #202302AO370011//Southwest United Graduate School/ ; },
mesh = {Animals ; *Gene Editing/methods ; *Zebrafish/genetics/embryology ; *Tamoxifen/analogs & derivatives/pharmacology ; *CRISPR-Cas Systems/genetics ; Mice ; Female ; *CRISPR-Associated Protein 9/genetics/metabolism ; Germ Cells/metabolism ; Organ Specificity ; Humans ; Receptors, Estrogen/genetics/metabolism ; Animals, Genetically Modified ; },
abstract = {Vertebrate genes function in specific tissues and stages, so their functional studies require conditional knockout or editing. In zebrafish, spatiotemporally inducible genome editing, particularly during early embryogenesis, remains challenging. Here, we establish inducible Cas9-based editing in defined cell types and stages. The nCas9ERT2 fusion protein, consisting of Cas9 and an estrogen receptor flanked by two nuclear localization signals, is usually located in the cytoplasm and efficiently translocated into nuclei upon 4-hydroxytamoxifen (4-OHT) treatment in cultured cells or embryos. As a proof of concept, we demonstrate that genes in primordial germ cells in embryos and germ cells in adult ovaries from a transgenic line with stable expression of nCas9ERT2 and gRNAs can be mutated by 4-OHT induction. The system also works in early mouse embryos. Thus, this inducible nCas9ERT2 approach enables temporospatial gene editing at the organismal level, expanding the tissue- and stage-specific gene-editing toolkit.},
}

Animals
*Gene Editing/methods
*Zebrafish/genetics/embryology
*Tamoxifen/analogs & derivatives/pharmacology
*CRISPR-Cas Systems/genetics
Mice
Female
*CRISPR-Associated Protein 9/genetics/metabolism
Germ Cells/metabolism
Organ Specificity
Humans
Receptors, Estrogen/genetics/metabolism
Animals, Genetically Modified

@article {pmid41479566,
year = {2025},
author = {Antipenko, ID and Venedyukhina, SA and Sorokina, NP and Kucherenko, IV and Smirnova, TS and Rogov, GN and Shkurnikov, MY},
title = {Whole-Genome Sequencing Uncovers Metabolic and Immune System Variations in Propionibacterium freudenreichii Isolates.},
journal = {Acta naturae},
volume = {17},
number = {4},
pages = {72-82},
pmid = {41479566},
issn = {2075-8251},
abstract = {Propionibacterium freudenreichii plays a crucial role in the production of Swiss-type cheeses; however, genomic variability among strains, which affects their technological traits, remains insufficiently explored. In this study, whole-genome sequencing and comparative analysis were performed on five industrial P. freudenreichii strains. Despite their overall high genomic similarity, the strains proved different in gas production and substrate metabolism. Phylogenetic analysis revealed a close relationship between strain FNCPS 828 and P. freudenreichii subsp. shermanii (z-score = 0.99948), with the latter being unable to reduce nitrates but being able to metabolize lactose. The narG gene encoding the nitrate reductase alpha subunit was detected in only one of the five analyzed strains - FNCPS 828 - and in 39% of previously described P. freudenreichii genomes, suggesting its potential as a marker of nitrate-reducing capability. Analysis of 112 genomes showed that the I-G CRISPR-Cas system was present in more than 90% of the strains, whereas the type I-E system was found in approximately 25%. All the five study strains harbored the type I-G system; strain FNCPS 3 additionally contained a complete type I-E system with the highest number of CRISPR spacers, some of which matched previously published bacteriophage sequences. The most prevalent anti-phage defense systems included RM I, RM IV, AbiE, PD-T4-6, HEC-06, and ietAS. These findings highlight the genetic diversity of P. freudenreichii strains, which is of great importance in their industrial applications. The identification of narG as a potential marker of nitrate-reducing activity, along with detailed mapping of CRISPR- Cas systems, boosts opportunities for the rational selection and engineering of starter cultures with tailored metabolic properties and increased resistance to bacteriophages.},
}

@article {pmid41479062,
year = {2026},
author = {Lee, NJ and Matsuoka, RL},
title = {CRISPR/Cas9-Based Mutagenesis Strategies for Efficient Biallelic Gene Inactivation and Consistent Phenotypic Detection in F0 Zebrafish.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2989},
number = {},
pages = {319-332},
pmid = {41479062},
issn = {1940-6029},
mesh = {Animals ; *Zebrafish/genetics ; *CRISPR-Cas Systems/genetics ; Phenotype ; *Mutagenesis ; Alleles ; *Gene Silencing ; Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; },
abstract = {The zebrafish is a valuable animal model for investigating the genetic basis of vertebrate evolution, development, behavior, and regeneration. However, the existence of numerous gene paralogs in the zebrafish genome represents a major challenge, complicating functional genomic research using reverse-genetics approaches. To facilitate reverse genetics-based phenotypic screens, we recently presented simple methods that enable efficient induction of biallelic gene disruptions in F0 zebrafish, providing a rapid avenue for screening potential gene functions through consistent phenotypic detection. Here, we describe detailed protocols for these CRISPR/Cas9-based mutagenesis strategies to achieve highly effective biallelic gene inactivation in F0 zebrafish. The high consistency of these strategies, combined with a streamlined workflow, offers a robust phenotypic screening platform for a quick and reliable functional assessment of genes of interest, both individually and in a scalable manner. These strategies enhance the efficacy of successful F0 zebrafish phenotypic screening, thereby accelerating functional genetic studies using this powerful model organism.},
}

Animals
*Zebrafish/genetics
*CRISPR-Cas Systems/genetics
Phenotype
*Mutagenesis
Alleles
*Gene Silencing
Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics

@article {pmid41389602,
year = {2026},
author = {Kumari, P and Gupta, V and Chhikara, A and Dalal, J},
title = {Revolutionizing forensic DNA analysis: The potential of CRISPR-Cas9 technology in genetic investigations.},
journal = {Journal of forensic and legal medicine},
volume = {117},
number = {},
pages = {103047},
doi = {10.1016/j.jflm.2025.103047},
pmid = {41389602},
issn = {1878-7487},
mesh = {Humans ; *CRISPR-Cas Systems ; *DNA Fingerprinting/methods ; *Forensic Genetics/methods ; *Gene Editing ; },
abstract = {The newest achievements in the field of molecular biology and gene-editing technologies have transformed the paradigm of forensic DNA analysis. However, there are still great difficulties in interpreting degraded, low-template, mixed genetic samples. The review critically evaluates the transformative potential of Clustered Regularly Interspaced Short Palindromic Repeats and an associated protein 9 (CRISPR-Cas9) as an accurate, effective, and cost-efficient system of genome-editing in the field of forensic science. Based on the evidence of the current literature, the paper critically analyzes the mechanisms of CRISPR-Cas9 activity, its RNA-guided specificity, dual-strand cleavage, and high-fidelity targeting, and compares its functionality with other standard methods like the STR and SNP profiling. The review also discusses more complex CRISPR-based diagnostic systems, such as SHERLOCK, DETECTR, and HOLMES that allow the analysis of DNA rapidly, without amplification, and in a portable format. Among major discoveries, there is the ability of CRISPR to increase the accuracy of DNA profiling, resolve mixture, recapitulate damaged genetic material, and reduce the possibility of contamination. In addition to genetic analysis, it has applications in forensic epigenetics, prediction of phenotypes, microbial forensics and environmental trace analysis. The review also covers the ethical, legal and governance implications of implementing CRISPR-based evidence in the judicial process especially in as far as data privacy; admissibility and equity of access are concerned. In general, CRISPR-Cas9 is a paradigm shift in forensic genomics, the one that has the potential to transform personal identification, reconstruction of the crime scene, and the interpretation of molecular evidence. Future efforts should focus on method validation, standardization, and ethical governance to ensure the responsible and sustainable implementation of this technology in forensic practice.},
}

Humans
*CRISPR-Cas Systems
*DNA Fingerprinting/methods
*Forensic Genetics/methods
*Gene Editing

@article {pmid41372414,
year = {2026},
author = {Habtewold, T and Lwetoijera, DW and Hoermann, A and Mashauri, R and Matwewe, F and Mwanga, R and Kweyamba, P and Maganga, G and Magani, BP and Mtama, R and Mahonje, MA and Tambwe, MM and Tarimo, F and Chennuri, PR and Cai, JA and Del Corsano, G and Capriotti, P and Sasse, P and Moore, J and Hudson, D and Manjurano, A and Tarimo, B and Vlachou, D and Moore, S and Windbichler, N and Christophides, GK},
title = {Gene-drive-capable mosquitoes suppress patient-derived malaria in Tanzania.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {442-448},
pmid = {41372414},
issn = {1476-4687},
mesh = {Tanzania/epidemiology ; Animals ; *Plasmodium falciparum/isolation & purification/genetics/drug effects/growth & development ; *Anopheles/genetics/parasitology ; Humans ; *Malaria, Falciparum/prevention & control/parasitology/transmission ; *Gene Drive Technology/methods ; *Mosquito Vectors/genetics/parasitology ; Female ; Animals, Genetically Modified/genetics ; Male ; Child ; CRISPR-Cas Systems/genetics ; },
abstract = {Gene drive technology presents a transformative approach to combatting malaria by introducing genetic modifications into wild mosquito populations to reduce their vectorial capacity. Although effective modifications have been developed, these efforts have been confined to laboratories in the global north. We previously demonstrated that modifying Anopheles gambiae to express two exogenous antimicrobial peptides inhibits the sporogonic development of laboratory-cultured Plasmodium falciparum, with models predicting substantial contributions to malaria elimination in Africa when integrated with gene drive[1-3]. However, the effectiveness of this modification against genetically diverse, naturally circulating parasite isolates remained unknown. To address this critical gap, we adapted our technology for an African context by establishing infrastructural and research capacity in Tanzania, enabling the engineering of local A. gambiae under containment. Here we report the generation of a transgenic strain equipped with non-autonomous gene drive capabilities that robustly inhibits genetically diverse P. falciparum isolates obtained from naturally infected children. These genetic modifications were efficiently inherited by progeny when supplemented with Cas9 endonuclease provided by another locally engineered strain. Our work brings gene drive technology a critical step closer to application, providing a locally tailored and powerful tool for malaria eradication through the targeted dissemination of beneficial genetic traits in wild mosquito populations.},
}

Tanzania/epidemiology
Animals
*Plasmodium falciparum/isolation & purification/genetics/drug effects/growth & development
*Anopheles/genetics/parasitology
Humans
*Malaria, Falciparum/prevention & control/parasitology/transmission
*Gene Drive Technology/methods
*Mosquito Vectors/genetics/parasitology
Female
Animals, Genetically Modified/genetics
Male
Child
CRISPR-Cas Systems/genetics

@article {pmid41313207,
year = {2026},
author = {Wang, Y and Qin, Z and Wang, Q and Yang, Y and Gu, C and Yu, F and Wu, Y and Zhang, Lx},
title = {An RPA-CRISPR/Cas12a-based rapid and sensitive nucleic acid method for detection of Toxoplasma gondii in tissue and blood samples.},
journal = {Microbiology spectrum},
volume = {14},
number = {1},
pages = {e0155025},
pmid = {41313207},
issn = {2165-0497},
support = {231111111500//Key Research and Development Special Project of Henan Province of China/ ; 2022YFD1800200, 2023YFD1801200//National Basic Research Program of China/ ; },
mesh = {*Toxoplasma/genetics/isolation & purification ; Animals ; *CRISPR-Cas Systems ; Humans ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; DNA, Protozoan/genetics ; *Toxoplasmosis/diagnosis/parasitology/blood ; Limit of Detection ; *Toxoplasmosis, Animal/diagnosis/parasitology ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Toxoplasma gondii is a zoonotic pathogen that can infect humans and a wide range of warm-blooded animals, posing a significant threat to human health and the livestock industry. The development of a time-saving, highly sensitive, and specific method for the detection of T. gondii in tissue and blood samples is crucial to the monitoring, prevention, and control of toxoplasmosis. In this study, we evaluated the efficiency of a previously described method, termed REPORT, that integrates recombinase polymerase amplification with CRISPR/Cas12a for the detection of T. gondii nucleic acids. We evaluated the limit of detection (LOD) and specificity of the extended REPORT method using prepared target DNA in addition to tissue and blood samples. Furthermore, we validated the accuracy of T. gondii detection in clinical samples using the REPORT-based method in comparison with nested PCR based on the B1 gene. Sensitivity tests showed that the LOD of the REPORT-based fluorescence method and the lateral flow strip method were 3.7 copies /μL for target DNA, 3.1 tachyzoites/g for tissue samples, and five tachyzoites/mL for blood samples. Specificity tests suggested that the REPORT method had good specificity and did not cross-react with several common parasites. The method performed well for clinical DNA samples, demonstrating its ability for use in on-site detection.IMPORTANCEToxoplasma gondii can infect over 200 species of warm-blooded animals, including humans, posing not only a significant threat to public health systems but also causing substantial economic losses to the global livestock industry. Current diagnostic methods are slow, equipment-dependent, and impractical for field use. This study addresses these limitations by developing REPORT, a rapid, ultrasensitive nucleic acid test combining recombinase polymerase amplification and CRISPR/Cas12a. The REPORT detects T. gondii in tissue and blood samples within 1 h at low cost, requiring only a portable heater. Its visual results (fluorescence or test strips) enable on-site use without specialized training, achieving 100% accuracy versus nested PCR. With a sensitivity of 3.1 parasites per gram of tissue and five parasites per milliliter of blood, this method revolutionizes toxoplasmosis screening in resource-limited clinics, farms, and food safety inspections, empowering timely interventions to curb transmission and improve public health outcomes.},
}

*Toxoplasma/genetics/isolation & purification
Animals
*CRISPR-Cas Systems
Humans
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
DNA, Protozoan/genetics
*Toxoplasmosis/diagnosis/parasitology/blood
Limit of Detection
*Toxoplasmosis, Animal/diagnosis/parasitology
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41225007,
year = {2026},
author = {Noh, H and Hashem, Z and Boms, E and Najafov, A},
title = {SIGLEC12 mediates plasma membrane rupture during necroptotic cell death.},
journal = {Nature},
volume = {649},
number = {8096},
pages = {460-466},
pmid = {41225007},
issn = {1476-4687},
mesh = {*Necroptosis/genetics ; Humans ; Animals ; *Cell Membrane/metabolism/pathology ; Mice ; Protein Kinases/metabolism ; Phosphorylation ; CRISPR-Cas Systems ; HEK293 Cells ; },
abstract = {Necroptosis is a form of lytic cell death that is overactivated during infections and in inflammatory pathologies[1]. NINJ1 was recently found to be a mediator of plasma membrane rupture (PMR) during pyroptosis, toxin-induced necrosis, apoptosis, and ferroptosis[2,3], but the mediator of PMR during necroptotic cell death remained unknown. Here, using a CRISPR-Cas9-based genome-wide knockout approach, we identify SIGLEC12 as a key mediator of necroptosis downstream of MLKL at the PMR step. Cells with knockdown or knockout of SIGLEC12 are defective in necroptosis-induced PMR and demonstrate ballooning morphology. During necroptosis, SIGLEC12 undergoes dephosphorylation, interacts with MLKL, forms cytosolic puncta and assembles into fibrils. Notably, SIGLEC12 is cleaved by TMPRSS4 during necroptosis to produce a 20-kDa fragment highly homologous to NINJ1, and this cleavage event is required and sufficient to induce PMR during necroptosis. A SIGLEC12 variant associated with cancer (Ser458Phe) and a variant found in the general human population (Arg528Trp) attenuate SIGLEC12 cleavage by TMPRSS4. Knockout of Siglec12 in mouse cells does not affect PMR, suggesting a species-specific role. Our identification of SIGLEC12 as a mediator of PMR expands our understanding of how programmed necrosis is executed and offers new approaches for targeting this proinflammatory form of cell death in human diseases.},
}

*Necroptosis/genetics
Humans
Animals
*Cell Membrane/metabolism/pathology
Mice
Protein Kinases/metabolism
Phosphorylation
CRISPR-Cas Systems
HEK293 Cells

@article {pmid40673390,
year = {2026},
author = {Guerra, F and De Rouck, S and Verhulst, EC},
title = {SYNCAS-mediated CRISPR-Cas9 genome editing in the Jewel wasp, Nasonia vitripennis.},
journal = {Insect molecular biology},
volume = {35},
number = {1},
pages = {48-55},
pmid = {40673390},
issn = {1365-2583},
support = {OCENW.M.22.140//Dutch Research Council (NWO)/ ; G035420N//Research Foundation Flanders (FWO)/ ; 101123162/ERC_/European Research Council/International ; },
mesh = {Animals ; *Wasps/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; Female ; Gene Knockout Techniques ; Insect Proteins/genetics ; },
abstract = {Genetic engineering is a formidable approach to studying biology. The development of CRISPR-Cas9 has allowed the genetic engineering of insect species from several orders, and in some species, this tool is used routinely for genetic research. However, insect gene editing often relies on the delivery of CRISPR-Cas9 components via embryo injection. This technique has a limitation: some species lay their eggs inside hard substrates or living hosts, making embryo collection impossible or labour intensive. Recently, a variety of techniques that exploit maternal injection of nucleases have been developed to circumvent embryo injection. Yet, despite this variety of maternal delivery techniques, some insects remain refractory to gene editing. One of these is the parasitoid wasp, Nasonia vitripennis, an important hymenopteran model species. In this study, a recently developed method termed SYNCAS was used to perform knock-out (KO) of the cinnabar gene in this wasp, obtaining KO efficiencies up to 10 times higher than reported for other maternal injection approaches. We found up to 2.73% of all offspring to display a KO phenotype, and we obtained up to 68 KO offspring per 100 injected mothers. The optimal timing of injection and provision of hosts for egg laying was determined. With this protocol, routine applications of CRISPR-Cas9 become feasible in this species, allowing reverse genetics studies of genes with unknown associated phenotypes and paving the way for more advanced editing techniques.},
}

Animals
*Wasps/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
Female
Gene Knockout Techniques
Insect Proteins/genetics

@article {pmid39901548,
year = {2025},
author = {Yang, F and Wang, H and Fan, S and Qiu, H and Li, X and Shi, G and Li, Z and Luan, X and Wu, H},
title = {Advances in Synthetic Lethality in Potential Oncology Therapeutic Approaches.},
journal = {Current topics in medicinal chemistry},
volume = {25},
number = {15},
pages = {1868-1881},
pmid = {39901548},
issn = {1873-4294},
support = {ZR2022MB140//Natural Science Foundation of Shandong Province/ ; ZYQR201810168//National funding of postdoctoral innovative talents support program of Henan Province/ ; },
mesh = {Humans ; *Neoplasms/genetics/drug therapy/pathology ; *Antineoplastic Agents/pharmacology/chemistry/therapeutic use ; *Synthetic Lethal Mutations/drug effects ; Molecular Targeted Therapy ; CRISPR-Cas Systems ; },
abstract = {Synthetic lethality represents a novel paradigm in molecular targeted cancer therapy. In synthetic lethality, perturbation of one gene alone does not hinder cell viability, yet simultaneous perturbation of both genes results in a loss of cellular viability. The presence of gene mutations in cancer cells, as opposed to normal cells, provides an opportunity for targeted therapies that mimic the effects of the second genetic mutation, enabling selective eradication of cancer cells. Recent advances in high-throughput screening technologies, such as CRISPR-Cas9 and RNA interference, have significantly enhanced the identification of synthetic lethal interactions, expanding the potential targets for therapeutic intervention. Challenges in exploiting synthetic lethality for cancer treatment include the complexities of tumor biology, limited comprehension of synthetic lethal interactions, drug resistance, and impediments in screening and clinical translation. Emerging strategies, such as combination therapies and novel drug designs, are being developed to overcome these obstacles. By virtue of its selective lethality towards cancer cells bearing specific genetic alterations, targeting synthetic lethal genes holds the promise to provide wider therapeutic windows compared to traditional cytotoxic chemotherapy. This review describes the current state of synthetic lethality applications in cancer treatment, encompassing both biological and methodological perspectives. It highlights the latest advancements in synthetic lethality with emerging interventional strategies. Furthermore, it explores future directions for research and clinical implementation, aiming to refine and expand the therapeutic potential of synthetic lethality in oncology.},
}

Humans
*Neoplasms/genetics/drug therapy/pathology
*Antineoplastic Agents/pharmacology/chemistry/therapeutic use
*Synthetic Lethal Mutations/drug effects
Molecular Targeted Therapy
CRISPR-Cas Systems

@article {pmid41478990,
year = {2026},
author = {Hu, O and Provvido, A and Zhu, Y},
title = {Generation of IL17RB Knockout Cell Lines Using CRISPR/Cas9-Based Genome Editing.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2983},
number = {},
pages = {361-370},
pmid = {41478990},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; *Gene Editing/methods ; Humans ; *Gene Knockout Techniques/methods ; *Receptors, Interleukin-17/genetics ; Cell Line ; HEK293 Cells ; },
abstract = {CRISPR/Cas9-based genome editing is an inexpensive and efficient tool for genetic modification. Here, we present a methodological approach for establishing interleukin-17 receptor B (IL17RB) knockout cell lines using CRISPR/Cas9-mediated genomic deletion. The IL17RB gene encodes for a cytokine receptor that specifically binds to IL17B and IL17E and is overexpressed in various cancers. The method involves CRISPR design, CRISPR cloning, delivery of the CRISPR clone into cells, and verification of IL17RB gene deletion by deletion screening primer design, genomic DNA extraction, and polymerase chain reaction (PCR). A similar approach can be used for generating mammalian cell lines with gene knockout for other genes of interest.},
}

*CRISPR-Cas Systems
*Gene Editing/methods
Humans
*Gene Knockout Techniques/methods
*Receptors, Interleukin-17/genetics
Cell Line
HEK293 Cells

@article {pmid41478989,
year = {2026},
author = {Huo, Z and Tu, J and Shoemaker, R and Lee, DF and Zhao, R},
title = {Engineering Mutation Clones in Mammalian Cells with CRISPR/Cas9.},
journal = {Methods in molecular biology (Clifton, N.J.)},
volume = {2983},
number = {},
pages = {343-359},
pmid = {41478989},
issn = {1940-6029},
mesh = {*CRISPR-Cas Systems ; Humans ; *Gene Editing/methods ; Cell Line ; *Tumor Suppressor Protein p53/genetics ; *Mutation ; RNA, Guide, CRISPR-Cas Systems/genetics ; Human Embryonic Stem Cells/metabolism/cytology ; Exons ; Gene Knock-In Techniques/methods ; *Genetic Engineering/methods ; },
abstract = {CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system, has been widely accepted and employed in gene editing of a vast range of cell types. Compared to zinc finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs), CRISPR shows a less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome wide screening. Here we will describe the procedure of generating a stable cell line with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.},
}

*CRISPR-Cas Systems
Humans
*Gene Editing/methods
Cell Line
*Tumor Suppressor Protein p53/genetics
*Mutation
RNA, Guide, CRISPR-Cas Systems/genetics
Human Embryonic Stem Cells/metabolism/cytology
Exons
Gene Knock-In Techniques/methods
*Genetic Engineering/methods

@article {pmid41478695,
year = {2026},
author = {Zhang, H and Han, M and Pang, Z and Li, W and Li, X and Sun, B},
title = {The lldD lactate dehydrogenase is a determinant of lactic acid tolerance in Pichia kudriavzevii by pyruvate metabolism pathway.},
journal = {Food microbiology},
volume = {136},
number = {},
pages = {104989},
doi = {10.1016/j.fm.2025.104989},
pmid = {41478695},
issn = {1095-9998},
mesh = {*Pichia/genetics/metabolism/enzymology/drug effects ; *Lactic Acid/metabolism/pharmacology ; Fermentation ; *L-Lactate Dehydrogenase/genetics/metabolism ; Metabolic Networks and Pathways ; *Pyruvic Acid/metabolism ; *Fungal Proteins/genetics/metabolism ; Ethanol/metabolism ; CRISPR-Cas Systems ; Glucose/metabolism ; Gene Knockout Techniques ; },
abstract = {Lactic acid stress is common in traditional fermented foods. Pichia kudriavzevii owes its industrial prevalence to its superior acid tolerance, making deciphering its response mechanism imperative for sustainable fermentation processes. This work explored the remarkable acid tolerance of P. kudriavzevii, which can tolerate 80 g/L lactic acid. The key lactic acid tolerance gene lldD of P. kudriavzevii was determined through transcriptomic analysis. Importantly, this work knocked out the key gene lldD related to lactic acid tolerance for the first time by using the CRISPR-Cas9 technology. Meanwhile, the lldD knockout strain (pk-ΔlldD) was successfully constructed. There were significant differences between pk-ΔlldD and P. kudriavzevii in terms of tolerance to lactic acid, metabolism of lactic acid, utilization of glucose and ethanol production. In addition, the deletion of the gene lldD has a significant impact on the ABC transporter and metabolites of the amino acid metabolic pathway in P. kudriavzevii. In conclusion, this work provides a theoretical basis for engineering high acid-tolerant industrial yeast strains through targeted genetic modification. It helps enhance the stability of fermentation processes under lactic acid stress and ultimately lays a foundation for promoting efficient and low-loss production in the fermentation industry.},
}

*Pichia/genetics/metabolism/enzymology/drug effects
*Lactic Acid/metabolism/pharmacology
Fermentation
*L-Lactate Dehydrogenase/genetics/metabolism
Metabolic Networks and Pathways
*Pyruvic Acid/metabolism
*Fungal Proteins/genetics/metabolism
Ethanol/metabolism
CRISPR-Cas Systems
Glucose/metabolism
Gene Knockout Techniques

@article {pmid41477891,
year = {2026},
author = {Luo, G and Song, J and Fu, Y and Jiang, Y and Gao, Y and Zhong, Z and Li, L and Wei, Y and Jia, HR and Guo, L and Fu, T and Wu, Q and Tan, W},
title = {SPARK-seq: A high-throughput platform for aptamer discovery and kinetic profiling.},
journal = {Science (New York, N.Y.)},
volume = {391},
number = {6780},
pages = {eadv6127},
doi = {10.1126/science.adv6127},
pmid = {41477891},
issn = {1095-9203},
mesh = {*Aptamers, Nucleotide/chemistry/genetics ; Kinetics ; Humans ; *High-Throughput Nucleotide Sequencing/methods ; *Single-Cell Analysis/methods ; *Membrane Proteins/metabolism/genetics/chemistry ; RNA, Messenger/genetics ; CRISPR-Cas Systems ; },
abstract = {Cell surface proteins are key disease biomarkers and therapeutic targets, yet high-throughput methods for aptamer discovery targeting these proteins in situ remain limited. We introduce single-cell perturbation-driven aptamer recognition and kinetics sequencing (SPARK-seq), a high-throughput platform integrating single-cell messenger RNA and aptamer sequencing with CRISPR-based surface protein perturbation. In a single experiment, SPARK-seq simultaneously mapped 5535 distinct aptamers to eight surface proteins, capturing interactions across more than two orders of magnitude in protein abundance and spanning diverse biophysical classes. The method discriminated closely related paralogous proteins with no detectable cross-reactivity and provided kinetic information that enabled the prioritization of aptamers with slow dissociation rates. Leveraging this kinetic diversity, we engineered variants with improved off-rate properties. SPARK-seq establishes a platform for high-efficiency discovery and rational variant design of aptamers and functional nucleic acids, unlocking possibilities in diagnostics and therapeutics.},
}

*Aptamers, Nucleotide/chemistry/genetics
Kinetics
Humans
*High-Throughput Nucleotide Sequencing/methods
*Single-Cell Analysis/methods
*Membrane Proteins/metabolism/genetics/chemistry
RNA, Messenger/genetics
CRISPR-Cas Systems

@article {pmid41477825,
year = {2026},
author = {Song, LCT and Alker, ATP and Oromí-Bosch, A and Swartz, SE and Martinson, JNV and Arora, J and Wang, AM and Rovinsky, R and Smith, SJ and Pierce, EC and Deutschbauer, AM and Doudna, JA and Cress, BF and Rubin, BE},
title = {Identification of proteins influencing CRISPR-associated transposases for enhanced genome editing.},
journal = {Science advances},
volume = {12},
number = {1},
pages = {eaea1429},
pmid = {41477825},
issn = {2375-2548},
mesh = {*Gene Editing/methods ; *Transposases/genetics/metabolism ; Escherichia coli/genetics ; Vibrio cholerae/genetics ; *CRISPR-Cas Systems ; *Genome, Bacterial ; *CRISPR-Associated Proteins/metabolism/genetics ; },
abstract = {CRISPR-associated transposases (CASTs) hold tremendous potential for microbial genome editing because of their ability to integrate large DNA cargos in a programmable, site-specific manner. However, their widespread application has been hindered by poorly understood host factor requirements for transposition. To address this gap, we conducted the first genome-wide screen for host factors affecting Vibrio cholerae CAST (VchCAST) activity using an Escherichia coli RB-TnSeq library and identified 15 genes affecting VchCAST transposition. Of these, seven factors were validated to improve VchCAST activity, and two were inhibitory. Guided by the identification of homologous recombination effectors, RecD and RecA, we tested the λ-Red recombineering system in our VchCAST editing vectors and increased editing efficiency by 55.2-fold in E. coli, 5.6-fold in Pseudomonas putida, and 10.8-fold in Klebsiella michiganensis while maintaining high target specificity and similar insertion arrangements. This study improves the understanding of factors affecting VchCAST activity and enhances its efficiency as a bacterial genome editor.},
}

*Gene Editing/methods
*Transposases/genetics/metabolism
Escherichia coli/genetics
Vibrio cholerae/genetics
*CRISPR-Cas Systems
*Genome, Bacterial
*CRISPR-Associated Proteins/metabolism/genetics

@article {pmid41406662,
year = {2026},
author = {Schalper, KT and Yang, R and Guan, X and Zhang, J and Schreiber, D and Moon, J and Liu, C},
title = {Programmable CRISPR-mediated gold nanoparticle adhesion for visual colorimetric detection.},
journal = {Biosensors & bioelectronics},
volume = {295},
number = {},
pages = {118319},
pmid = {41406662},
issn = {1873-4235},
support = {U01 CA269147/CA/NCI NIH HHS/United States ; },
mesh = {*Colorimetry/methods ; *Gold/chemistry ; *Biosensing Techniques/methods ; Humans ; *Metal Nanoparticles/chemistry ; *CRISPR-Cas Systems/genetics ; *Papillomavirus Infections/diagnosis/virology ; *DNA, Viral/isolation & purification/genetics/analysis ; *Papillomaviridae/isolation & purification/genetics ; Female ; Limit of Detection ; DNA, Single-Stranded/chemistry ; },
abstract = {While the aggregation behavior of gold nanoparticles (AuNPs) has been extensively studied in biosensing, catalysis, and nanomedicine, their potential for programmable surface adhesion via tunable surface chemistry remains largely untapped. Here, a programmable CRISPR-mediated hydrophobic adhesion phenomenon using streptavidin-coated AuNPs functionalized with Cy5-ssDNA-biotin probes is introduced. Hydrophobic Cy5 moieties on the AuNP surface induce localized aggregation and strong adhesion to hydrophobic surfaces. This unique behavior was leveraged by coupling CRISPR-Cas12a-mediated ssDNA cleavage with Cy5-labeled ssDNA-coated AuNPs to develop a simple, visual-readout colorimetric assay for nucleic acid detection. When combined with recombinase polymerase amplification, the method achieved ultrasensitive detection of human papillomavirus (HPV) DNA down to 10 aM, without the need for complex instrumentation. The platform's clinical utility was validated by detecting HPV DNA in cervical swab samples, highlighting its promise for low-cost, sensitive, and accessible point-of-care diagnostics in resource-limited settings. Unlike conventional aggregation-dispersion systems, this platform introduces a fundamentally distinct signal transduction mechanism based on surface adhesion, defining a new modality within CRISPR-based colorimetric diagnostics and offering a simple, low-cost solution for point-of-care testing.},
}

*Colorimetry/methods
*Gold/chemistry
*Biosensing Techniques/methods
Humans
*Metal Nanoparticles/chemistry
*CRISPR-Cas Systems/genetics
*Papillomavirus Infections/diagnosis/virology
*DNA, Viral/isolation & purification/genetics/analysis
*Papillomaviridae/isolation & purification/genetics
Female
Limit of Detection
DNA, Single-Stranded/chemistry

@article {pmid41402283,
year = {2025},
author = {Sivanandan, S and Leitmann, B and Lubeck, E and Sultan, MM and Stanitsas, P and Ranu, N and Ewer, A and Mancuso, JE and Phillips, ZF and Kim, A and Bisognano, JW and Cesarek, J and Ruggiu, F and Feldman, D and Koller, D and Sharon, E and Kaykas, A and Salick, MR and Chu, C},
title = {A pooled Cell Painting CRISPR screening platform enables de novo inference of gene function by self-supervised deep learning.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {77},
pmid = {41402283},
issn = {2041-1723},
mesh = {*Deep Learning ; Humans ; *CRISPR-Cas Systems/genetics ; Gene Regulatory Networks ; *Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; },
abstract = {Pooled CRISPR screening enables large-scale interrogation of gene functions but typically measures simple phenotypes such as fitness. High-content methods like Perturb-seq extend dimensionality to transcriptomics but are costly and limited in scope. Optical pooled screening (OPS) combines pooled CRISPR screening with imaging to yield scalable, information-rich readouts, yet existing implementations remain pathway-specific. Here we describe an OPS-compatible Cell Painting platform that enables hypothesis-free reverse genetic screening through multiplexed morphological profiling. We validate this technique using a well-defined morphological gene set, compare classical image analysis to self-supervised learning methods using a mechanism-of-action library, and perform discovery screening with a druggable genome library. By combining rich morphological data with deep learning, gene networks emerge without the need for target-specific biomarkers, leading to unbiased discovery of gene functions.},
}

*Deep Learning
Humans
*CRISPR-Cas Systems/genetics
Gene Regulatory Networks
*Clustered Regularly Interspaced Short Palindromic Repeats/genetics

@article {pmid41381248,
year = {2026},
author = {Adamopoulos, PG and Athanasopoulou, K and Scorilas, A},
title = {A versatile type VI CRISPR-based approach for targeted m[6]A demethylation in mRNAs.},
journal = {Genome research},
volume = {36},
number = {1},
pages = {169-182},
pmid = {41381248},
issn = {1549-5469},
mesh = {Humans ; *RNA, Messenger/genetics/metabolism ; *Adenosine/analogs & derivatives/metabolism ; HeLa Cells ; AlkB Homolog 5, RNA Demethylase/genetics/metabolism ; Demethylation ; *CRISPR-Cas Systems ; *RNA Processing, Post-Transcriptional ; },
abstract = {Epitranscriptomics, a rapidly evolving field mainly driven by massive parallel sequencing technologies, explores post-transcriptional RNA modifications. N [6]-methyladenosine (m[6]A) has emerged as the most prominent and dynamically regulated modification in human mRNAs, being implicated in the regulation of diverse biological processes, including spermatogenesis, heat shock response, ultraviolet-induced DNA damage response and maternal mRNA clearance. Despite the recognized significance of m[6]A in mRNA regulation, limited studies have focused on the targeted and efficient manipulation of this modification in mRNAs. Here, we present Dem6A-Vec, an "all-in-one" plasmid vector designed for site-specific m[6]A demethylation in human mRNAs. Dem6A-Vec integrates the expression of a catalytically inactive RfxCas13d fused to the m[6]A demethylase ALKBH5 and a U6-driven customizable guide RNA in a single construct, simplifying experimental workflows and enhancing targeting efficiency. Using nanopore direct RNA sequencing, we identify high-confident m[6]A sites in HeLa cells, which serve as targets for Dem6A-Vec. We validate the targeted demethylation of m[6]A sites in the EEF2 and RRAGA genes using the established SELECT-qPCR method, confirming the impacts on mRNA stability and highlighting the tool's precision and versatility. The presented approach is implemented in multiple mRNA sites with diverse methylation stoichiometries, underscoring its adaptability to various transcriptomic contexts. This study provides a robust and scalable method for investigating the functional roles of m[6]A modifications, offering a transformative platform for advancing epitranscriptomic research and potential therapeutic applications.},
}

Humans
*RNA, Messenger/genetics/metabolism
*Adenosine/analogs & derivatives/metabolism
HeLa Cells
AlkB Homolog 5, RNA Demethylase/genetics/metabolism
Demethylation
*CRISPR-Cas Systems
*RNA Processing, Post-Transcriptional

@article {pmid41380236,
year = {2026},
author = {Yin, Z and Yin, H and Zhou, Y and Liu, H},
title = {CRISPR/Cas13a-mediated photoelectrochemical-colorimetric dual-mode biosensor for RNA N-acetyltransferase 10 detection based on Bi2O2S/Ag2S and laccase-like nanozyme.},
journal = {Biosensors & bioelectronics},
volume = {295},
number = {},
pages = {118298},
doi = {10.1016/j.bios.2025.118298},
pmid = {41380236},
issn = {1873-4235},
mesh = {*Biosensing Techniques/methods ; Laccase/chemistry ; Electrochemical Techniques/methods ; Colorimetry/methods ; *Acetyltransferases/isolation & purification/analysis/chemistry ; Humans ; CRISPR-Cas Systems ; Limit of Detection ; Bismuth/chemistry ; },
abstract = {N-Acetyltransferase 10 (NAT10) is a crucial protein that catalyzes RNA acetylation modification and plays a significant role in biological activities. Accurate detection of NAT10 is of great importance in clinical testing and drug development. To achieve this goal, a novel biosensing platform was developed for NAT10 detection, relying on an RNA acetylation-inhibited CRISPR/Cas13a system, a Bi2O2S/Ag2S type-II heterojunction, and a laccase-mimetic nanocatalyst, using a photoelectric-colorimetric dual-mode strategy. Based on the catalysis effect of laccase-mimetic nanocatalyst, its substrates (hydroquinone and 2,4-dichlorophenol) were oxidized to form p-benzoquinone and 2,4-dichloroquinone, respectively. This oxidation process not only impaired the electron-donating ability of hydroquinone but also induced the coupling of 2,4-dichloroquinone with 4-antipyrine to generate a red product-enabling both photoelectrochemical and colorimetric detection of the NAT10 protein. The biosensor exhibited wide linear ranges of 0.1-1000 μg/L (photoelectrochemical mode) and 1-1000 μg/L (colorimetric mode), and the low detection limit of 0.056 and 0.348 μg/L for the two modes, respectively. Additionally, this biosensor was used to investigate the inhibitory effects of plasticizers and phosphorus-containing flame retardants on NAT10. Furthermore, molecular docking simulations were employed to explore the underlying inhibition mechanism. The developed biosensor not only provided a novel detection technique for NAT10, but also offers an alternative method for NAT10 inhibitor screening and a new tool for evaluating the ecotoxicological effects of organic pollutants.},
}

*Biosensing Techniques/methods
Laccase/chemistry
Electrochemical Techniques/methods
Colorimetry/methods
*Acetyltransferases/isolation & purification/analysis/chemistry
Humans
CRISPR-Cas Systems
Limit of Detection
Bismuth/chemistry

@article {pmid41330302,
year = {2026},
author = {Xu, W and Lin, Y and Huang, Z and Li, Y and Lu, Y and Liu, M and Cui, S and Zhang, T and Shi, N and Sheng, Y and Hu, J},
title = {Split proximity circuit initiated CRISPR-Cas12a system profiling exosomal surface proteins for early cancer detection.},
journal = {Biosensors & bioelectronics},
volume = {295},
number = {},
pages = {118280},
doi = {10.1016/j.bios.2025.118280},
pmid = {41330302},
issn = {1873-4235},
mesh = {Humans ; *Exosomes/chemistry/genetics ; *CRISPR-Cas Systems/genetics ; *Biosensing Techniques/methods ; *Breast Neoplasms/diagnosis/blood/genetics ; *Early Detection of Cancer/methods ; Female ; Epithelial Cell Adhesion Molecule/genetics/isolation & purification ; *Biomarkers, Tumor/genetics/blood ; Limit of Detection ; Mucin-1/genetics ; Aptamers, Nucleotide/chemistry ; },
abstract = {Early diagnosis of breast cancer is critical for improving prognosis, but traditional methods have limitations. Herein, we propose an SPC-CRISPR system for the sensitive and specific detection of multiple breast cancer exosomal proteins without prior exosome isolation. This system couples CRISPR system with an enzyme-free amplification method to achieve dual-signal amplification. SPC-CRISPR is based on a split proximity circuit (SPC) that triggers catalytic hairpin assembly (CHA), converting protein signals on the surface of exosomes into nucleic acid signals, and the CRISPR-Cas12a system enabling further signal amplification and output. The system targets phosphatidylserine (PS), MUC1, and EpCAM on exosomes: Tim4-modified magnetic beads capture PS-expressing exosomes, and dual-aptamers recognize MUC1 and EpCAM, enabling SPC assembly and subsequent amplification. In buffer and cell-derived exosomes, the SPC-CRISPR system showed a detection limit of 10 particles/μL (R[2] = 0.990). Clinical tests utilizing merely 1 μL of serum samples successfully distinguished breast cancer patients from healthy donors (AUC = 0.9778, accuracy = 91.23 %), detected stage 0 breast cancer patients against healthy controls (accuracy = 92.59 %), and differentiated metastatic from non-metastatic cases (p < 0.001). The combination of high sensitivity, minimal sample requirements, and an exosome isolation-free workflow positions the SPC-CRISPR system as a promising tool for the clinical early detection and classification of breast cancer, with broader applicability to other cancers by swapping the corresponding aptamers.},
}

Humans
*Exosomes/chemistry/genetics
*CRISPR-Cas Systems/genetics
*Biosensing Techniques/methods
*Breast Neoplasms/diagnosis/blood/genetics
*Early Detection of Cancer/methods
Female
Epithelial Cell Adhesion Molecule/genetics/isolation & purification
*Biomarkers, Tumor/genetics/blood
Limit of Detection
Mucin-1/genetics
Aptamers, Nucleotide/chemistry

@article {pmid41318543,
year = {2025},
author = {Pernaci, C and Johnson, A and Gillette, S and Warden, AS and McCormick, C and Weiser-Novak, S and Ramirez, G and Broersma, EH and Mishra, P and Sivakumar, A and Cherqui, S and Coufal, NG},
title = {Microgliopathy as a primary mediator of neuronal death in models of Friedreich's Ataxia.},
journal = {Nature communications},
volume = {17},
number = {1},
pages = {81},
pmid = {41318543},
issn = {2041-1723},
support = {TRAN1-13983//California Institute for Regenerative Medicine (CIRM)/ ; EDUC2-08388//California Institute for Regenerative Medicine (CIRM)/ ; R01NS135162//Foundation for the National Institutes of Health (Foundation for the National Institutes of Health, Inc.)/ ; },
mesh = {*Friedreich Ataxia/pathology/genetics/metabolism ; Animals ; *Microglia/pathology/metabolism ; Humans ; Induced Pluripotent Stem Cells/metabolism ; Mice ; Frataxin ; Disease Models, Animal ; Iron-Binding Proteins/genetics/metabolism ; Cell Death ; Mitochondria/metabolism/pathology ; *Neurons/pathology/metabolism ; Purkinje Cells/pathology/metabolism ; Trinucleotide Repeat Expansion ; Iron/metabolism ; Male ; Female ; White Matter/pathology ; CRISPR-Cas Systems ; },
abstract = {Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by a GAA repeat expansion in the frataxin (FXN) gene, leading to a severe reduction of the mitochondrial FXN protein, crucial for iron metabolism. While microglial inflammation is observed in FRDA, it remains unclear whether immune dysfunction is a primary disease mediator or a secondary reactionary phenotype. Utilizing patient-derived induced pluripotent stem cells (iPSCs), we report an intrinsic microglial phenotype of stark mitochondrial defects, iron overload, lipid peroxidation, and lysosomal abnormalities. These factors drive a pro-inflammatory state that contributes to neuronal death in co-culture systems. In a murine xenograft model, transplanted human FRDA microglia accumulate in white matter and the Purkinje cell layer, resulting in Purkinje neuron loss in otherwise healthy brains. Notably, CRISPR/Cas9-mediated correction of the GAA repeat reverses microglial defects and mitigates neurodegeneration. Here, we suggest that microglial dysfunction serve as a disease driver and a promising therapeutic target in FRDA.},
}

*Friedreich Ataxia/pathology/genetics/metabolism
Animals
*Microglia/pathology/metabolism
Humans
Induced Pluripotent Stem Cells/metabolism
Mice
Frataxin
Disease Models, Animal
Iron-Binding Proteins/genetics/metabolism
Cell Death
Mitochondria/metabolism/pathology
*Neurons/pathology/metabolism
Purkinje Cells/pathology/metabolism
Trinucleotide Repeat Expansion
Iron/metabolism
Male
Female
White Matter/pathology
CRISPR-Cas Systems

@article {pmid41277417,
year = {2026},
author = {Choi, MY and Li, C and Choi, JH and Choi, JW},
title = {Intracellular biosensors by functional nanomaterial-integrated CRISPR technologies for real-time molecular sensing.},
journal = {Chemical communications (Cambridge, England)},
volume = {62},
number = {1},
pages = {58-70},
doi = {10.1039/d5cc05016b},
pmid = {41277417},
issn = {1364-548X},
mesh = {*Biosensing Techniques/methods ; Humans ; *CRISPR-Cas Systems ; *Nanostructures/chemistry ; Gene Editing ; Nanotechnology ; },
abstract = {CRISPR technology, originally developed as a genome-editing tool, has recently emerged as a powerful platform for intracellular biosensing. By harnessing the programmability and target specificity of CRISPR-associated proteins, such as Cas9, Cas12, and Cas13, researchers have engineered biosensors capable of detecting a wide array of intracellular signals, including nucleic acids, non-coding RNAs, and small-molecule metabolites. This review discusses the recent advancements in CRISPR-powered biosensors for real-time, dynamic monitoring of cellular processes and molecular events. Particular focus is given to the integration of nanotechnology, which plays a crucial role in enhancing the delivery efficiency, signal amplification, and sensor stability. Nanomaterials such as gold nanoparticles, quantum dots, DNA nanostructures, and upconversion nanoparticles have been strategically employed to improve the intracellular transport of CRISPR components, facilitate signal readouts, and enable multimodal sensing in complex cellular environments. Additionally, we delve into how CRISPR-nanotechnology hybrids can be adapted for multiplex analysis and single-cell resolution. This review also addresses the current challenges in intracellular biosensing, including precise delivery, biocompatibility, and long-term monitoring, and outlines future directions for the application of these systems in precision medicine, synthetic biology, and advanced therapeutic monitoring. Through the convergence of gene-editing systems and nanotechnology, CRISPR-based intracellular biosensors are anticipated to revolutionize next-generation diagnostic and therapeutic strategies.},
}

*Biosensing Techniques/methods
Humans
*CRISPR-Cas Systems
*Nanostructures/chemistry
Gene Editing
Nanotechnology

@article {pmid41261774,
year = {2026},
author = {Sun, W and Zhu, Y and Zhang, X and Meng, X and Xia, K and Zhang, X and Han, X and Pan, W and Guo, J and Li, J and Zhang, H},
title = {A mortise-tenon joint system facilitates precise targeted DNA insertion and replacement in rice.},
journal = {Molecular plant},
volume = {19},
number = {1},
pages = {36-47},
doi = {10.1016/j.molp.2025.11.006},
pmid = {41261774},
issn = {1752-9867},
mesh = {*Oryza/genetics ; *Gene Editing/methods ; *Mutagenesis, Insertional/methods ; CRISPR-Cas Systems/genetics ; Genome, Plant/genetics ; DNA, Plant/genetics ; },
abstract = {Precise and scarless DNA insertion and replacement represent two of the major challenges in plant genome editing. Numerous tools have been developed, including homology-directed repair-dependent tools and prime-editing-mediated systems. However, none has fully resolved these challenges. In this study, we develop the "mortise-tenon joint system" (MT), a novel strategy that enables precise and efficient targeted DNA insertion and replacement. By leveraging the APOBEC-Cas9-uracil DNA glycosylase/(apurinic or apyrimidinic site) lyase within our previously reported APOBEC-Cas9 fusion-induced deletion system, which performs single-strand cleavage on the non-target strand and double-strand cleavage on the target strand, we generated a unique "mortise" structure, consisting of double-strand breaks with single or double non-complementary 5' overhangs. We further designed "tenons," double-stranded DNA donors containing 5' sticky ends precisely matching the 5' overhangs of the mortises. The end-capture interaction between mortises and tenons facilitates precise targeted insertion and replacement, achieving frequencies of 16.30%-59.47% across seven tested targets using 21-85 bp donor inserts in rice. If long DNA donors with sticky ends complementary to the mortise structure can be generated, the MT system may enable highly precise targeted insertion and replacement of large DNA fragments.},
}

*Oryza/genetics
*Gene Editing/methods
*Mutagenesis, Insertional/methods
CRISPR-Cas Systems/genetics
Genome, Plant/genetics
DNA, Plant/genetics

@article {pmid41148632,
year = {2026},
author = {Jin, Z and Yang, Y and Chen, C and Zhang, Z and Ren, Q and Cui, Z and Huang, CZ and Zuo, H},
title = {Gene silencing regulated by aggregates of Corn aptamer at 3' UTR of mRNA.},
journal = {Nanoscale horizons},
volume = {11},
number = {1},
pages = {232-242},
doi = {10.1039/d5nh00510h},
pmid = {41148632},
issn = {2055-6764},
mesh = {*RNA, Messenger/genetics/chemistry/metabolism ; *3' Untranslated Regions/genetics ; *Gene Silencing ; *Aptamers, Nucleotide/chemistry/genetics ; Humans ; CRISPR-Cas Systems ; *Zea mays/genetics/chemistry ; },
abstract = {Gene therapy, as a cutting-edge approach for disease intervention, relies heavily on advancements in gene silencing techniques. For instance, CRISPR-Cas9 has emerged as a leading gene-editing tool due to its ability to introduce precise cuts at specific genomic loci, enabling targeted gene insertion, deletion, or modification. In this study, we developed a simple and effective gene silencing strategy by introducing a nucleic acid self-assembly module into the 3' untranslated region (UTR) of mRNA. This module demonstrated significant gene silencing efficacy in eukaryotic cells through the formation of RNA aggregates. To systematically investigate its regulatory mechanism on translation efficiency through the formation of higher-order RNA structures, we quantitatively analyzed both mRNA and protein expression levels. Furthermore, our modular 3' UTR sequences can be integrated with classical 5' UTR elements (e.g., TOP sequences) to construct a multidimensional post-transcriptional regulatory network. This technology expands the diversity of existing UTR element libraries and offers a reservoir of programmable regulatory elements for applications in synthetic biology. It enables the construction of orthogonal combinations of multidimensional elements, tailored to specific gene expression regulation needs.},
}

*RNA, Messenger/genetics/chemistry/metabolism
*3' Untranslated Regions/genetics
*Gene Silencing
*Aptamers, Nucleotide/chemistry/genetics
Humans
CRISPR-Cas Systems
*Zea mays/genetics/chemistry

@article {pmid41121528,
year = {2026},
author = {Horsley, N and Sythoff, AVB and Delgado, M and Liu, S and Cabernard, C},
title = {CrisprBuildr: an open-source application for CRISPR-mediated genome engineering in Drosophila melanogaster.},
journal = {G3 (Bethesda, Md.)},
volume = {16},
number = {1},
pages = {},
pmid = {41121528},
issn = {2160-1836},
support = {R35GM148160/NH/NIH HHS/United States ; },
mesh = {Animals ; *Drosophila melanogaster/genetics ; *CRISPR-Cas Systems ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Software ; *Genome, Insect ; *Genetic Engineering/methods ; Clustered Regularly Interspaced Short Palindromic Repeats ; },
abstract = {CRISPR/Cas9 is a powerful tool for targeted genome editing experiments. Using CRISPR/Cas9, genes can be deleted or modified by inserting specific DNA sequences, encoding for fluorescent proteins, small peptide tags, or other modifications. Such experiments are essential for detailed gene and protein characterization. However, designing and cloning the corresponding constructs can be repetitive, time-consuming, and laborious. To assist users in CRISPR/Cas9-based genome engineering, we developed CrisprBuildr, an open-source, web-based application for designing modifications to their target genes. CrisprBuildr guides users through creating guide RNAs and repair template vectors to generate cloning maps. The application is designed for the Drosophila melanogaster genome but can serve as a template for other available genomes. We also created new tagging vectors using EGFP and mCherry combined with the small peptide SspB-Q73R for use in iLID-based optogenetic experiments.},
}

Animals
*Drosophila melanogaster/genetics
*CRISPR-Cas Systems
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Software
*Genome, Insect
*Genetic Engineering/methods
Clustered Regularly Interspaced Short Palindromic Repeats

@article {pmid41477711,
year = {2025},
author = {Rozov, SM and Deineko, EV},
title = {[Recombinase-Based Engineering of Plant Genomes in the Era of Genome Editing].},
journal = {Molekuliarnaia biologiia},
volume = {59},
number = {6},
pages = {873-890},
doi = {10.7868/S3034555325060016},
pmid = {41477711},
issn = {0026-8984},
mesh = {*Gene Editing/methods ; *Genome, Plant ; *CRISPR-Cas Systems ; *DNA Nucleotidyltransferases/genetics/metabolism ; *Recombinases/genetics/metabolism ; *Plants/genetics ; DNA Transposable Elements ; },
abstract = {The rapidly evolving CRISPR/Cas-based genome editing technologies, which have dominated nearly all areas of molecular biology over the past decade, still face several unresolved challenges. One of the major limitations of current genome editing tools is the low efficiency of targeted long-sequence insertions. This issue is particularly critical in plant systems, where genome editing efficiency is hindered by specific cellular characteristics. Site-specific recombinases (SSRs), which have long been employed in genetic engineering to mediate various genomic rearrangements-including deletions, duplications, insertions, and inversions-are limited in their application by the requirement for preexisting recombination recognition sites in the genome. However, CRISPR/Cas and recombinase tools complement each other, and their combined use offers a powerful strategy to overcome key limitations of genome editing. The discovery of CRISPR-associated transposons such as CAST and OMEGA, which naturally utilize their own recombinases, marks a significant advance in genome engineering, providing an elegant example of the natural convergence between CRISPR and recombinase technologies.},
}

*Gene Editing/methods
*Genome, Plant
*CRISPR-Cas Systems
*DNA Nucleotidyltransferases/genetics/metabolism
*Recombinases/genetics/metabolism
*Plants/genetics
DNA Transposable Elements

@article {pmid41475352,
year = {2025},
author = {Martinho, C and Hoshino, M and Raphalen, M and Bukhanets, V and Kerur, A and Bogaert, KA and Luthringer, R and Coelho, SM},
title = {Efficient CRISPR-Cas genome editing in brown algae.},
journal = {Cell reports methods},
volume = {},
number = {},
pages = {101273},
doi = {10.1016/j.crmeth.2025.101273},
pmid = {41475352},
issn = {2667-2375},
abstract = {Brown algae represent the third most complex lineage to have independently evolved multicellularity, distinct from plants and animals. Yet, functional studies of their development and evolution have been limited by the absence of efficient genome editing tools. Here, we present a robust, high-efficiency, and transgene-free CRISPR-based genome editing platform applicable across four ecologically and biotechnologically important brown algal species. Using Ectocarpus as a model, we optimized a polyethylene glycol (PEG)-mediated ribonucleoprotein (RNP) delivery system that achieves reproducible editing across multiple loci without cloning or specialized equipment. As proof of concept, we recreated the hallmark imm mutant phenotype by precisely editing the IMMEDIATEUPRIGHT (IMM) locus. APT/2-fluoroadenine (2-FA) selection further enhanced specificity with minimal false positives. The method was easily transferable to other species, including kelps. This platform now enables functional genomics in brown algae, providing powerful tools for investigating development, life cycle regulation, and the independent evolution of complex multicellularity.},
}

@article {pmid41475348,
year = {2025},
author = {Gao, X and Zhu, K and Zhang, W and Wang, L and Wang, L and Hua, L and Niu, T and Qin, B and Yu, X and Zhu, H and Cui, S},
title = {RNA anti-CRISPRs deplete Cas proteins to inhibit the CRISPR-Cas system.},
journal = {Molecular cell},
volume = {},
number = {},
pages = {},
doi = {10.1016/j.molcel.2025.12.005},
pmid = {41475348},
issn = {1097-4164},
abstract = {RNA-based anti-CRISPRs (Racrs) interfere with the type I-F CRISPR-Cas system by mimicking the repeats found in CRISPR arrays. Here, we determined the cryo-electron microscopy (cryo-EM) structures of the type I-F crRNA-guided surveillance complex (Csy complex) from Pectobacterium atrosepticum and three RacrIF1-induced aberrant subcomplexes. Additionally, we observed that Cas7f proteins could bind to non-specific nucleic acids, forming right-handed superhelical filaments composed of different Cas7 copies. Mechanistically, RacrIF1 lacks the specific S-conformation observed in the corresponding position of the 5' handle in canonical CRISPR complexes, and it instead adopts a periodic "5 + 1" pattern. This conformation creates severe steric hindrance for Cas5f-Cas8f heterodimer and undermines their binding. Furthermore, Cas7f nonspecifically binds nucleic acids and can form infinite superhelical filaments along Racrs molecules. This oligomerization sequesters Cas6f and Cas7f from binding, therefore blocking the formation of functional CRISPR-Cas effector complexes and ultimately blocking antiviral immunity. Our study provides a structural basis underlying Racrs-mediated CRISPRs inhibition.},
}

@article {pmid41475278,
year = {2025},
author = {Kolanchi, P and Saminathan, N and Selvaraj, D and Krishnamoorthy, A and Palanivelu, K and Aruchalam, A},
title = {Bioprocess and genetic advances enhancing Beauveria bassiana biocontrol efficacy.},
journal = {Microbial pathogenesis},
volume = {211},
number = {},
pages = {108272},
doi = {10.1016/j.micpath.2025.108272},
pmid = {41475278},
issn = {1096-1208},
abstract = {Beauveria bassiana is a widely exploited entomopathogenic fungus that has emerged as a central component of ecologically sustainable pest management. Recent years have witnessed rapid progress across its biological understanding, technological development, and application potential. This review synthesizes contemporary advances spanning infection biology, secondary metabolite biosynthesis, strain development, bioprocess engineering, formulation science, and genetic improvement. At the molecular level, multi-omics studies have elucidated the coordinated regulation of surface adhesion, cuticular penetration, host immune modulation, dimorphic transitions, and toxin production, revealing gene networks that govern virulence, stress tolerance, and ecological adaptation. These insights have informed improved strategies for strain isolation and high-throughput phenotypic screening, enabling the selection of isolates with enhanced pathogenicity, environmental robustness, and endophytic competence. Parallel advances in solid-state and submerged fermentation, supported by agro-industrial substrates and data-driven optimization, have strengthened large-scale production of infective propagules with consistent quality. Such gains are further reinforced by modern formulation approaches, including oil-based dispersions, encapsulation systems, nanoemulsions, and seed-coating technologies, which collectively improve spore stability, persistence, and delivery under heterogeneous field conditions. More recently, CRISPR/Cas-based genome editing and pathway engineering have opened new avenues for precision enhancement of virulence traits, metabolic output, and abiotic stress resilience. Despite these achievements, the broader adoption of B. bassiana remains constrained by variable field performance, slower speed of action relative to chemical insecticides, strain-dependent efficacy, and regulatory and quality-control challenges. By integrating fundamental biology with technological innovation and practical limitations, this review provides a coherent framework for advancing B. bassiana from laboratory optimization to reliable field implementation, underscoring its promise as a next-generation, environmentally aligned biocontrol agent in modern agriculture.},
}

@article {pmid41474879,
year = {2025},
author = {Cheng, M and Wang, Y and Lin, W and Ye, J and Wu, M and Xiang, B and Liu, L and Sun, B},
title = {A Universal Light-Activated CRISPR-RNA Based on Split Direct Repeat for One-Pot Cas12a Nucleic Acid Detection.},
journal = {Analytical chemistry},
volume = {},
number = {},
pages = {},
doi = {10.1021/acs.analchem.5c05722},
pmid = {41474879},
issn = {1520-6882},
abstract = {Spatiotemporal regulation of CRISPR-Cas systems holds significant promise for precision gene editing and molecular diagnostics. While photochemical strategies for CRISPR activity control have advanced, a universal regulatory approach remains elusive. Here, we report a modular light-activated CRISPR-RNA design through splitting conventional crRNA within the direct repeat (DR) into two functional domains: a conserved 5' split direct repeat (5' SDR) and a variable 3' split direct repeat (SDR) + spacer (3' SDR-Spacer) module. Double-stranded extensions were introduced at the cleavage site to preserve functional integrity. Through screening of light-sensitive caging group modification sites in the universal 5' SDR, a novel light-activated CRISPR-RNA system was developed. This system only requires spacer redesign of the 3' SDR-Spacer for new targets, while the caged 5' SDR is universal. Thereupon, we established a universal light-activated CRISPR-RNA assisted one-pot RAA-Cas12a detection system (UniLight-CRISPR). When applied to Mycoplasma pneumoniae detection using qPCR-validated clinical samples, UniLight-CRISPR demonstrated 95.45% sensitivity and 100% specificity, matching the performance of conventional two-step Cas12a assays. This universal photo regulation strategy not only addresses current limitations in CRISPR diagnostics but also provides a blueprint for adapting other Cas enzymes. We anticipate broad applications of our universal light-activated CRISPR-RNA system, extending from molecular diagnostics to gene-editing research.},
}

@article {pmid41048051,
year = {2026},
author = {Huang, LT and Gao, RJ and Zhang, D and Nian, C and Martzke, W and Shapiro, AMJ and Kin, T and Tahamtani, Y and Lynn, FC},
title = {Truncated CD19 as a selection marker for the isolation of stem cell-derived β-cells.},
journal = {Disease models & mechanisms},
volume = {19},
number = {1},
pages = {},
doi = {10.1242/dmm.052376},
pmid = {41048051},
issn = {1754-8411},
support = {5-SRA-2020-1059-S-B, 3-COE-2022-1103-M-B//Juvenile Diabetes Research Foundation United States of America/ ; ASD-173663//Institute of Nutrition, Metabolism and Diabetes/ ; 5238 BIOM//Michael Smith Health Research BC/ ; IGAP//BC Children's Hospital/ ; 5-SRA-2020-1059-S-B//Breakthrough T1D Canada/ ; 3-COE-2022-1103-M-B//Breakthrough T1D Canada/ ; ASD-173663/CAPMC/CIHR/Canada ; //BC Children's Hospital/ ; 6563//National Science and Engineering Research Council of Canada/ ; },
mesh = {Humans ; *Insulin-Secreting Cells/cytology/metabolism ; *Antigens, CD19/metabolism ; Biomarkers/metabolism ; *Cell Separation/methods ; Human Embryonic Stem Cells/cytology/metabolism ; CRISPR-Cas Systems/genetics ; Insulin/metabolism ; C-Peptide/metabolism ; Cell Differentiation ; },
abstract = {Stem cell-derived β-cells (SCβ-cell) are a renewable and scalable alternative to cadaveric islets as a cell-replacement therapy for type 1 diabetes (T1D). However, heterogeneity within SCβ-cell cultures remains problematic for graft safety and function. Magnetic selection of SCβ-cells expressing a unique cell-surface marker may help deplete undesirable cell types and facilitate functional maturation. Here, we explored the transmembrane glycoprotein CD19 as a potential cell-surface marker for the enrichment of insulin-expressing SCβ-cells. Using CRISPR/Cas9 technology, we created a knock-in add-on of CD19-mScarlet downstream of insulin (INS) coding sequence exon 2 in human embryonic stem cells (hESCs). We developed and optimized a magnetic-activated cell sorting protocol for CD19-mScarlet-expressing cells, forming enriched SCβ-cell clusters with improved glucose-stimulated C-peptide secretion. This strategy holds promise to facilitate large-scale production of functional SCβ-cells for disease modeling and cell-replacement therapy.},
}

Humans
*Insulin-Secreting Cells/cytology/metabolism
*Antigens, CD19/metabolism
Biomarkers/metabolism
*Cell Separation/methods
Human Embryonic Stem Cells/cytology/metabolism
CRISPR-Cas Systems/genetics
Insulin/metabolism
C-Peptide/metabolism
Cell Differentiation

@article {pmid41316351,
year = {2025},
author = {Qin, Z and Wang, Y and Sun, M and Wang, Q and Duan, J and Gu, C and Zhang, X and Yu, F and Wu, Y and Xu, H and Li, J and Zhang, L},
title = {Development of a field-deployable RPA-CRISPR/Cas12a assay for the detection of Cyclospora cayetanensis in human feces.},
journal = {Parasites & vectors},
volume = {19},
number = {1},
pages = {4},
pmid = {41316351},
issn = {1756-3305},
support = {232102110088//Henan Province Scientific and Technological Project/ ; 2023YFD1801200//National Key Research and Development Program of China/ ; 231111111500//Key Research and Development Project of Henan Province/ ; },
mesh = {*Cyclospora/isolation & purification/genetics ; Humans ; *Feces/parasitology ; *Cyclosporiasis/diagnosis/parasitology ; *CRISPR-Cas Systems ; *Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; Recombinases/genetics/metabolism ; Biosensing Techniques/methods ; *Molecular Diagnostic Techniques/methods ; },
abstract = {BACKGROUND: Cyclospora is an emerging intestinal pathogenic protozoan transmitted through foodborne and waterborne routes. At least 19 countries in the world have recorded outbreaks of cyclosporiasis, mainly associated with the consumption of contaminated fresh agricultural products. The lack of a sensitive immediate test is one of the major obstacles to the rapid diagnosis of cyclosporiasis. The target interference mechanisms of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) protein systems have been adapted into versatile and efficient genome manipulation and disease-curing technologies, while also being promising for point-of-care testing (POCT) applications. It can serve as an excellent rapid and specific detection tool.

METHODS: The recombinase polymerase amplification (RPA) and the CRISPR/Cas12a system were combined to develop a detection method for C. cayetanensis (termed RECCT-Cay) via visual observation of fluorescent readings under blue light and field diagnosis using lateral flow strip (LFS) biosensors.

RESULTS: The detection limit of the established RECCT-Cay was 7 copies/μL. Under simulated clinical conditions, the detection limit was 30 oocysts per gram of stool. At the same time, the established detection platform can distinguish C. cayetanensis from the closely related Eimeria spp. The results of our constructed assay were compared with nested PCR, and the detection results of 30 clinical stool samples were consistent, with three samples positive for C. cayetanensis. Based on the RECCT-Cay detection principle, a portable suitcase-sized device has been designed, which can conduct rapid on-site detection of clinical samples.

CONCLUSIONS: The RECCT-Cay platform features rapid speed, high sensitivity, and the capability for field detection, making it a promising tool for use in remote areas.},
}

*Cyclospora/isolation & purification/genetics
Humans
*Feces/parasitology
*Cyclosporiasis/diagnosis/parasitology
*CRISPR-Cas Systems
*Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
Recombinases/genetics/metabolism
Biosensing Techniques/methods
*Molecular Diagnostic Techniques/methods

@article {pmid41474623,
year = {2025},
author = {Banh, DV and Goldberg, GW and Marraffini, LA},
title = {Phage induction of Staphylococcus aureus pathogenicity islands promotes the CRISPR-Cas adaptive immune response.},
journal = {Cell reports},
volume = {45},
number = {1},
pages = {116776},
doi = {10.1016/j.celrep.2025.116776},
pmid = {41474623},
issn = {2211-1247},
abstract = {Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements carrying virulence genes that spread upon infection by helper phages that induce their transfer. Staphylococci also carry type II and III CRISPR-Cas systems that mount an adaptive immune response against phages through the acquisition of spacer sequences from viral genomes, directing Cas nucleases to their targets. Whether and how SaPIs and CRISPR interact with each other during helper phage infection is not known. Here we report that, as a result of the packaging of incomplete helper phage genomes into SaPI particles, defective viral DNA delivered into new hosts stimulates spacer acquisition in both CRISPR types. Once immunized, staphylococci target the helper phage and prevent SaPI mobilization. Our work reveals an unexpected synergy between CRISPR-Cas systems and SaPIs that enhances antiphage immunity and could favor the retention of beneficial elements within the population.},
}

@article {pmid41474367,
year = {2026},
author = {Rasool, HMH and Gong, X and Jin, Y and Liu, M and Yanyan, W and Qu, M and Anxiang, Z and Lingling, X and Li, Z and Zhou, J and Chen, Q},
title = {Development of Visual and Fluorescence Detection Method of Brucella by RPA-CRISPR/Cas12a Assay.},
journal = {FASEB journal : official publication of the Federation of American Societies for Experimental Biology},
volume = {40},
number = {1},
pages = {e71397},
doi = {10.1096/fj.202503610R},
pmid = {41474367},
issn = {1530-6860},
support = {2022YFC2304001//MOST | National Key Research and Development Program of China (NKPs)/ ; 2024BEG-02029//The Key Research and Development Program of Ningxia Hui Autonomous Region/ ; 2024312885//Comprehensive Control Technology For Brucellosis and Hydatic Disease in Xinjiang/ ; CAAS-ZDRW202410//The Agricultural Science and Technology Innovation Program (ASTIP)/ ; },
mesh = {*Brucella/genetics/isolation & purification ; Animals ; *Brucellosis/diagnosis/microbiology ; *CRISPR-Cas Systems/genetics ; Sensitivity and Specificity ; Cattle ; Fluorescence ; Limit of Detection ; Humans ; },
abstract = {Brucella is a significant pathogen in the livestock industry, causing Brucellosis, a zoonotic disease that leads to considerable health and economic losses in both humans and animals. Current diagnostic methods for Brucella, including culture, serological assays, and PCR/qPCR, are valuable tools but have inherent limitations. These include the requirement for BSL-3 laboratories, trained personnel, complex procedures, expensive equipment, issues with sensitivity and specificity, and the time-consuming nature of assays, making them unsuitable for large-scale epidemiological screening. Therefore, there is a critical need to develop a rapid, portable, and cost-effective diagnostic method with high specificity and sensitivity. In this study, we established a rapid, portable, reliable, and inexpensive detection method for Brucella genus identification based on RPA-CRISPR/Cas12a technology. Specific RPA primers and crRNA sequences were designed targeting the bcsp31 gene of Brucella. Subsequently, both a fluorescence assay and a lateral flow strip (LFS) assay were developed after optimizing the conditions using the RPA-CRISPR/Cas12a system. The limit of detection (LoD) was 1 copy/μL for RPA-CRISPR/Cas12a-F and 10 copies/μL for RPA-CRISPR/Cas12a-LFS and the entire assay was completed in less than 30 min. This method demonstrated excellent specificity in distinguishing Brucella from other closely related pathogens. Moreover, the RPA-CRISPR/Cas12a assay showed high concordance with classical quantitative real-time PCR when testing diverse clinical samples (blood, serum, milk, semen, vaginal secretions). Together, these findings make this method a promising tool for Brucella detection, with potential applications in both field surveillance and clinical diagnostics.},
}

*Brucella/genetics/isolation & purification
Animals
*Brucellosis/diagnosis/microbiology
*CRISPR-Cas Systems/genetics
Sensitivity and Specificity
Cattle
Fluorescence
Limit of Detection
Humans

@article {pmid41472258,
year = {2025},
author = {Gladue, DP and O'Mahony, A},
title = {CRISPR Treatments for AI-Designed Synthetic Viruses: Rapid Programmable Countermeasures for Emerging and Engineered Viruses.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472258},
issn = {1999-4915},
mesh = {Humans ; *CRISPR-Cas Systems ; Genome, Viral ; *Artificial Intelligence ; *Synthetic Biology/methods ; *Gene Editing/methods ; RNA, Guide, CRISPR-Cas Systems/genetics ; *Viruses/genetics ; Animals ; Genetic Engineering/methods ; },
abstract = {The convergence of artificial intelligence and synthetic biology is innovating and accelerating the design of novel viral genomes, expanding both therapeutic opportunities and dual-use risk. This review articulates a countermeasure strategy for emerging and engineered viruses leveraging the programmable CRISPR modality. Building on mounting in vitro and in vivo evidence that Cas9 degrades DNA viruses (e.g., Orthopoxviruses, HSV-1, ASFV), while Cas13 targets RNA viral genomes (e.g., Influenza A, Dengue, RSV), both leading to reduced viremia, diminished disease burden, and alleviated symptoms. Here, we outline a rapid-response pipeline to position CRISPR-based countermeasures in translational and pandemic-response frameworks, linking real-time sequencing to AI-assisted gRNA selection and multiplexed cassette design to achieve viral targeting efficacy. To minimize resistance and off-target risk, we emphasize multi-gRNA cocktails, continuous genomic surveillance, and adaptive gRNA rotation. We also propose governance mechanisms, such as pre-cleared gRNA repositories, transparent design logs, standardized off-target/safety screening, and alignment with evolving nucleic-acid-synthesis screening frameworks to enable emergency deployment while preserving security. Furthermore, compressing the time from sequence to treatment and complementary to vaccines and small-molecule antivirals, CRISPR represents a technologically agile and strategically essential capability to combat both natural outbreaks and AI-enabled biothreats. Collectively, programmable CRISPR antivirals represent an auditable, rapidly adaptable foundation for next-generation biodefense preparedness.},
}

Humans
*CRISPR-Cas Systems
Genome, Viral
*Artificial Intelligence
*Synthetic Biology/methods
*Gene Editing/methods
RNA, Guide, CRISPR-Cas Systems/genetics
*Viruses/genetics
Animals
Genetic Engineering/methods

@article {pmid41472250,
year = {2025},
author = {Tan, C and Xing, S and Xie, X and Chen, X and Liu, X and Wang, W and Liu, L and Zhou, X and Wu, J and Li, C},
title = {Development and Application of a Rapid Field Detection Technology for DENV-2 Based on the HUDSON Nucleic Acid Extraction-Free/RT-RAA/CRISPR-Cas12a System.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472250},
issn = {1999-4915},
support = {2024YFC2607800//Chunxiao-Li/ ; },
mesh = {*Dengue Virus/genetics/isolation & purification/classification ; *Dengue/diagnosis/virology ; *CRISPR-Cas Systems ; Humans ; Sensitivity and Specificity ; Animals ; *Nucleic Acid Amplification Techniques/methods ; RNA, Viral/genetics ; Bacterial Proteins ; Endodeoxyribonucleases ; CRISPR-Associated Proteins ; },
abstract = {Dengue fever has become a major global public health challenge due to its rapidly in-creasing incidence. Rapid on-site detection of dengue virus (DENV) is critical for early diagnosis, timely patient isolation, and outbreak control. In this study, dengue virus serotype 2 (DENV-2), the predominant strain circulating in tropical and subtropical regions, was selected as the target pathogen. We established a one-tube rapid detection assay that integrates the HUDSON nucleic acid extraction-free protocol, reverse transcription recombinase-aided amplification (RT-RAA), and CRISPR/Cas12a-mediated trans cleavage activity. The method achieved a detection limit of 1 × 10[2] copies/μL for simulated infected samples and exhibited no cross-reactivity with other DENV serotypes (DENV-1, DENV-3, DENV-4) or with other arboviruses, including Zika, Japanese encephalitis, yellow fever, and chikungunya viruses. The assay demonstrated high sensitivity and specificity across various sample types, including mosquitoes, rodents, blood, and cultured cells, with results consistent with quantitative PCR (qPCR). Requiring only basic equipment such as a water bath, the system enables on-site detection of DENV-2 within 1 h. This simple, cost-effective, and reliable assay provides a practical tool for field-based DENV-2 surveillance and supports effective public health responses in resource-limited settings.},
}

*Dengue Virus/genetics/isolation & purification/classification
*Dengue/diagnosis/virology
*CRISPR-Cas Systems
Humans
Sensitivity and Specificity
Animals
*Nucleic Acid Amplification Techniques/methods
RNA, Viral/genetics
Bacterial Proteins
Endodeoxyribonucleases
CRISPR-Associated Proteins

@article {pmid41472204,
year = {2025},
author = {Dong, J and He, X and Bao, S and Wei, Z},
title = {Diagnostic Methods for Bovine Coronavirus: A Review of Recent Advancements and Challenges.},
journal = {Viruses},
volume = {17},
number = {12},
pages = {},
pmid = {41472204},
issn = {1999-4915},
support = {KJZC-2024-15//Gansu Provincial Department of Agriculture and Rural Affairs Science and Technology Support Project/ ; },
mesh = {Animals ; Cattle ; *Coronavirus, Bovine/genetics/isolation & purification ; *Cattle Diseases/diagnosis/virology ; *Molecular Diagnostic Techniques/methods ; *Coronavirus Infections/diagnosis/veterinary/virology ; Nucleic Acid Amplification Techniques/methods ; Sensitivity and Specificity ; CRISPR-Cas Systems ; },
abstract = {Bovine coronavirus(BCoV) is a significant pathogen causing substantial economic losses in the cattle industry through increased calf mortality, reduced growth performance, and decreased milk yield. Rapid and accurate diagnostic methods are therefore essential for controlling BCoV transmission. Current diagnostic methods comprise two primary categories: conventional techniques and cutting-edge innovations. Conventional approaches, including molecular methods like RT-PCR/qRT-PCR and immunological assays such as ELISA and neutralization tests, remain the main diagnostic methods. However, they are limited by laboratory dependency as well as the necessary balance between speed and sensitivity. These limitations have promoted the development of innovative methods, including isothermal amplification, CRISPR/Cas systems, droplet digital PCR, and integrated platforms. This review comprehensively analyzes the advantages, limitations, and applications of current diagnostic methods, highlighting integrated platforms such as RPA-CRISPR-LFA and microfluidics-based LFA. These innovations bridge critical performance gaps by enhancing sensitivity and specificity while enabling field application, demonstrating significant potential as next-generation point-of-care diagnostics for managing this economically critical pathogen.},
}

Animals
Cattle
*Coronavirus, Bovine/genetics/isolation & purification
*Cattle Diseases/diagnosis/virology
*Molecular Diagnostic Techniques/methods
*Coronavirus Infections/diagnosis/veterinary/virology
Nucleic Acid Amplification Techniques/methods
Sensitivity and Specificity
CRISPR-Cas Systems

@article {pmid41472049,
year = {2025},
author = {Slukin, PV and Fursov, MV and Volkov, DV and Sizova, AA and Detushev, KV and Dyatlov, IA and Fursova, NK},
title = {Diversity of CRISPR-Cas Systems Identified in Urological Escherichia coli Strains.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472049},
issn = {2076-2607},
support = {075-15-2025-525 of 30.05.2025.//Ministry of Science and Higher Education of the Russian Federation/ ; },
abstract = {Type I-E and I-F CRISPR-Cas systems were identified in 237 E. coli strains isolated from patients with urinary tract infections (UTIs) between 2004 and 2019. The strains were classified into nine distinct groups (I-IX) based on the presence or absence of cas genes and repeat regions (RRs). Within the type I-E systems, two sequence variants were identified, distinguished by polymorphisms in the casB, cas3, cas7, cas5, and cas6 genes. The direct repeats (DRs) also differed, with I-E-associated RRs ranging from 26 to 32 bp and I-F-associated RRs consistently being 28 bp. We identified 762 unique spacers (29-35 bp in length) across the strain collection, while the number of spacers per strain varied from 1 to 47, and potential DNA targets were determined for 65 spacers, targeting 38 bacteriophage genomes, 19 plasmids, and 8 cas genes of the I-F type CRISPR-Cas system. Multilocus sequence typing (MLST) revealed 68 sequence types and 24 clonal complexes (CCs), with the most prevalent being ST131, CC10, CC69, CC405, CC14, CC38, CC73, and CC648. Significant correlations were observed between specific phylogroups/CCs, the type of CRISPR-Cas system present, and distinct profiles of virulence and antibiotic resistance genes.},
}

@article {pmid41472024,
year = {2025},
author = {Yi, M and Hu, Y and Fan, B and Pan, Y and Pan, B and Wang, J and Liu, Z},
title = {Advances in Novel Detection Technologies for Occult Hepatitis B Virus Infection: Building an Ultra-Sensitive Barrier for Transfusion Safety.},
journal = {Microorganisms},
volume = {13},
number = {12},
pages = {},
pmid = {41472024},
issn = {2076-2607},
abstract = {Occult hepatitis B virus infection (OBI), characterized by extremely low viral loads and the persistent intrahepatic presence of cccDNA, poses a profound challenge to global public health security. With a prevalence ranging from 0.06% to over 15% in different donor populations, OBI maintains a risk of transmission and can progress to hepatocellular carcinoma. Its prevention and control have long been limited by the sensitivity constraints of conventional detection methods, highlighting the urgent need for more sensitive diagnostic innovations. Emerging technologies offer distinct breakthroughs: ddPCR facilitates absolute quantification; CRISPR-Cas systems coupled with isothermal amplification enable rapid, point-of-care testing; third-generation sequencing resolves viral integration and mutations; and nanomaterials enhance the signal detection. This review synthesises advancements in OBI diagnostic technologies and provides a comparative overview of their strengths, limitations, and transfusion safety implications, as well as their potential applications in blood transfusion. Recommendations are also proposed to inform the advancement of OBI risk control in blood transfusion and to guide the development of novel diagnostic technologies, particularly relevant to regions with high HBV endemicity, such as China.},
}

@article {pmid41251053,
year = {2026},
author = {Shrestha, M and Kim, Y and Park, S and Jiang, HL and Lee, JY and Kim, D and Jeong, JH},
title = {eVLP-Mediated Cas9 Delivery for Preventing IBMIR in Islet Transplantation.},
journal = {Small (Weinheim an der Bergstrasse, Germany)},
volume = {22},
number = {1},
pages = {e08128},
doi = {10.1002/smll.202508128},
pmid = {41251053},
issn = {1613-6829},
support = {//National Research Foundation of Korea/ ; RS-2025-02303064//Korean government/ ; RS-2024-02507183//Ministry of Health and Welfare/ ; 22A0205L1//Ministry of Health and Welfare/ ; 23A0205L1//Ministry of Health and Welfare/ ; 21A0202L1//Ministry of Health and Welfare/ ; 21A0202L1//Ministry of Science and ICT, South Korea/ ; },
mesh = {Animals ; *Islets of Langerhans Transplantation/adverse effects/methods ; Thromboplastin/metabolism/genetics ; Plasminogen Activator Inhibitor 1/metabolism/genetics ; Rats ; Diabetes Mellitus, Experimental/therapy ; Male ; *Inflammation/prevention & control ; *CRISPR-Associated Protein 9/metabolism ; Mice ; Islets of Langerhans ; *CRISPR-Cas Systems/genetics ; },
abstract = {Islet transplantation is a promising strategy for effective β-cell replacement in patients with type 1 diabetes. However, its success is hindered significantly by instant blood-mediated inflammatory reaction (IBMIR), which leads to rapid graft loss. IBMIR is triggered when the transplanted islets come in contact with blood, activating the coagulation cascade, complement pathways, and innate immune responses. Tissue factor (TF), abundantly expressed on the islet surface, initiates the coagulation cascade, leading to thrombin formation, platelet activation, and neutrophil infiltration. Plasminogen activator inhibitor-1 (PAI-1) plays a critical role in IBMIR by inhibiting fibrinolysis and causing ischemic injury in the graft. TF and PAI-1 contribute significantly to IBMIR, thus making them critical targets for genetic interventions to prevent IBMIR. In this study, an engineered virus-like particle (eVLP)-mediated Cas9 nuclease is employed to knock out TF and PAI-1 genes in rat islets. TF and PAI-1 expression are effectively downregulated without inducing any off-target effects or without compromising the viability and functionality of the islets. Streptozotocin-induced diabetic mice transplanted with TF- and PAI-1-knockout islets exhibited improved glycemic control and a significant reduction in the plasma levels of thrombin-antithrombin (TAT) complex and complement component 3a (C3a), indicating the successful inhibition of IBMIR post-transplantation.},
}

Animals
*Islets of Langerhans Transplantation/adverse effects/methods
Thromboplastin/metabolism/genetics
Plasminogen Activator Inhibitor 1/metabolism/genetics
Rats
Diabetes Mellitus, Experimental/therapy
Male
*Inflammation/prevention & control
*CRISPR-Associated Protein 9/metabolism
Mice
Islets of Langerhans
*CRISPR-Cas Systems/genetics

@article {pmid40899441,
year = {2026},
author = {Patel, UA and Shi, MY and Kazan, JM and Nixon, KCJ and Ran, X and Nair, SN and Huang, O and Song, L and Aparnathi, MK and He, MY and Bakhtiari, M and Krishnan, R and Hessenow, RK and Philip, V and Ketela, T and Jendrossek, V and Hakem, R and He, HH and Kridel, R and Lok, BH},
title = {CRISPR Screen Identifies HDAC3 as a Novel Radiosensitizing Target in Small Cell Lung Cancer.},
journal = {Molecular cancer therapeutics},
volume = {25},
number = {1},
pages = {183-195},
pmid = {40899441},
issn = {1538-8514},
support = {U01 CA253383/CA/NCI NIH HHS/United States ; UL1 TR000457/TR/NCATS NIH HHS/United States ; //Terry Fox Research Institute (TFRI)/ ; //Canada Foundation for Innovation (CFI)/ ; //Cancer Research Society (CRS)/ ; //Canadian Institutes of Health Research (CIHR)/ ; U01CA253383//National Cancer Institute (NCI)/ ; UL1TR00457//Clinical and Translational Science Center, Weill Cornell Medicine (CTSC)/ ; GRK 2762/1//Deutsche Forschungsgemeinschaft (DFG)/ ; },
mesh = {Humans ; Animals ; *Small Cell Lung Carcinoma/genetics/pathology/radiotherapy/drug therapy ; *Histone Deacetylases/genetics/metabolism ; Mice ; *Lung Neoplasms/genetics/pathology/radiotherapy ; *Radiation-Sensitizing Agents/pharmacology ; Cell Line, Tumor ; Xenograft Model Antitumor Assays ; CRISPR-Cas Systems ; Radiation Tolerance/genetics/drug effects ; *Clustered Regularly Interspaced Short Palindromic Repeats ; Acrylamides ; Phenylenediamines ; },
abstract = {Small cell lung cancer (SCLC) is an aggressive malignancy, with most patients presenting with prognostically poor extensive-stage disease. Limited progress in standard care stresses the urgent need for novel therapies. Radiotherapy offers some survival benefit for selected patients with SCLC but could be enhanced with radiosensitizers. In this study, we identify HDAC3 as a novel radiosensitizing target in SCLC using a CRISPR knockout screen and demonstrate its efficacy and mechanism. SBC5 cells were transduced with a custom EpiDrug single-guide RNA library and treated with ionizing radiation (IR) to identify radiosensitizing genes. HDAC3 emerged as a candidate and was validated through genetic knockdown and pharmacologic inhibition (RGFP966) in multiple SCLC cell lines. Both approaches enhanced radiosensitivity, as shown by cell viability (dose modification factor10 = 1.14-1.69) and clonogenic assays (dose modification factor10 = 1.16-1.41). We assessed changes in chromatin accessibility by assay for transposase-accessible chromatin using sequencing and IR-induced DNA damage and repair using γH2AX foci detection, double-strand break (DSB) repair assays, and immunoblotting of repair proteins. HDAC3-deficient cells exhibited increased chromatin accessibility, greater IR-induced DSBs, and impaired repair capacity, resulting in persistent DNA damage. This repair defect sensitized cells to PARP inhibitors, for which combining RGFP966 with olaparib or talazoparib produced additive to synergistic effects. In SCLC xenograft models, HDAC3 knockdown or RGFP966, combined with IR, achieved significant tumor growth inhibition. Collectively, we identified HDAC3 as a novel radiosensitizing target in SCLC. Its functional loss increased the generation and persistence of IR-induced DNA DSBs, effectively sensitizing SCLC cell lines and xenografts to IR, providing a potential radiosensitization strategy to treat SCLC.},
}

Humans
Animals
*Small Cell Lung Carcinoma/genetics/pathology/radiotherapy/drug therapy
*Histone Deacetylases/genetics/metabolism
Mice
*Lung Neoplasms/genetics/pathology/radiotherapy
*Radiation-Sensitizing Agents/pharmacology
Cell Line, Tumor
Xenograft Model Antitumor Assays
CRISPR-Cas Systems
Radiation Tolerance/genetics/drug effects
*Clustered Regularly Interspaced Short Palindromic Repeats
Acrylamides
Phenylenediamines

@article {pmid41471222,
year = {2025},
author = {Nass, NM and Zaher, KA},
title = {From Methylomes to CRISPR Epigenetic Editing: New Paths in Antibiotic Resistance.},
journal = {Pathogens (Basel, Switzerland)},
volume = {14},
number = {12},
pages = {},
doi = {10.3390/pathogens14121267},
pmid = {41471222},
issn = {2076-0817},
mesh = {*Gene Editing/methods ; *Epigenesis, Genetic ; *Bacteria/genetics/drug effects ; *CRISPR-Cas Systems ; Anti-Bacterial Agents/pharmacology ; *Drug Resistance, Bacterial/genetics ; Humans ; DNA Methylation ; *Epigenome ; *Drug Resistance, Microbial/genetics ; Gene Expression Regulation, Bacterial ; Epigenome Editing ; },
abstract = {Antibiotic resistance (AR) has long been interpreted through the lens of genetic mutations and horizontal gene transfer. Yet, mounting evidence suggests that epigenetic regulation, including DNA and RNA methylation, histone-like proteins, and small non-coding RNAs, plays a similarly critical role in bacterial adaptability. These reversible modifications reshape gene expression without altering the DNA sequence, enabling transient resistance, phenotypic heterogeneity, and biofilm persistence under antimicrobial stress. Advances in single-molecule sequencing and methylome mapping have uncovered diverse DNA methyltransferase systems that coordinate virulence, efflux, and stress responses. Such epigenetic circuits allow pathogens to survive antibiotic exposure, then revert to susceptibility once pressure subsides, complicating clinical treatment. Parallel advances in CRISPR-based technologies now enable direct manipulation of these regulatory layers. CRISPR interference (CRISPRi) and catalytically inactive dCas9-fused methyltransferases can silence or reactivate genes in a programmable, non-mutational manner, offering a new route to reverse resistance or sensitize pathogens. Integrating methylomic data with transcriptomic and proteomic profiles further reveals how epigenetic plasticity sustains antimicrobial tolerance across environments. This review traces the continuum from natural bacterial methylomes to engineered CRISPR-mediated epigenetic editing, outlining how this emerging interface could redefine antibiotic stewardship. Understanding and targeting these reversible, heritable mechanisms opens the door to precision antimicrobial strategies that restore the effectiveness of existing drugs while curbing the evolution of resistance.},
}

*Gene Editing/methods
*Epigenesis, Genetic
*Bacteria/genetics/drug effects
*CRISPR-Cas Systems
Anti-Bacterial Agents/pharmacology
*Drug Resistance, Bacterial/genetics
Humans
DNA Methylation
*Epigenome
*Drug Resistance, Microbial/genetics
Gene Expression Regulation, Bacterial
Epigenome Editing